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An Arabidopsis ABC Transporter Mediates Phosphate Deficiency-Induced Remodeling of Root Architecture by Modulating Iron Homeostasis in Roots
Accepted Manuscript An Arabidopsis ABC transporter mediates phosphate deficiency-induced remodeling of root architecture by modulating iron homeostasis in roots Jinsong Dong, Miguel A. Piñeros, Xiaoxuan Li, Haibing Yang, Yu Liu, Angus S. Muphy, Leon V. Kochian, Dong Liu PII: DOI: Reference:
S1674-2052(16)30270-2 10.1016/j.molp.2016.11.001 MOLP 388
To appear in: MOLECULAR PLANT Accepted Date: 5 November 2016
Please cite this article as: Dong J., Piñeros M.A., Li X., Yang H., Liu Y., Muphy A.S., Kochian L.V., and Liu D. (2016). An Arabidopsis ABC transporter mediates phosphate deficiency-induced remodeling of root architecture by modulating iron homeostasis in roots. Mol. Plant. doi: 10.1016/ j.molp.2016.11.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in MOLECULAR PLANT are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs.
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Full title:
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An Arabidopsis ABC transporter mediates phosphate deficiency-induced remodeling
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of root architecture by modulating iron homeostasis in roots
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Full names and affiliations of authors:
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Jinsong Dong1, Miguel A. Piñeros2, Xiaoxuan Li1, Haibing Yang3, Yu Liu4, Angus S.
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Muphy5, Leon V. Kochian2, Dong Liu1*
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1. Ministry of Education Key Laboratory of Bioinformatics, Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China 2. USDA-ARS, Robert Holley Center for Agriculture and Health, Cornell University, Ithaca, NY 14580 USA 3. Department of Horticulture, Purdue University, West Lafayette, Indiana 47907-2010 4. State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, China 5. Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD 20742, USA
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*Corresponding author
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E-mail: [email protected]
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Running title:
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Pi deficiency-regulated root development
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ACCEPTED MANUSCRIPT Short summary
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The Arabidopsis ALUMINUM SENSITIVE3 (ALS3) and AtSTAR1 form an ABC transporter
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complex in the tonoplasts. It acts with LOW PHOSPHATE ROOT1/2 (LPR1/2), two
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ferroxidases, to mediates phosphate deficiency-induced inhibition of primary root growth by
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modulating root iron homeostasis. The functional disruption of this ABC transporter may also
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affect integrity of cell wall architecture.
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ABSTRACT
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The remodeling of root architecture is a major developmental response of plants to
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phosphate (Pi) deficiency and is thought to enhance a plant’s ability to forage Pi in
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topsoil. The underlying mechanism controlling this response, however, is poorly
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understood. In this work, we identified an Arabidopsis mutant, hps10 (hypersensitive
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to Pi starvation 10), that is morphologically normal under Pi sufficiency but shows
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increased inhibition of primary root growth and enhanced production of lateral roots
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under Pi deficiency. hps10 is a previously identified allele (als3-3) of the
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ALUMINUM SENSITIVE3 (ALS3) gene, which is involved in plant tolerance to
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aluminum toxicity. Our results show that ALS3 and its interacting protein AtSTAR1
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form an ABC transporter complex in tonoplasts. This protein complex mediates a
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highly electrogenic transport in Xenopus oocytes. Under Pi deficiency, als3
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accumulates higher levels of Fe3+ in its roots than the wild type. In Arabidopsis, LPR1
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(LOW PHOSPHATE ROOT1) and LPR2 encode ferroxidase, which when mutated
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reduces Fe3+ accumulation in roots and causes root growth to be insensitive to Pi
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deficiency. Here, we provide compelling evidence that ALS3 acts with LPR1/2 to
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regulate Pi deficiency-induced remodeling of root architecture by modulating Fe
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homeostasis in roots.
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Keywords:
phosphate deficiency,
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transporter
ALUMINUM SENSITIVE3,
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root architecture, AtSTAR1 2
iron homeostasis,
ABC
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INTRODUCTION Root development is a postembryonic process that is highly plastic in responding
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to fluctuations in nutrient levels in the environment. Root system architecture (RSA)
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is a major determinant of the plant’s ability to acquire water and nutrients from the
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soil (Lynch, 1995). Phosphate (Pi), the major form for phosphorus (P) uptake and
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assimilation by roots, decreases with soil depth. When grown under Pi deficiency,
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plants decrease their primary root growth but increase the production of lateral roots
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(Lopez-Bucio et al., 2003; Desnos, 2008). Such remodeling of RSA is thought to
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maximize a plant’s potential to exploit the Pi resource in topsoil. Although the Pi
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deficiency-induced remodeling of RSA has been well documented in a variety of plant
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species (Vance et al., 2003), the underlying molecular mechanism for this adaptive
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response remains largely unknown.
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Pi deficiency-induced remodeling of root development has been shown to be an
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active cellular process that is determined by an internal genetic program rather than a
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consequence of reduced metabolic activity due to nutrient shortage (Péret et al., 2014).
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This remodeling process is triggered by a decrease in local, external Pi levels, with the
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root tip playing a key role in the sensing of change in Pi status in the environment
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(Ticconi et al., 2004, 2009; Svistoonoff et al., 2007; Thibaud et al., 2010). Forward
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genetics has identified several key molecular components involved in the control of
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primary root growth under Pi deficiency. The Arabidopsis pdr2 (phosphate deficiency
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responses2) mutant is hypersensitive to Pi deficiency-induced inhibition of primary
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root growth (Ticconi et al., 2004). In contrast, the primary root growth of lpr1 (low
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phosphate root1) is insensitive to Pi deficiency (Svistoonoff et al., 2007). PDR2
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encodes an endoplasmic reticulum (ER)-localized P5-type ATPase (Ticconi et al.,
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2009), and LPR1 and its close homolog LPR2 belong to a large family of multicopper
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oxidases (Svistoonoff et al., 2007). Genetic analysis indicates that PDR2 and
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LPR1/LPR2 act in the same pathway, with LPR1/LPR2 being epistatic to PDR2
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(Ticconi et al., 2009). However, how these proteins interact to control the Pi
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deficiency-induced remodeling of RSA is still unclear.
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Interestingly, iron (Fe) is essential for Pi deficiency-induced inhibition of 3
ACCEPTED MANUSCRIPT primary root growth, with Pi-deficient plants accumulating a high level of Fe in the
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roots (Misson et al., 2005; Hirsch et al., 2006; Svistoonoff et al., 2007; Ward et al.,
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2008; Zheng et al., 2009). When plants are grown on a Pi-deficient (P-) medium
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without Fe, the inhibition of primary root growth is abolished (Ward et al., 2008).
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Subsequent studies demonstrated that both LPR1 and LPR2 proteins possess
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ferroxidase activity (Müller et al., 2015). On P- medium, the accumulation of Fe3+ in
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lpr1 was significantly reduced; in contrast, pdr2 accumulated a higher level of Fe3+
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than the wild type (WT). Müller et al (2015) proposed that plants subjected to Pi
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deficiency over-accumulate Fe3+, which generates a high level of reactive oxygen
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species (ROS), resulting in increased deposition of callose in cell walls and
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plasmodesmata (PD). The enhanced callose deposition in PD then interferes with the
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intercellular movement of SHR (SHORT ROOT) protein, a key transcription factor
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involved in the maintenance of the root stem cell niche (SCN), and thus impairs
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primary root growth. However, researchers have yet to identify the other components
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of the PDR2-LPR1/2 pathway that regulates Pi deficiency-induced remodeling of
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RSA by modulating Fe homeostasis in roots.
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In this work, we characterized an Arabidopsis mutant, hps10, which is
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hypersensitive to Pi deficiency-induced inhibition of primary root growth and
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enhanced lateral root formation. We show that hps10 is a previously identified allele
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(als3-3) of the ALUMINUM SENSITIVE3 (ALS3) gene that is implicated in plant
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tolerance to aluminium (Al) toxicity (Larsen et al., 2005). We further demonstrate that
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ALS3 and its interacting protein, AtSTAR1, form an ABC transporter complex in
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tonoplasts. Based on the results obtained, we propose that this transporter acts with
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LPR1/LPR2 to regulate Pi deficiency-induced remodeling of RSA through
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modulation of Fe homeostasis in roots.
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RESULTS
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als3-3 is Hypersensitive to Pi deficiency-Induced Remodeling of RSA An Arabidopsis mutant, hps10 (hypersensitive to Pi starvation10), was 4
ACCEPTED MANUSCRIPT identified from a T-DNA insertion library (Alonso et al., 2003). hps10 was found to be
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the Salk_004094 line. This T–DNA line was previously characterized as als3-3
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(aluminum sensitive3-3), which displays a hypersensitive response to Al toxicity
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(Larsen et al., 2005). When grown on a Pi-sufficient (P+) medium (1/2 MS medium
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with 1% sucrose and 1.2% agar), the roots of the als3-3 mutant did not differ
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morphologically from those of the WT (Figure 1A). In contrast, when grown on a P-
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medium, the length of als3-3 primary root was about 50% shorter than that of the WT.
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Accordingly, the length of root epidermal cells in the maturation zone were half as
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long in als3-3 as in the WT (Supplemental Figure 1A). At 11 DAG (days after
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germination), the root apical meristem (RAM) of the WT was 50% smaller on P-
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medium than on P+ medium, with the cellular organization of the RAM remaining
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well maintained (Figure 1B). At that time, the root meristematic cells of Pi-deficient
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als3-3 had lost their identity, becoming enlarged and highly vacuolated, and had
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prematurely differentiated into root hairs. Under Pi deficiency, lateral roots appeared
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at 5 DAG for als3-3 but at 7 DAG for the WT. By 8 DAG, lateral root density was two
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times greater for als3-3 than for the WT (Supplemental Figure 1B). On P- medium,
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although the primary root of als3-3 stopped growing by 4 DAG, its lateral roots
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continued to elongate. Under Pi deficiency, als3-3 also formed more and longer root
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hairs than the WT (Supplemental Figure 1C). A Pi concentration-dependent analysis
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indicated that Pi levels < 100 µM were required to distinguish root phenotypic
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differences between als3-3 and the WT (Supplemental Figure 2).
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When als3-3 was backcrossed to the WT, all F1 plants showed WT phenotypes,
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and F2 progeny derived from selfed F1 plants segregated into mutant and WT
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phenotypes in a ratio of 1:3 (52:169), indicating that the als3-3 mutant phenotype was
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caused by a single recessive mutation. als3-3 was backcrossed to the WT four times
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before further characterization.
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als3-3 Over-accumulates Fe3+ in Roots Under Pi Deficiency
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We then examined the level of Fe in the roots of 8-day-old seedlings using the
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Perls staining method, which mainly stains labile (non-heme) Fe3+ (Merguro et al., 5
ACCEPTED MANUSCRIPT 2007). In both the WT and als3-3 grown on P+ medium, the blue staining of Fe3+ was
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mainly localized in the SCN, which includes the quiescent center (QC) and its
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surrounding initials, as well as in the cortex of the root apex (Figure 2A). This was
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consistent with the observations of Müller et al (2015). On P- medium, Fe3+
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accumulation in the SCN and cortex of WT roots was dramatically reduced and was
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undetectable in most samples; instead, a light-blue staining was evident proximal to
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the root apex, mostly in the maturation zone. For als3-3 on P- medium, however,
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there was a light-blue staining in the elongation zone and a dark-blue staining in the
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maturation zone. A similar Fe accumulation pattern was observed when a more
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sensitive Perls/DAB (diaminobenzidine) staining method was used (Figure 2B). In
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this method, the Perls staining was intensified with the addition of DAB, which stains
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both Fe2+ and Fe3+ (Roschzttardtz et al., 2009). With the Perls/DAB staining method,
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a low level of Fe accumulation was detected in the SCN and in all root cell layers in
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the root apex except the epidermis. Quantitative analysis indicated that the total Fe
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content was about four times higher in P- roots than in P+ roots of the WT (Figure 3).
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The Fe content in the roots of P- als3-3 was, however, only 25% higher than that in
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the roots of P- WT, indicating that the overstaining of Fe in P- als3-3 was mainly due
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to the conversion of Fe 2+ to Fe3+ rather than to an increase in total Fe content.
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When grown on P- medium in the absence of Fe, the root morphology of als3-3
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did not differ from that of the WT (Supplemental Figure 3), and Fe staining was not
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detected in either als3-3 or WT roots by the Perls method (Figure 2A). A very weak
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staining pattern was observed for roots of als3-3 and WT using the Perls/DAB
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method (Figure 2B). These results suggested that the enhanced inhibition of primary
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root growth of Pi-deficient als3-3 might be due to the over-accumulation of Fe3+ in its
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roots.
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Mutation in ALS3 Causes A Hypersensitive Root Response to Pi Deficiency
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als3-3 carries a T-DNA insertion near the end of the second exon of the gene
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AT2G37330 (Figure 4A). No transcript of AT2G37330 was detected in this line by
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RT-PCR analysis, indicating that als3-3 is a strong allele (Supplemental Figure 4A). 6
ACCEPTED MANUSCRIPT The AT2G37330 gene is 1,050 bp long and contains three exons and two introns.
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AT2G37330 was previously identified as ALS3 (ALUMINUM SENSITIVE 3), because
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its mutation resulted in plants being hypersensitive to Al toxicity (Larsen et al., 2005).
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ALS3 encodes a half-ABC (ATP-binding cassette) transporter-like protein that has
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seven predicted transmembrane domains (TMD) but lacks nucleotide-binding
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domains (NBDs). We then examined the Pi deficiency responses of als3-1 and als3-2
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(Larsen et al., 2005) and another T-DNA insertion allele (SALK_061074, designated
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als3-4). als3-1 carries a substitution of T for C at nucleotide 335, resulting in the
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conversion of a serine to a leucine (Larsen et al., 2005), and als3-2 and als3-4 contain
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T-DNA insertions in the 5’ UTR and the third exon of the AT2G37330 gene,
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respectively (Figure 4A). Like als3-3, all other als3 alleles were morphologically
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normal under Pi sufficiency but exhibited enhanced inhibition of primary root growth
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under Pi deficiency (Figure 4C). The root growth inhibition was less for als3-2 than
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for the other three als3 alleles. This was probably due to a residual expression of the
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ALS3 gene in this SALK line (Supplemental Figure 4A). To confirm that the T-DNA
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insertion in the AT2G37330 gene was responsible for the mutant phenotypes of
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Pi-deficient als3-3, we introduced the genomic sequence of the WT AT2G37330 gene
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into als3-3 under the control of the CaMV 35S promoter or its own promoter. Both
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gene constructs could fully complement the mutant root phenotypes (Figure 4D),
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demonstrating that the T-DNA insertion in the AT2G37330 gene was responsible for
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the Pi-hypersensitive phenotype of als3-3.
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The growth phenotypes of the WT and als3-3 were also examined under nitrogen
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(N), potassium (K), and Fe deficiencies, as well as under low pH (4.2). None of these
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stress treatments resulted in a growth difference between als3-3 and WT
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(Supplemental Figure 5), suggesting ALS3 specificity in the processes underlying Pi
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deficiency and Al toxicity responses.
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AtSTAR1 and ALS3 Function Similarly in Plant Responses to Pi Deficiency
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ABC transporters represent a large family in Arabidopsis; they are localized in
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different organelles, transport a diverse array of substrates, and thereby function in a 7
ACCEPTED MANUSCRIPT wide range of physiological processes (Kang et al., 2011). A functional ABC
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transporter is composed of at least one TMD and one NBD. Given that ALS3 lacks an
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NBD, it must act with a partner protein to transport its substrate(s). OsSTAR1 and
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OsSTAR2 were previously reported to form a functional ABC transporter that is
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required for Al tolerance in rice (Huang et al., 2009). OsSTAR1 contains only an
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NBD, while OsSTAR2, the counterpart of ALS3 in rice, contains only a TMD.
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Knockout of AtSTAR1 (ABCI17, AT1G67940), the counterpart of OsSTAR1 in
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Arabidopsis, also resulted in plants that were hypersensitive to Al toxicity (Huang et
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al., 2010). These observations, in conjunction with the phenotypes described above
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for als3-3, suggested that AtSTAR1 and ALS3 might form a protein complex that
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functions as an ABC transporter in Arabidopsis. Consequently, if AtSTAR1 and
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ALS3 form a functional protein complex, the knockout of AtSTAR1 should also result
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in plants showing a hypersensitive phenotype to Pi deficiency. The strong allele of the
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AtSTAR1 gene, atstar1 (ABRC stock no. CS384144) contains a T-DNA insertion in
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its second exon that strongly decreases the transcription of the AtSTAR1 gene (Figure
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4B and Supplemental Figure 4B). We then examined the atstar1 phenotype in
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response to Pi deficiency. The root growth inhibition in P- medium was significantly
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greater for atstar1 than for the WT (Figure 4E) and was similar in degree to that
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exhibited by als3-3. Like als3-3, atstar1 also over-accumulated Fe3+ in its roots on P-
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medium (Figure 5). These phenotypic similarities indicated that AtSTAR1 and ALS3
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components of common physiological mechanism involved in plant responses to
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both Pi deficiency and Al toxicity. Furthermore, on our standard P- medium (1/2 MS
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medium with 1% sucrose and 1.2% agar without Pi), the growth of plants
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overexpressing either ALS3 or AtSTAR1 alone was similar to that of plants
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concurrently overexpressing AtSTAR1 and ALS3. This was potentially due to residual
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Pi (about 10 µM Pi) remaining in the agar, which was not stressful enough to
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distinguish the Pi deficiency responses among the plants with different genotypes. We
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then replaced agar in our standard P- medium with agarose so that the plants grown
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on this modified medium would be more Pi starved. On this agarose-containing
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medium, the transgenic plants concurrently overexpressing AtSTAR1 and ALS3 grew
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significantly better than the plants overexpressing either ALS3 or AtSTAR1 alone
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(Supplemental Figure 6), thereby providing further evidence for ALS3 and AtSTAR1
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interactions.
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ALS3 and AtSTAR1 Directly Interact at the Tonoplasts
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Several approaches were taken to validate the inferred ALS3 and AtSTAR1
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interactions. First, a yeast split-ubiquitin two-hybrid approach (Johnsson and
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Varshavsky, 1994) was used, where ALS3 was fused to the C-terminal half of the
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ubiquitin gene (Cub), and AtSTAR1 was fused to the mutated N-terminal half of the
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ubiquitin gene (NubG). As the negative controls, the co-transformation of ALS3-Cub
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with the NubG empty vector or co-transformation of the Cub empty vector and
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NubG-AtSTAR1 did not enable the yeast cells to grow on the selective medium. In
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contrast, the yeast cells co-transformed with ALS3-Cub and NubG-AtSTAR1
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constructs were able to grow on the selective medium, indicating that ALS3 and
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AtSTAR1 interacted to activate the expression of the reporter gene for growth
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selection (Figure 6A).
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The ALS3 and AtSTAR1 interaction in planta was then validated in the leaves
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of
Nicotiana
benthamiana
via
two
independent
approaches:
luciferase
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complementation imaging (LCI) assays (Chen et al., 2008) and bimolecular
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fluorescence complementation (BiFC) assays. For LCI, the coding sequences (CDSs)
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of ALS3 and AtSTAR1 were fused to the N- and C-terminal half of the luciferase
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(LUC) gene, respectively, resulting in the gene constructs ALS3-nLUC and
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cLUC-AtSTAR1. Transient co-expression of these chimeras under the control of the
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CaMV 35S promoter resulted in strong LUC activity (Figure 6B). In contrast,
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co-expression of nLUC and cLUC, nLUC and cLUC-AtSTAR1, or ALS3-nLUC and
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cLUC did not result in LUC activity. For BiFC analysis of the ALS3-AtSTAR1
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interaction, the CDSs of ALS3 and AtSTAR1 were fused to the N- and C-terminal
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halves, respectively, of the yellow fluorescence protein (YFP) gene. Transient
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co-expression of ALS3-nYFP with cYFP-AtSTAR1 under the control of the CaMV 35S
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promoter restored the YFP fluorescence (Figure 6C). The fluorescence signal in intact
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ACCEPTED MANUSCRIPT leaf tissue was observed predominantly in the periphery of leaf cells, with an
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additional band surrounding the nucleus. Released vacuoles from the isolated
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mesophyll protoplasts from transformed leaves exhibited the YFP signal, which was
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associated with the tonoplast (Figure 6D). The vacuolar localization of the
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ALS3-AtSTAR1 interaction was further confirmed by transient expression of the
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BiFC constructs in protoplasts isolated from mesophyll cells of Arabidopsis, which
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resulted in a YFP signal that did not co-localize with the plasma membrane-specific
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dye, FM4-64 (Figure 6E). The absence of the fluorescence signal in specific regions
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of the cell periphery (usually near the places where the chloroplasts were located)
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provided additional evidence that ALS3 and AtSTAR1 interact at the tonoplast.
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Co-expression of either ALS3-nYFP or cYFP-AtSTAR1 with the corresponding half of
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the YFP empty vector did not result in a fluorescence signal in any of the above BiFC
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assays.
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To further confirm the tonoplast localization of the ALS3/AtSTAR1 protein complex, the HA-tagged ALS3 (ALS3-HA) and FLAG-tagged
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(AtSTAR1-FLAG) were transiently co-expressed in the leaves of N. benthamiana by
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Agrobacterium infection. After 48 h, the total proteins were isolated from the infected
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leaves and subjected to Western blot using HA- and FLAG-specific antibodies. The
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results indicated that both proteins were successfully expressed in the leaves of N.
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benthamiana (Figure 6F). Next, the total proteins were separated into soluble and
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membrane fractions. The ALS3-HA was exclusively detected in the membrane
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fraction whereas AtSTAR1-FLAG was found in both soluble and membrane fractions
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(Figure 6G). Finally, we performed sucrose density-gradient centrifugation using
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tonoplast- and plasma membrane-specific markers to determine the subcellular
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localization of the ALS3/AtSTAR1 complex. The results showed that the distributions
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of ALS3-HA and AtSTAR1-FLAG completely overlapped with the distribution of the
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tonoplast-specific marker, V-ATPase, but not with distribution of the plasma
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membrane-specific marker, H+-ATPase (Figure 6H).
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Expression of ALS3 and AtSTAR1 Is Induced by Pi Deficiency 10
ACCEPTED MANUSCRIPT To determine whether the expression of ALS3 is affected by Pi availability, we
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first performed quantitative real-time PCR (qPCR) assays using mRNA extracted
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from 8-day-old WT seedlings. Pi deficiency induced upregulation of ALS3 in both
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shoots and roots (Supplemental Figure 7A). To identify the tissues where ALS3
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expression is induced, we fused a DNA fragment upstream of its transcription start
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site and its genomic sequence to a GUS reporter gene (ALS3::ALS3-GUS) according
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to Larsen et al. (2010) and transformed this gene construct into WT plants. On P+
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medium, GUS activity was only detected in the vascular tissues of roots and
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cotyledons of 8-day-old seedlings (Supplemental Figure 7B). In the root tips of P-
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seedlings, the GUS expression domain extended from vascular tissues to all layers of
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roots cells, including root hairs. No GUS expression was detected in the root
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meristem under either P+ or P- conditions.
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The expression of AtSTAR1 was also slightly induced by Pi deficiency as
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determined by qPCR (Supplemental Figure 7C). To determine its tissue-specific
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expression patterns, we constructed an AtSTAR1::AtSTAR1-GUS gene chimera
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according to Huang et al. (2010) and transformed it into WT plants. In the 8-day-old
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AtSTAR1::AtSTAR1-GUS seedlings, the GUS gene was expressed in all parts of the
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root except for the meristem (Supplemental Figure 7D). GUS staining was further
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enhanced by Pi deficiency in the roots.
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previously investigated in Xenopus laevis oocytes, and the research indicated that the
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protein complex transports UDP-glucose (UDP-Glc) but not UDP-glucuronic acid
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(UDP-GlcA) or UDP-galactose (UDP-Gal) (Huang et al., 2009). Because
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exogenously applied UDP-Glc alleviated the toxic effect of Al on the root growth of
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rice star1 seedlings (Huang et al., 2009), we determined whether UDP-Glc and its
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related compounds had similar effects on als3-3. The results showed that inclusion of
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500 µM UDP-Glc or UDP-GlcA, but not UDP, glucose, or UDP-Gal, completely
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rescued the short-root phenotype of als3-3 on P- medium (Figure 7). The lowest 11
ACCEPTED MANUSCRIPT 1
effective concentration of UDP-Glc was 100 µM (Supplemental Figure 8). UDP-Glc
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could also rescue the root phenotypes of atstar1 grown on P- medium (Figure 7).
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AtSTAR1 and ALS3 Interact to Mediate a Highly Electrogenic Transport in
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Xenopus Oocytes
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we investigated their electrophysiological properties when expressed in Xenopus
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oocytes. The ALS3-AtSTAR1 interaction was first validated via BiFC, by coinjection
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of cRNA chimeras. YFP signal was detected upon co-injection of ALS3::nYFP and
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cYFP::AtSTAR1 (Figure 8A), while no background YFP signal was detected in cells
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co-injected with ALS3::nYFP and the cRNA encoding the complementary cYFP.
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Having validated the ALS3-AtSTAR1 structural interactions, we then determined the
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functional interactions by examining the transport characteristics of ALS3, AtSTAR1,
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and the protein complex via two-electrode voltage clamp (TEVC) of Xenopus oocytes.
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Cells injected only with ALS3 or AtSTAR1 cRNA had resting membrane potentials
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(RMPs) resembling the values in control cells (i.e., cells injected with water). In
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contrast, cells co-injected with ALS3 and AtSTAR1 cRNA had significantly less
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negative RMPs than control cells (Figure 8B). Under voltage clamp conditions, cells
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expressing only ALS3 or AtSTAR1 did not show a significant difference in
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electrogenic transport compared to that recorded in control cells. In contrast, cells
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co-expressing ALS3 and AtSTAR1 showed large, slowly activating inward (negative)
22
currents (Figure 8C and 8D), which reversed (i.e., change in current sign) at a
23
potential significantly less negative than those recorded in cells expressing only ALS3
24
or AtSTAR1, or in control cells. By convention, the ALS3/AtSTAR1-mediated inward
25
(negative) currents are the product of net positive charge influx (e.g., Na+ or H+) or
26
net negative charge efflux (e.g., organic or inorganic anions), This inference is
27
consistent with the depolarization state (i.e., reduction of the net internal negative
28
charge) recorded in ALS3+AtSTAR1, relative to that found in control cells or in cells
29
expressing only ALS3 or AtSTAR1.
30
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We also determined whether the ALS3/AtSTAR1 complex could mediate Pi or 12
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2
was insensitive to increases in intracellular Pi concentrations (i.e., loading up to 3
3
days) or UDP-Glc (i.e., microinjected) (Supplemental Figure 9). Overall, the above
4
results validated a functional interaction between ALS3 and AtSTAR1, and indicated
5
that this protein complex mediates significant transport across the membrane;
6
nonetheless, the nature of the endogenous (in oocytes) and/or in planta substrate(s)
7
for the ALS3/AtSTAR1 transporter remains unknown.
8
ALS3 and LPR1/2 Act in the Same Regulatory Pathway
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In Arabidopsis, LPR1 and its homolog LPR2 play important roles in regulating Pi
11
deficiency-induced inhibition of primary root growth (Svistoonoff et al., 2007). These
12
two proteins possess ferroxidase activity (Müller et al., 2015). lpr1 and lpr2 mutants
13
accumulated less Fe3+ in their roots than the WT and were insensitive to Pi
14
deficiency-induced inhibition of primary root growth. To determine whether ALS3
15
and LPR1/2 act in the same regulatory pathway, we constructed the triple mutant
16
als3-3lpr1lpr2. In our standard agar-containing P- medium, the primary root growth
17
of lpr1lpr2 was similar to that of the WT. Therefore, we used a modified
18
agarose-containing P- medium as mentioned earlier to test plant growth phenotypes.
19
In this modified medium, lpr1lpr2 was less sensitive to Pi deficiency than the WT, as
20
previously reported by Svistoonoff et al. (2007). The root growth phenotypes of
21
als3-3lpr1lpr2 resembled those of lpr1lpr2 (Figure 9A). In addition, we identified a
22
genetic suppressor of als3-3 (from EMS-mutagenized als3-3 plants) whose root
23
phenotype was similar to that of lpr1 (Figure 9B). Sequencing of the LPR1 gene in
24
this suppressor indicated a single point mutation in its second exon at position 1157,
25
with a G to A substitution resulting in a conversion of a glycine to an arginine (Figure
26
9C). We refer to this suppressor as als3-3/lpr1. Together, these results suggest that
27
ALS3 and LPR1/2 act in the same signalling pathway that regulates Pi
28
deficiency-mediated inhibition of primary root growth, with LPR1/2 being epistatic to
29
ALS3.
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Additional Evidence That the Over-accumulation of Fe3+ Is Responsible for
2
als3-3 Mutant Phenotypes As noted earlier, our results suggested that the over-accumulation of Fe3+ in roots
4
is directly linked to the als3-3 root phenotypes (Figure 2). To further test this
5
hypothesis, we examined Fe3+ accumulation in the roots of als3-3 grown on P-
6
medium supplemented with UDP-Glc. UDP-Glc treatment completely reversed the
7
over-accumulation of Fe3+ in als3-3 (Figure 10 and Supplemental Figure 10). Also, in
8
the als3-3lpr1hpr2 triple mutant and als3-3 suppressor als3-3/lpr1, the levels of Fe3+
9
accumulation were similar to that of lpr1hpr2, both being lower than that of the WT
10
(Figure 10 and Supplemental Figure 10). These results provided additional evidence
11
that the over-accumulation of Fe3+ in roots is responsible for the root phenotypes of
12
als3-3.
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DISCUSSION
The remodeling of RSA is a major developmental response of plants to Pi
16
deficiency and is believed to enhance a plant’s ability to acquire Pi. Although this
17
phenomenon has been well documented in a variety of plant species, the underlying
18
molecular mechanism(s) remain unclear. In this work, we identified the Arabidopsis
19
mutant hps10 as being hypersensitive to inhibition of primary root growth and
20
stimulation of lateral root production induced by Pi deficiency (Figure 1). hps10 was
21
further found to be a previously identified allele (als3-3) of ALS3, which is a key
22
determinant of plant tolerance to Al toxicity (Larsen et al., 2005). A similar result was
23
reported by Belal et al. (2015) during the course of this research. In their brief report,
24
the latter authors also found that als3-3 was hypersensitive to Pi deficiency-induced
25
remodeling of root architecture, and that the sensitivity of als3-3 to Pi deficiency was
26
sucrose-dependent. Our work corroborated their results, and together the results
27
suggest that the mechanism used to sense external Pi levels is impaired in als3-3. To
28
date, all of the reported Arabidopsis mutants with altered root responses to Pi
29
deficiency, except for lpr1 and lpr2, showed developmental abnormalities under
30
normal growth conditions (for examples, see Miura et al., 2005; Nacry et al., 2005;
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Jiang et al., 2007; Mayzlish-Gati et al., 2012; Singh et al., 2014). This indicates that
2
the effects of these mutations are not specific to Pi sensing and signaling. In contrast,
3
the root phenotype of als3-3 does not
4
growth conditions or in response to other types of nutrient deficiency (such as N, K,
5
and Fe deficiency) or to low pH conditions (Supplemental Figure 5). These results
6
suggest that ALS3 is not involved in the regulation of intrinsic developmental
7
programs but is an important component of specific pathways
8
responses to Pi deficiency. These results also suggest that als3-3 will be particularly
9
useful for studying Pi sensing mechanisms in higher plants.
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controlling plant
ALS3 encodes a TMD of a bacterial-type ABC transporter. In rice, OsSTAR1
11
(which contains only an NBD) interacts with OsSTAR2 (which contains only a TMD),
12
forming a transport complex in cytosolic vesicle membranes of root cells (Huang et
13
al., 2009). AtSTAR1 and ALS3 are the Arabidopsis counterparts of OsSTAR1 and
14
OsSTAR2 (Huang et al., 2009, 2010). Our work and that of Belal et al (2015) showed
15
that like als3-3, the atstar1 mutant displays hypersensitive root responses to Pi
16
deficiency, suggesting that AtSTAR1 and ALS3 might interact to form a common
17
transporter complex (Figure 4E). The similarity in the over-accumulation of Fe3+ in
18
roots of atstar1 and als3-3 (Figure 5) also indicated that AtSTAR1 and ALS3 may
19
co-exist in the same protein complex. Before the current study, however, experimental
20
evidence for the direct interaction between ALS3 and AtSTAR1 in Arabidopsis was
21
lacking. In this study, we provided compelling evidence for the interaction between
22
ALS3 and AtSTAR1. Based on yeast two-hybrid, LCI, and BiFC assays and
23
membrane fractionation experiments, the current study demonstrated that ALS3 and
24
AtSTAR1 physically interact in the tonoplasts (Figure 6). This is consistent with the
25
finding that AtSTAR1 has been detected in the proteome of tonoplasts (Jaquinod et al.,
26
2007). The detection of AtSTAR1-FLAG in the soluble fraction in our experiments
27
was probably due to the overexpression of AtSTAR1-FLAG proteins, some of which
28
were not associated with ALS3 in the tonoplasts. Expression in Xenopus oocytes
29
further demonstrated that the AtSTAR1 and ALS3 interaction leads to the formation
30
of a functional transport complex, i.e., the co-expression of ALS3 and AtSTAR1 but
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not the expression of either protein alone resulted in a significant increase in
2
electrogenic transport activity (Figure 8). Arabidopsis lpr1 and lpr2 mutants are insensitive to Pi deficiency-induced
4
inhibition of primary root growth (Svistoonoff et al., 2007). Like als3-3, these two
5
mutants are also morphologically normal under Pi sufficiency. Through genetic
6
analysis, we showed that ALS3 and LPR1/LPR2 act in the same pathway in
7
regulating Pi deficiency-induced inhibition of primary root growth and that
8
LPR1/LPR2 mutations are epistatic to ALS3 (Figure 9). LPR1 is localized in both the
9
ER and cell walls and has ferroxidase activity that oxidizes Fe2+ to Fe3+ in the root
10
apoplast (Müller et al., 2015). In the WT under P+ conditions, Fe3+ mainly
11
accumulates in the SCN and cortex of the root apex, as revealed by histochemical
12
staining (Figure 2 and 5). This Fe3+ accumulation pattern agrees with the
13
tissue-specific expression pattern of the LPR1 gene as reported by Müller et al (2015).
14
Under P- conditions, the level of Fe3+ in the SCN and cortex of the root apex is
15
dramatically reduced, and the accumulation of Fe3+ is shifted to the upper part of the
16
root. In lpr1, the accumulation of Fe3+ was barely detected by Perls staining under
17
either P+ or P- conditions (Figure 10 and Supplemental Figure 10). For als3-3 on P+
18
medium, the Fe3+ accumulation pattern is similar to that of the WT; for als3-3 on P-
19
medium, however, Fe3+ accumulation is greatly increased behind the root apex
20
(Figure 2). This change in the Fe3+ accumulation pattern correlates with the root
21
phenotypes of als3-3 under both P+ and P- conditions and suggests that the
22
over-accumulation of Fe3+ is the cause of the enhanced inhibition of primary root
23
growth in Pi-deficient als3-3. This inference was further supported by the following
24
experimental evidence: 1) the hypersensitive root phenotype of als3-3 is no longer
25
evident on P-Fe- medium, and over-accumulation of Fe3+ in the root is also
26
diminished (Supplemental Figure 3 and Figure 2); 2) in the als3-3lpr1lpr1 triple
27
mutant and in a genetic suppressor of als3-3 (als3-3/lpr1), neither the Pi
28
hypersensitive phenotype nor the over-accumulation of Fe3+ is observed (Figure 9 and
29
10, Supplemental Figure 10); and 3) in the UDP-Glc-treated als3-3, whose
30
root-growth phenotypes are restored to those of the WT on P- medium, the level of
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ACCEPTED MANUSCRIPT Fe3+ accumulation is similar to that of the WT (Figure 7 and 10, Supplemental Figure
2
10). Taken together, these observations lead us to hypothesize that the
3
ALS3/AtSTAR1 protein complex mediates Pi deficiency-induced remodeling of RSA
4
by modulating Fe homeostasis in roots. The hypothesis could explain why
5
LPR1/LPR2 is epistatic to ALS3 in regulating Pi deficiency-induced inhibition of
6
primary root growth, i.e., the over-accumulation of Fe3+ in the root apoplast of als3-3
7
may be overcome by the loss of ferroxidase activity encoded by LPR1.
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The mutant phenotypes of both als3-3 and atstar1 are restored by exogenously
9
applied UDP-Glc or UDP-GlcA (Figure 7). LPR1 is associated with cell walls, and
10
the oxidation of Fe2+ to Fe3+ by LPR1 apparently occurs in the apoplast of roots
11
(Müller et al., 2015). The rescue effect of UDP-Glc and UDP-GlcA might result from
12
the chelation of Fe3+ by the Pi released from the UDP moiety as catalyzed by secreted
13
acid phosphatases. However, this does not seem to be the case because UDP and
14
UDP-Gal did not show the rescue effect. UDP-Glc is the active form of Glc, which is
15
the substrate for the biosynthesis of cellulose, the major structural element of cell
16
walls. UDP-Glc can also be converted to UDP-GlcA by a UDP-Glc dehydrogenase,
17
which will further produce UDP-xylose (UDP-Xyl) by a UDP-GlcA carboxylase.
18
UDP-Xyl is the active form of Xyl, which is the substrate for the biosynthesis of
19
xyloglucan. Another major component of the cell wall, pectin, is also rich in GlcA. In
20
plants, cellulose and xyloglucans together form a carbohydrate network that is
21
embedded in a matrix consisting of pectins, hemicellulose, and cell wall proteins.
22
Therefore, the rescue effect of UDP-Glc and UDP-GlcA on als3-3 root phenotypes
23
suggests that the root cells of als3-3 may have defects in the formation of xyloglucan
24
or pectins and that these defects alter cell wall architecture. Cell wall architecture
25
profoundly affects the function of cell wall-associated enzymes by restricting the
26
location of these enzymes in cell walls or by altering their enzymatic activity through
27
the electrostatic interactions between the enzyme molecules and pectins, which carry
28
a large number of negative charges. Using comparative transcriptome and proteome
29
analyses, a recent study found that the genes involved in cell wall modification and
30
ROS formation were overrepresented in the list of the genes whose expression was
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ACCEPTED MANUSCRIPT deregulated in pdr2 and lpr1lpr2 mutants (Hoehenwarter et al., 2016). This study also
2
reported that roots of pdr2 had increased pectin content and that the Arabidopsis
3
mutant qua1-2, which had reduced pectin content, was hypersensitive to Pi starvation.
4
Therefore, the over-accumulation of Fe3+ in als3-3 might result from the elevated
5
LPR1 activity in the root apoplast due to reduced pectin content. And, the
6
exogenously supplied UDP-Glc or UDP-GlcA could compensate the reduced pectin
7
biosynthesis in als3-3 and atstar1, which alleviates their hypersensitive root
8
phenotypes in responding to Pi deficiency. This notion was further supported by our
9
quantitative analysis of Fe content, which showed that the over-accumulation of Fe3+
10
in P- als3-3 might result from over-conversion of Fe2+ to Fe3+ due to the elevated
11
LPR1/LPR2 activities rather than from the overload of Fe from the growth medium
12
(Figure 3). Alternatively, the mutation in the ALS3/AtSTAR1 complex may elevate
13
the enzymatic activity of LPR1/LPR2 through an unknown mechanism that results in
14
the over-accumulation of Fe3+. The increased Fe3+ content, in turn, as proposed by
15
Müller (2015), generates more ROS, which greatly enhances the crosslinking of the
16
cell walls, leading to the hypersensitive root phenotypes of als3-3 and atstar1.
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The rice OsSTAR1/OsSTAR2 complex is localized in cytoplasmic membrane
18
vesicles and transports UDP-Glc but not UDP-GlcA in Xenopus oocytes (Huang et al.,
19
2009). The latter study suggested this was the underlying reason as to why
20
exogenously applied UDP-Glc but not UDP-GlcA alleviated the toxic effect of Al on
21
the growth of rice seedling roots. Huang et al. (2009) proposed that in the WT, the
22
Golgi-synthesized UDP-Glc is delivered to the cell surface to modify the cell walls,
23
thereby masking sites of Al3+ binding, resulting in increased Al tolerance in rice. In
24
rice osstar1 or osstar2 mutant, such delivery is impaired, making the rice seedlings
25
more sensitive to Al toxicity. This explanation does not seem to apply to Arabidopsis
26
for the following reasons: 1) OsSTAR1/OsSTAR2 is located in cytoplasmic vesicle
27
membranes whereas ALS3/AtSTAR1 is located in tonoplasts (Figure 6); 2) OsSTAR1
28
and OsSTAR2 are mainly expressed in roots while ALS3 and AtSTAR1 are expressed
29
in roots as well as in the vascular tissues of other organs (Supplemental Figure 7); 3)
30
the als3-3 mutant phenotypes can be rescued by both UDP-Glc and UDP-GlcA
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2
(Huang et al., 2009); and 4) ALS3/AtSTAR1 does not transport UDP-Glc in oocytes
3
(Supplemental Figure 9). Thus, ALS3/AtSTAR1 most likely uses a different
4
mechanism from that of OsSTAR1/OsSTAR2 to regulate root response to Pi
5
deficiency. Because both OsSTAR1 and AtSTAR1 show sequence similarity to the
6
bacterial Pi transporter pstB (Huang et al., 2009; Chan and Torriani, 1996), we also
7
attempted to determine whether ALS3/AtSTAR1 can transport Pi. Functional analysis
8
in oocytes demonstrated that Pi is not a substrate for the ALS3/AtSTAR1 transporter,
9
at least under the experimental ionic conditions in our studies (Supplemental Figure 9).
10
A future challenge is to understand how the defect in the tonoplast-localized
11
ALS3/AtSTAR1 transporter complex affects cell wall integrity, which in turn
12
interferes with the Fe homeostasis in root cells.
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Identification of HPS10 as ALS3 also suggested that ALS3 might play a critical
14
role in the cross-talk between P and Al. It has long been known that P interacts with
15
Al in regulating plant growth and metabolism (Hartwell and Pember, 1918; Clarkson,
16
1967), and that the inhibitory effect of Al on root growth can be ameliorated by
17
increasing the supply of Pi (Tan and Keltjens, 1990; Liao et al., 2006; Sun et al.,
18
2008). A high supply of Pi alters Al-induced exudation of organic acids and the
19
transcription of some Al-responsive genes (Liang et al., 2013). Such interactions have
20
been thought to occur through chemical or biochemical reactions because Pi can
21
deplete Al3+ levels by forming an Al(PO4)3 complex. However, by maintaining the
22
same level of free Al3+ activity in nutrient solutions, Liao et al. (2006) found that
23
increasing the Pi supply still suppressed Al-induced exudation of citrate. Thus, the
24
antagonistic effect of high Pi on Al-induced citrate exudation might not simply be due
25
to the chelation of Al by Pi. Instead, Pi and Al may use some common signaling
26
mechanisms to elicit plant responses to both Pi deficiency and Al toxicity. Perhaps
27
ALS3 is a key component that is shared by Pi and Al for their cross-talk. It follows
28
that als3-3 could be useful for studying the molecular mechanism of P and Al
29
interactions.
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METHODS
3
Plant Materials All Arabidopsis thaliana plants used in this study were in the Columbia-0
5
background. als3-1 (CS3848), the three T-DNA insertion lines for ALS3, i.e.,
6
SALK_011435 (als3-2), SALK_004094 (als3-3), and SALK_061074 (als3-4), and a
7
T-DNA insertion line for AtSTAR1, i.e., CS384144 (atstar1), were obtained from
8
ABRC. Genomic DNA was extracted from each T-DNA line and was analyzed via
9
PCR using the primers specific for each T-DNA insertion. The mRNA expression of
10
the corresponding gene in the T-DNA insertion lines was analyzed by a
11
semi-quantitative RT-PCR method. The lpr1lpr2 mutant was a kind gift from Dr.
12
Thierry Desnos (CEA Cadarach, France). The als3-3lpr1lpr2 triple mutant was
13
generated by genetic cross between als3-3 and lpr1lpr2. The sequences of all primers
14
used are listed in Supplemental Table S1.
15 16
Plant Growth Conditions
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Half-strength MS medium with 1% (w/v) sucrose, 0.1% MES (w/v), and 1.2%
18
(w/v) agar (Sigma Cat. No. A1296) or 0.8% agarose (Biowest Regular Agarose G-10)
19
was used as the standard Pi-sufficient (P+) medium. The Pi-deficient medium (P-)
20
was prepared by replacing KH2PO4 with K2SO4 in the P+ medium. For P+Fe- and
21
P-Fe- media, the 50 µM Fe-EDTA was omitted from P+ and P- media. For Pi
22
dose-response analysis, various amounts of KH2PO4 were added to the P- medium.
23
The N-, and K- media were prepared as described (Jung et al., 2009; Yong et al.,
24
2010).
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In some cases (the experiments shown in Figure 9 and Supplemental Figure 7),
26
the agar in the standard media was replaced with agarose to further reduce the amount
27
of residual Pi in the growth medium.
28
Seeds were surface-sterilized in 20% bleach for 10 min and washed three times
29
with sterile-distilled water before they were placed on plates containing media. After
30
the seeds were stratified at 4 °C in the dark for 2 days, the plates were placed 20
ACCEPTED MANUSCRIPT 1
vertically in a growth room with a photoperiod of 16-h light/8-h dark at 21 to 23°C.
2
The light intensity was 100 µmol m–2 s–1.
3 4
Analyses of Root Growth Phenotypes Seedlings grown on the plates with media were photographed with a digital
6
camera (Sony DSC-W50). The length of primary roots was measured with Digimize
7
software. The lateral roots were counted with the aid of an SZ61 stereomicroscope. To
8
examine the cellular organization of the root meristem, the root tips were excised and
9
mounted in 30 µl of HCG clearing solution (chloroacetaldehyde : water : glycerol
10
=8:3:1) under a coverslip. The root tips were then visualized and imaged with a
11
differential interference contrast (DIC) microscope (Olympus BX51) equipped with a
12
camera (Olympus DP71). The length of root cells was determined by measuring the
13
length of root epidermal cells in the maturation zone. For observation of root hair
14
patterns, the first 5-mm segment of the root tip of 8-day-old seedlings was
15
photographed with a stereomicroscope (Olympus SZ61) equipped with a digital
16
camera (Olympus DP72).
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Quantitative Real-time RT-PCR (qPCR)
qPCR analyses of the expression of ALS3 and AtSTAR1 were performed as
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Vector Construction and Plant Transformation To generate plant expression vectors for genetic complementation, a DNA
24
fragment containing the genomic sequence of the ALS3 gene and its 1.2-kb promoter
25
region was PCR amplified from the genomic DNA isolated from WT Arabidopsis
26
seedlings. The amplified DNA fragment was cloned into EcoR I and Kpn I sites of the
27
binary vector pCAMBIA1300, resulting in the construct ALS3::ALS3 gDNA. To
28
generate the ALS3 overexpression vector, the genomic sequence of ALS3 was cloned
29
into the plant expression vector pZH01 using the Gibson assembly cloning method
30
(Gibson et al., 2009), resulting in the construct 35S::ALS3 gDNA. To generate the 21
ACCEPTED MANUSCRIPT AtSTAR1 overexpression vector, the genomic sequence of AtSTAR1 was PCR
2
amplified from genomic DNA and used to replace the GUS gene on the plant vector
3
pBI121, resulting in the construct 35S::AtSTAR1 gDNA. For analyses of
4
tissue-specific expression patterns of ALS3 and AtSTAR1, the translational fusion
5
constructs ALS3::ALS3-GUS and AtSTAR1::AtSTAR1-GUS were generated according
6
to Larsen et al. (2005) and Huang et al. (2010), respectively. The sequences of the
7
primers used for PCR amplification of the ALS3 and AtSTAR1 genes are listed in
8
Supplemental Table S1.
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The above constructs were transferred into Agrobacterium tumefaciens strain
10
GV3101 and transformed into als3-3, atstar1, or WT plants using the floral dip
11
method (Clough and Bent, 1998).
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The histochemical analyses of GUS activity were performed as described by Jefferson et al. (1987).
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Yeast Two-hybrid (Y2H) Assay
The Y2H assay was performed using the DUALmembrane System (Biotech
19
Dualsystems). The coding sequence (CDS) of HPS10 was PCR amplified from cDNA
20
of WT Arabidopsis plants and fused with the C-terminal half of the ubiquitin gene
21
(Cub) to generate the construct pDHB-ALS3-Cub. The CDS of AtSTAR1 was PCR
22
amplified and was fused with the mutated N-terminal half of ubiquitin (NubG),
23
resulting in the construct pPR3-NubG-AtSTAR1. The sequences of the primers used
24
for Y2H assay are listed in Supplemental Table S1. Various combinations of plasmids
25
were then co-transformed into NMY51 yeast cells according to the manufacturer’s
26
instructions. The transformants were selected on SD medium without Leu and Trp.
27
The yeast cells were spotted on the indicated plates for growth assay as described
28
previously (Huang et al., 2009).
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Luciferase Complementation Imaging (LCI) Assays 22
ACCEPTED MANUSCRIPT For vector construction for LCI assays, the CDSs of the ALS3 and AtSTAR1
2
genes were PCR amplified from Arabidopsis cDNA and inserted into the vectors
3
pCAMBIA1300-nLUC and pCAMBIA1300-cLUC (Chen et al., 2008), respectively,
4
to generate the constructs ALS3-nLUC and cLUC-AtSTAR1. The sequences of the
5
primers used to amplify the CDSs of ALS3 and AtSTAR1 are listed in Supplemental
6
Table S1. The LCI assays were performed in the leaves of N. benthamiana according
7
to Sun et al. (2016).
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BiFC Assays
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For BiFC assays in the leaves of N. benthamiana, the CDSs of ALS3 and
11
AtSTAR1 were cloned into the plant binary expression vectors pCAMBIA1300-nYFP
12
and pCAMBIA1300-cYFP, respectively, to generate constructs ALS3-nYFP and
13
cYFP-AtSTAR1. The BiFC assays in N. benthamiana were carried out according to
14
Sun et al. (2016). The vacuoles were isolated from mesophyll protoplasts of the leaves
15
of N. benthamiana transformed with the BiFC vectors as described (Robert et al.,
16
2007).
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18
AtSTAR1 were fused to the C-terminal and N-terminal fragments of YFP gene in the
19
plasmids pVYNE and pVYCER, respectively, resulting in the constructs ALS3-nYFP
20
and cYFP-AtSTAR1. Various combinations of BiFC constructs were co-transformed
21
into Arabidopsis mesophyll protoplasts via a PEG-mediated method as described
22
(Yoo et al., 2007). Transformed protoplasts were stained with a culture medium
23
supplemented with 5 µM FM4-64 (Invitrogen) for 15 min, and were observed with
24
confocal microscopy.
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Sucrose Density-Gradient Centrifugation
27
For the 35S::ALS3-HA and 35S::AtSTAR1-FLAG constructs, the CDSs of ALS3
28
and AtSTAR1 were PCR amplified and cloned into the modified plant binary
29
expression
30
pCAMBIA1300-3×Flag (Lu et al., 2016), respectively. The constructs were
vectors
pCAMBIA1300-3×HA 23
(Ma
et
al.,
2013)
and
ACCEPTED MANUSCRIPT 1
co-transformed into leaves of N. benthamiana by Agrobacterium-mediated infiltration.
2
After 48 h, three grams of fresh leaves was harvested. Total, soluble, and microsomal protein fractions were extracted from the leaves
4
as described (Michael Weaver et al., 2006), with minor modification. In brief, the
5
leaves were ground in the pre-chilled homogenization buffer [50 mM Tris.HCl (pH
6
7.5), 20% glycerol, 5 mM DTT, 2 mM EDTA, 7 mM MgCl2 and protease inhibitors
7
(Roche) ] on ice, and centrifuged at 10,000 × g for 15 min at 4°C. The supernatant
8
was then collected and centrifuged at 100,000 × g for 1 h at 4°C to obtain soluble (S)
9
and microsomal membrane (M) fractions. The microsomal pellet was suspended in
10
the suspension buffer [25 mM Tris.HCl (pH 7.5), 10% (w/w) sucrose, 1 mM DTT, 2
11
mM EDTA, 7 mM MgCl2, and protease inhibitors (Roche)]. For sucrose
12
density-gradient centrifugation, the suspended samples were then layered on a
13
20-50% (w/w) sucrose gradient formed by the Gradient Master 108 (Biocomp),
14
followed by centrifugation at 210, 000 × g for 16 h using a SW41Ti rotor (Beckman).
15
The 600-µl density gradient fractions were collected for Western blot analysis.
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Western Blot Analyses
Proteins were separated on 8% SDS-polyacrylamide gels, transferred to a PVDF
19
membrane, and then subjected to Western blot analyses with the indicated antibodies.
20
Rabbit polyclonal antibodies against subunit E of plant V-ATPase (AS07 213,
21
Agrisera) or plant H+-ATPase (AS07 260-100, Agrisera) were used at a dilution of
22
1:3000. Mouse monoclonal antibodies against HA (CWBIO) and FLAG (Abmart)
23
were used at a dilution of 1:2000. Secondary goat anti-rabbit antibody (CWBIO) or
24
goat anti-mouse antibody (Abmart) conjugated with horseradish peroxidase was used
25
at a dilution of 1:5000. The signal was detected by the chemiluminescence reaction
26
using the eECL Western Blot Kit (CWBIO).
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Confocal Microscopy
29
The fluorescence signals generated in BiFC assays were observed with an
30
LSM710 META laser scanning microscope (Zeiss). The wavelengths of 24
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excitation/emission were 514 nm/520-550 nm for YFP protein and 543 nm/575-630
2
nm for FM4-64. Images were analyzed with ZEN 2009 Light Edition software.
3 4
Fe Histochemical Staining Assay The accumulation of Fe in roots was detected using the Perls staining method as
6
described (Roschzttardtz et al., 2009). In brief, roots were excised from 8-day-old
7
seedlings and vacuum infiltrated with Perls staining solution containing equal
8
volumes of 4% (v/v) HCl and 4% (w/v) K-ferrocyanide for 15 min. After infiltration,
9
the samples were kept in the same solution for another 30 min at room temperature
10
before they were rinsed with ultrapure water to stop the reaction. Stained samples
11
were cleared in the HCG clearing solution (1 g/ml chloral hydrate in 15% glycerol)
12
and examined with a DIC microscope.
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For the DAB intensification, Perls-stained samples were incubated for 60 min in
14
methanol containing 0.01 M NaN3 and 0.3% H2O2 (30%). Then, the samples were
15
washed with 0.1 M Na-phosphate buffer (pH7.2) and incubated for 5 min in the same
16
buffer containing 0.025% (w/v) DAB (Sigma-Aldrich) and 0.005% H2O2. To stop the
17
reaction, the samples were washed several times with phosphate buffer. Stained
18
samples were cleared by mounting them in HCG clearing solution on glass slides and
19
were examined with DIC microscopy.
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Quantification of Fe Content in Roots Fe content of root tissues was determined by the spectrophotometric BPS method
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according to Tamarit et al. (2006).
Genetic Screening for als3-3 Suppressors A 2.5-g
quantity of
als3-3
seeds
was
mutagenized
with
a
0.4%
27
ethylmethanesulfonate (EMS) solution as described (Kim et al., 2006). The
28
mutagenized M1 seeds were grown in soil and self-pollinated to yield M2 seeds. The
29
M2 seeds were then sown on 1.2% agar-solidified P- medium to screen for mutants
30
with the same primary root length as the WT. The phenotypes of the putative hps10 25
ACCEPTED MANUSCRIPT 1
suppressors were reconfirmed in the M3 generation.
2 3
Protein-protein Interaction and Electrophysiological Assays in Xenopus Oocytes The CDSs of ALS3 and AtSTAR1 were cloned into the T7TS vector (Pineros et al.,
5
2008) and into BiFC expression vectors suitable for electrophysiological and
6
protein-protein interaction analysis, respectively (Nour-Eldin et al., 2006). The BiFC
7
chimeras consisted of i) ALS3::nYFP, with ALS3 fused to the N-terminal half of a
8
split YFP; and ii) cYFP::AtSTAR1, with the C-terminal half of a split YFP fused to
9
AtSTAR1. After the respective cDNAs were linearized, cRNA was synthesized using
10
an mMessage mMachine in vitro transcription kit (Ambion) according to the
11
manufacturer’s recommendations.
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For electrophysiological experiments, stage V-VI Xenopus laevis oocytes were
13
harvested and microinjected with 50 nl of H2O for control cells, 50 nl of 250 ng/µl of
14
either AtSTAR1 or ALS3 cRNA, or 50 nl of 250 ng/µl of a 1:1 ALS3/AtSTAR1
15
mixture (500 ng/µl total cRNA). Oocytes used for BiFC were injected with a 1:1
16
ALS3::nYFP+cYFP::AtSTAR1 cRNA mixture or ALS3::nYFP+cYFP mixture (250
17
ng/µl total cRNA). Microinjected oocytes were incubated in ND88 at 18°C for 2 days
18
before they were examined with confocal microscopy or were subjected to
19
electrophysiological analysis. For experiments evaluating the effect of increased
20
intracellular P concentration on ALS3-AtSTAR1 activity, cRNA-microinjected
21
oocytes were incubated for an additional 48 to 96 h in a modified ND88 solution with
22
or without 5 mM NaH2PO4 as described (Wang et al., 2015). YFP signals in Xenopus
23
oocytes were visualized using a TCS SP5 confocal laser-scanning microscope (Leica)
24
at excitation and emission wavelengths of 514 and 525–550 nm, respectively. The
25
standard ND96 bath solution used for electrophysiological recordings contained 96
26
mM NaCl, 1 mM KCl, and 1.8 mM CaCl2, with the pH adjusted to 7.5 or 5.5 with 5
27
mM HEPES-NaOH or HCl.
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In experiments concerning permeability to UDP-Glc, cells were microinjected
29
with 50 nl of 5 M HEPES buffer (pH 7.5, control) or with 50 nl of a 50 mM UDP-Glc
30
stock solution prepared in the latter buffer; microelectodes were inserted into cells 2 h 26
ACCEPTED MANUSCRIPT after microinjection. The 50-nl microinjection results in an approximately 4 mM
2
intracellular increase in UDP-Glc, assuming a 1.2-mm cell diameter (see Piñeros et al.,
3
[2008] for intracellular determinations). All electrophysiological measurements were
4
performed under two-electrode voltage-clamp (TEVC) using a GeneClamp 500
5
amplifier (Axon Instruments; www.moleculardevices.com) as described in Piñeros et
6
al. (2008). Cells were allowed to recover at least 10 minutes following microelectrode
7
impalement. Currents were elicited by voltage pulses stepped between values
8
indicated in the figures with a 10-sec rest at 0 or –20 mV between each pulse.
9
Steady-state current-voltage (I/V) relationships were constructed by measuring the
10
current amplitude at the end (i.e., at 2 sec) of the test pulse. Liquid junction potentials
11
were measured and corrected accordingly. Mean current values represent the average
12
of at least n (indicated in each figure legend) oocytes and two to three donor frogs.
13
Error bars denote SEM.
14
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ACCESSION NUMBERS
16
Sequence data from this article can be found in the TAIR database under the
17
following accession numbers: ALS3 (AT2G37330), AtSTAR1 (AT1G67940), LPR1
18
(AT1G23010), and LPR2 (AT1G71040).
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SUPPLEMENTAL INFORMATION
21
Supplemental information is available at Molecular Plant Online.
22
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FUNDING
24
This work was supported by funds from the Ministry of Science and Technology of
25
China (grant no. 2016YFD0100700 )
26
China (grant no. 31170238), and the Ministry of Agriculture of China (grant no.
27
2014ZX0800932B).
the National Natural Science Foundation of
28 29
AUTHOR CONTRIBUTIONS 27
ACCEPTED MANUSCRIPT 1
D.L., J.D., M.A.P., H.Y., A.S.M., and L.V. K. designed the research. J.D., M.A.P.,
2
X.L., H.Y., Y.L., and A.S.M. performed the research. D.L., J.D., M.A.P., H.Y., and
3
A.S.M., analyzed the data. D.L., J.D., M.A.P., and L.V. K. wrote the article.
4
ACKNOWLEDGEMENTS
6
We thank the Arabidopsis Biological Resource Center for providing the seed stocks of
7
the mutant lines, and Dr. Thierry Desnos for providing the lpr1lpr2 mutant.
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Figure 1. Effect of Pi availability on the root phenotype of WT and als3-3 seedlings.
21
(A) Morphology of WT and als3-3 seedlings grown on a Pi-sufficient (P+) or
22
Pi-deficient (P-) medium at 8 and 11 DAG. Inset: A magnified view of the root tip of
23
a P- als3-3 seedling. In the inset, the red arrow indicates the tip of the primary root. (B)
24
Close view of the root tips of 11-day-old seedlings shown in (A). The arrows indicate
25
the boundary between meristematic and elongation zones.
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Figure 2. Fe accumulation patterns as indicated by Perls (A) and Perls/DAB (B)
28
staining in the roots of 8-day-old WT and als3-3 seedlings grown on P+Fe+ (P+),
29
P-Fe+ (P-), and P-Fe- media.
30
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Figure 3. Fe contents in the roots of 8-day-old WT and als3-3 seedlings grown on P+
32
and P- media. The values are means±SD. The asterisk indicates a significant
33
difference from the WT (t-test, P<0.05).
34 35
Figure 4. Molecular characterization of ALS3 and AtSTAR1 genes and root
36
phenotypes of als3 and atstar1. (A) and (B) Diagrams showing the structure of the 32
ACCEPTED MANUSCRIPT ALS3 and AtSTAR1 genes, the positions of the point mutation in als3-1, and the
2
position of the T-DNA insertions (the empty triangles) in als3-2, als3-3, als3-4, and
3
atstar1. The black box, grey box, and the horizontal line represent the coding regions,
4
untranslated regions, and introns, respectively. (C) Root phenotypes of the WT, als3-1,
5
als3-2, als3-3, and als3-4 on P- medium. (D) Root phenotypes of the WT, als3-3, and
6
two complementation lines on P- medium. (E) Root phenotypes of WT, als3-3, and
7
atstar1 seedlings on P+ and P- media. In (C) to (E), the photographed seedlings were
8
8 days old.
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Figure 5. Fe accumulation patterns as indicated by Perls (A) and Perls/DAB (B)
11
staining in the roots of the 8-day-old WT and atstar1 seedlings grown on P+ and P-
12
media.
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Figure 6. Yeast two-hybrid (A), LCI (B), and BiFC (C-E) assays indicated physical
15
interaction between ALS3 and AtSTAR1. (A) Yeast cells co-transformed with various
16
combinations of the plasmids were grown on synthetic dropout (SD) medium without
17
Leu or Trp (left panel) or SD medium without Leu, Trp, His, or Ade (right panel) for 3
18
days at 30ºC. The activation of the reporter genes by the SV40 large T antigen-Cub
19
(large T-Cub) and NubG-the N-terminally truncated tumor suppressor protein p53
20
(NubG-∆p53) or ALS3-Cub and NubI was used as a positive control. (B) The
21
ALS3-nLUC and cLUC-AtSTAR1 fusion genes were co-expressed in the leaves of N.
22
benthamiana, and bioluminescence was captured with a CCD camera. (C) The
23
ALS3-nYFP and cYFP-AtSTAR1 fusion genes were co-expressed in the leaves of N.
24
benthamiana, and fluorescence signals in the pavement cells were analyzed by
25
confocal microscopy. (D) The fluorescence signals in the vacuoles isolated from the
26
mesophyll protoplasts of the transformed leaves of N. benthamiana as shown in (C).
27
(E) The ALS3-nYFP and cYFP-AtSTAR1 fusion genes were co-expressed in
28
Arabidopsis protoplasts. Plasma membranes are stained red by FM4-64. The signal
29
from the interaction between ALS3 and AtSTAR1 is shown in green. Inset: A close
30
view of part of the cell periphery showing the non-overlapping of plasma membrane
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2
in tobacco leaves by Western blot using HA and FLAG antibodies. 1 and 2: total
3
proteins extracted from uninfiltrated and Agrobacterium-infiltrated leaves of N.
4
benthamiana. Rubsico was used as the protein loading control. (G) Detection of
5
ALS3-HA and AtSTAR1-FLAG proteins in different cellular fractions. T: total
6
proteins; S: soluble proteins; M: microsomal membrane fractions. V-ATPase: a
7
tonoplast-specific marker. Rubisco was used as a soluble protein marker. (H) Sucrose
8
density-gradient centrifugation. Microsomal membranes were fractionated over a
9
20-50% (w/w) sucrose gradient, and samples of each fraction (20 µl each) were
10
analyzed by Western blot using specific antibodies. H+-ATPase: a plasma
11
membrane-specific marker.
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Figure 7. UDP-Glc and UDP-GlcA rescue the mutant phenotype of als3-3 and atstar1.
14
The growth characteristics of 7-day-old als3-3 and atstar1 seedlings grown on P+, P-,
15
or P- media supplemented with various compounds (500 µM).
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Figure 8. AtSTAR1 and ALS3 interact to form a functional transporter. (A) BiFC
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assays in Xenopus oocytes showing fluorescence (left panel) and bright field-merged
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(right panel) images resulting from the protein-protein interaction between AtSTAR1
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and ALS3 (top row). No BiFC fluorescence was observed in the controls in which
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oocytes were co-injected with ALS3::nYFP and the complementary cYFP (bottom
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row). (B) Resting membrane potentials (measured in a standard ND96 bath solution)
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of cells injected with water (control), ALS3, or AtSTAR1, or co-injected with both
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AtSTAR1 and ALS3 (n = 12 cells in each case). (C) Electrogenic transport in control
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cells, cells injected with either AtSTAR1 or ALS3, or cells co-injected with AtSTAR1
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and ALS3. Example of currents elicited in response to holding potentials ranging from
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0 to -130 mV (in 10-mV increments with an inter-episode holding potential of -20 mV
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for 10 sec) in AtSTAR1+ALS3 co-injected cells (right panel), or cells injected only
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with water, AtSTAR1, or ALS3 (left panel). Symbols of each injection correspond to
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those shown in (B). Cells were bathed in standard ND96 (pH 7.5) solution. (D) Mean
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current/voltage (I/V) relationships from recordings like those shown in (C). The
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straight and dotted arrows above the X-axis indicate the reversal potential (Erev)
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recorded in cells co-injected with AtSTAR1 and ALS3 (full circles) and all other
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control and single injections. Symbols correspond to those in (B and C) (n = 10 cells).
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Figure 9. Root phenotypes of the WT and various mutants grown on
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agarose-containing P+ and P- media. (A) 8-day-old seedlings of the WT, als3-3,
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lpr1lpr2, and als3-3lpr1lpr2. (B) 8-day-old seedlings of the WT, als3-3, lpr1, and a
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genetic suppressor of als3-3, als3-3/lpr1. (C) Diagram illustrating the position of the
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point mutation in the LPR1 gene in als3-3/lpr1.
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Figure 10. Fe accumulation patterns as indicated by Perls (A) and Perls/DAB (B)
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staining in the maturation zones of the roots of 8-day-old seedlings of the WT,
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UDP-Glc-treated als3-3, and various mutants grown on P- medium.
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Figure 1
Figure 1. Effect of Pi Availability on the Root Phenotype of WT and als3-3 Seedlings.
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(A) Morphology of WT and als3-3 seedlings grown on a Pi-sufficient (P+) or Pi-deficient (P-) medium at 8 and 11 DAG. Inset: A magnified view of the root tip of a P- als3-3 seedling. In the inset, the red arrow
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indicates the tip of the primary root. (B) Close view of the root tips of 11-day-old seedlings shown in (A). The arrows indicate the boundary between meristematic and elongation zones.
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Figure 2. Fe Accumulation Patterns as Indicated by Perls (A) and Perls/DAB (B) Staining in the Roots
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of 8-Day-Old WT and als3-3 Seedlings Grown on P+Fe+ (P+), P-Fe+ (P-), and P-Fe- Media.
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Figure 3. Fe Contents in the Roots of 8-Day-Old WT and als3-3 Seedlings Grown on P+ and P- Media. The values are means±SD. The asterisk indicates a significant difference from
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the WT (t-test, P<0.05).
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Figure 4. Molecular Characterization of ALS3 and AtSTAR1 Genes and Root Phenotypes of als3
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and atstar1.
(A) and (B) Diagrams showing the structure of the ALS3 and AtSTAR1 genes, the position of the point
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mutation in als3-1, and the positions of the T-DNA insertions (the empty triangles) in als3-2, als3-3, als3-4, and atstar1. The black box, grey box, and the horizontal line represent the coding regions, untranslated regions, and introns, respectively. (C) Root phenotypes of the WT, als3-1, als3-2, als3-3, and als3-4 on Pmedium. (D) Root phenotypes of the WT, als3-3, and two complementation lines on P- medium. (E) Root phenotypes of WT, als3-3, and atstar1 seedlings on P+ and P- media. In (C) to (E), the photographed seedlings were 8 days old.
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Figure 5. Fe Accumulation Patterns as Indicated by Perls (A) and Perls/DAB (B) Staining in the
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Roots of the 8-Day-Old WT and atstar1 Seedlings Grown on P+ and P- Media.
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H+-ATPase
Figure 6. Yeast Two-Hybrid (A), LCI (B), and BiFC (C-E) Assays Indicated Physical Interaction between ALS3 and AtSTAR1.
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(A) Yeast cells co-transformed with various combinations of the plasmids were grown on synthetic dropout (SD) medium without Leu or Trp (left panel) or SD medium without Leu, Trp, His, or Ade (right panel) for 3
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days at 30ºC. The activation of the reporter genes by the SV40 large T antigen-Cub (large T-Cub) and NubGthe N-terminally truncated tumor suppressor protein p53 (NubG-Δp53) or ALS3-Cub and NubI was used as a positive control. (B) The ALS3-nLUC and cLUC-AtSTAR1 fusion genes were co-expressed in the leaves of N. benthamiana, and bioluminescence was captured with a CCD camera. (C) The ALS3-nYFP and cYFPAtSTAR1 fusion genes were co-expressed in the leaves of N. benthamiana, and fluorescence signals in the pavement cells were analyzed by confocal microscopy. (D) The fluorescence signals in the vacuoles isolated from the mesophyll protoplasts of the transformed leaves of N. benthamiana as shown in (C). (E) The ALS3nYFP and cYFP-AtSTAR1 fusion genes were co-expressed in Arabidopsis protoplasts. Plasma membranes are stained red by FM4-64. The signal from the interaction between ALS3 and AtSTAR1 is shown in green. Inset: A close view of part of the cell periphery showing the non-overlapping of plasma membrane and tonoplast. (F) Detection of expression of ALS3-HA and AtSTAR1-FLAG proteins in tobacco leaves by Western
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blot using HA and FLAG antibodies. 1 and 2: total proteins extracted from uninfiltrated and Agrobacteriuminfiltrated leaves of N. benthamiana. Rubsico was used as the protein loading control. (G) Detection of ALS3HA and AtSTAR1-FLAG proteins in different cellular fractions. T: total proteins; S: soluble proteins; M: microsomal membrane fractions. V-ATPase: a tonoplast-specific marker. Rubisco was used as a soluble protein marker. (H) Sucrose density-gradient centrifugation. Microsomal membranes were fractionated over a 20-50% (w/w) sucrose gradient, and samples of each fraction (20 μl each) were analyzed by Western blot
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using specific antibodies. H+-ATPase: a plasma membrane-specific marker.
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Figure 7. UDP-Glc and UDP-GlcA Rescue the Mutant Phenotype of als3-3 and atstar1.
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The growth characteristics of 7-day-old als3-3 and atstar1 seedlings grown on P+, P-, or P- media
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supplemented with various compounds (500 μM).
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Figure 8. AtSTAR1 and ALS3 Interact to Form a Functional Transporter.
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(A) BiFC assays in Xenopus oocytes showing fluorescence (left panel) and bright field-merged (right panel) images resulting from the protein-protein interaction between AtSTAR1 and ALS3 (top row). No BiFC fluorescence was observed in the controls in which oocytes were co-injected with ALS3::nYFP and the complementary cYFP (bottom row). (B) Resting membrane potentials (measured in a standard ND96 bath
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solution) of cells injected with water (control), ALS3, or AtSTAR1, or co-injected with both AtSTAR1 and ALS3 (n = 12 cells in each case). (C) Electrogenic transport in control cells, cells injected with either AtSTAR1 or
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ALS3, or cells co-injected with AtSTAR1 and ALS3. Example of currents elicited in response to holding potentials ranging from 0 to -130 mV (in 10-mV increments with an inter-episode holding potential of -20 mV for 10 sec) in AtSTAR1+ALS3 co-injected cells (right panel), or cells injected only with water, AtSTAR1, or ALS3 (left panel). Symbols of each injection correspond to those shown in (B). Cells were bathed in standard ND96 (pH 7.5) solution. (D) Mean current/voltage (I/V) relationships from recordings like those shown in (C). The straight and dotted arrows above the X-axis indicate the reversal potential (Erev) recorded in cells coinjected with AtSTAR1 and ALS3 (full circles) and all other control and single injections. Symbols correspond to those in (B and C) (n = 10 cells).
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Media.
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Figure 9. Root Phenotypes of the WT and Various Mutants Grown on Agarose-Containing P+ and P-
(A) 8-day-old seedlings of the WT, als3-3, lpr1lpr2, and als3-3lpr1lpr2. (B) 8-day-old seedlings of the WT, als3-3, lpr1, and a genetic suppressor of als3-3, als3-3/lpr1. (C) Diagram illustrating the position of the point mutation in the LPR1 gene in als3-3/lpr1.
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Figure 10. Fe Accumulation Patterns as Indicated by Perls (A) and Perls/DAB (B) Staining in the Maturation Zones of the Roots of 8-Day-Old Seedlings of the WT, UDP-Glc-treated als3-3, and
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Various Mutants Grown on P- Medium.
S1674-2052(16)30270-2 10.1016/j.molp.2016.11.001 MOLP 388
To appear in: MOLECULAR PLANT Accepted Date: 5 November 2016
Please cite this article as: Dong J., Piñeros M.A., Li X., Yang H., Liu Y., Muphy A.S., Kochian L.V., and Liu D. (2016). An Arabidopsis ABC transporter mediates phosphate deficiency-induced remodeling of root architecture by modulating iron homeostasis in roots. Mol. Plant. doi: 10.1016/ j.molp.2016.11.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in MOLECULAR PLANT are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs.
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Full title:
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An Arabidopsis ABC transporter mediates phosphate deficiency-induced remodeling
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of root architecture by modulating iron homeostasis in roots
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Full names and affiliations of authors:
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Jinsong Dong1, Miguel A. Piñeros2, Xiaoxuan Li1, Haibing Yang3, Yu Liu4, Angus S.
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Muphy5, Leon V. Kochian2, Dong Liu1*
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1. Ministry of Education Key Laboratory of Bioinformatics, Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China 2. USDA-ARS, Robert Holley Center for Agriculture and Health, Cornell University, Ithaca, NY 14580 USA 3. Department of Horticulture, Purdue University, West Lafayette, Indiana 47907-2010 4. State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, China 5. Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD 20742, USA
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*Corresponding author
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E-mail: [email protected]
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Running title:
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Pi deficiency-regulated root development
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ACCEPTED MANUSCRIPT Short summary
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The Arabidopsis ALUMINUM SENSITIVE3 (ALS3) and AtSTAR1 form an ABC transporter
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complex in the tonoplasts. It acts with LOW PHOSPHATE ROOT1/2 (LPR1/2), two
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ferroxidases, to mediates phosphate deficiency-induced inhibition of primary root growth by
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modulating root iron homeostasis. The functional disruption of this ABC transporter may also
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affect integrity of cell wall architecture.
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ABSTRACT
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The remodeling of root architecture is a major developmental response of plants to
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phosphate (Pi) deficiency and is thought to enhance a plant’s ability to forage Pi in
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topsoil. The underlying mechanism controlling this response, however, is poorly
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understood. In this work, we identified an Arabidopsis mutant, hps10 (hypersensitive
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to Pi starvation 10), that is morphologically normal under Pi sufficiency but shows
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increased inhibition of primary root growth and enhanced production of lateral roots
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under Pi deficiency. hps10 is a previously identified allele (als3-3) of the
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ALUMINUM SENSITIVE3 (ALS3) gene, which is involved in plant tolerance to
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aluminum toxicity. Our results show that ALS3 and its interacting protein AtSTAR1
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form an ABC transporter complex in tonoplasts. This protein complex mediates a
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highly electrogenic transport in Xenopus oocytes. Under Pi deficiency, als3
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accumulates higher levels of Fe3+ in its roots than the wild type. In Arabidopsis, LPR1
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(LOW PHOSPHATE ROOT1) and LPR2 encode ferroxidase, which when mutated
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reduces Fe3+ accumulation in roots and causes root growth to be insensitive to Pi
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deficiency. Here, we provide compelling evidence that ALS3 acts with LPR1/2 to
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regulate Pi deficiency-induced remodeling of root architecture by modulating Fe
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homeostasis in roots.
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Keywords:
phosphate deficiency,
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transporter
ALUMINUM SENSITIVE3,
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root architecture, AtSTAR1 2
iron homeostasis,
ABC
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INTRODUCTION Root development is a postembryonic process that is highly plastic in responding
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to fluctuations in nutrient levels in the environment. Root system architecture (RSA)
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is a major determinant of the plant’s ability to acquire water and nutrients from the
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soil (Lynch, 1995). Phosphate (Pi), the major form for phosphorus (P) uptake and
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assimilation by roots, decreases with soil depth. When grown under Pi deficiency,
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plants decrease their primary root growth but increase the production of lateral roots
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(Lopez-Bucio et al., 2003; Desnos, 2008). Such remodeling of RSA is thought to
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maximize a plant’s potential to exploit the Pi resource in topsoil. Although the Pi
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deficiency-induced remodeling of RSA has been well documented in a variety of plant
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species (Vance et al., 2003), the underlying molecular mechanism for this adaptive
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response remains largely unknown.
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Pi deficiency-induced remodeling of root development has been shown to be an
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active cellular process that is determined by an internal genetic program rather than a
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consequence of reduced metabolic activity due to nutrient shortage (Péret et al., 2014).
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This remodeling process is triggered by a decrease in local, external Pi levels, with the
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root tip playing a key role in the sensing of change in Pi status in the environment
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(Ticconi et al., 2004, 2009; Svistoonoff et al., 2007; Thibaud et al., 2010). Forward
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genetics has identified several key molecular components involved in the control of
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primary root growth under Pi deficiency. The Arabidopsis pdr2 (phosphate deficiency
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responses2) mutant is hypersensitive to Pi deficiency-induced inhibition of primary
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root growth (Ticconi et al., 2004). In contrast, the primary root growth of lpr1 (low
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phosphate root1) is insensitive to Pi deficiency (Svistoonoff et al., 2007). PDR2
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encodes an endoplasmic reticulum (ER)-localized P5-type ATPase (Ticconi et al.,
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2009), and LPR1 and its close homolog LPR2 belong to a large family of multicopper
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oxidases (Svistoonoff et al., 2007). Genetic analysis indicates that PDR2 and
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LPR1/LPR2 act in the same pathway, with LPR1/LPR2 being epistatic to PDR2
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(Ticconi et al., 2009). However, how these proteins interact to control the Pi
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deficiency-induced remodeling of RSA is still unclear.
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Interestingly, iron (Fe) is essential for Pi deficiency-induced inhibition of 3
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roots (Misson et al., 2005; Hirsch et al., 2006; Svistoonoff et al., 2007; Ward et al.,
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2008; Zheng et al., 2009). When plants are grown on a Pi-deficient (P-) medium
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without Fe, the inhibition of primary root growth is abolished (Ward et al., 2008).
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Subsequent studies demonstrated that both LPR1 and LPR2 proteins possess
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ferroxidase activity (Müller et al., 2015). On P- medium, the accumulation of Fe3+ in
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lpr1 was significantly reduced; in contrast, pdr2 accumulated a higher level of Fe3+
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than the wild type (WT). Müller et al (2015) proposed that plants subjected to Pi
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deficiency over-accumulate Fe3+, which generates a high level of reactive oxygen
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species (ROS), resulting in increased deposition of callose in cell walls and
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plasmodesmata (PD). The enhanced callose deposition in PD then interferes with the
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intercellular movement of SHR (SHORT ROOT) protein, a key transcription factor
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involved in the maintenance of the root stem cell niche (SCN), and thus impairs
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primary root growth. However, researchers have yet to identify the other components
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of the PDR2-LPR1/2 pathway that regulates Pi deficiency-induced remodeling of
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RSA by modulating Fe homeostasis in roots.
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In this work, we characterized an Arabidopsis mutant, hps10, which is
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hypersensitive to Pi deficiency-induced inhibition of primary root growth and
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enhanced lateral root formation. We show that hps10 is a previously identified allele
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(als3-3) of the ALUMINUM SENSITIVE3 (ALS3) gene that is implicated in plant
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tolerance to aluminium (Al) toxicity (Larsen et al., 2005). We further demonstrate that
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ALS3 and its interacting protein, AtSTAR1, form an ABC transporter complex in
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tonoplasts. Based on the results obtained, we propose that this transporter acts with
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LPR1/LPR2 to regulate Pi deficiency-induced remodeling of RSA through
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modulation of Fe homeostasis in roots.
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RESULTS
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als3-3 is Hypersensitive to Pi deficiency-Induced Remodeling of RSA An Arabidopsis mutant, hps10 (hypersensitive to Pi starvation10), was 4
ACCEPTED MANUSCRIPT identified from a T-DNA insertion library (Alonso et al., 2003). hps10 was found to be
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the Salk_004094 line. This T–DNA line was previously characterized as als3-3
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(aluminum sensitive3-3), which displays a hypersensitive response to Al toxicity
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(Larsen et al., 2005). When grown on a Pi-sufficient (P+) medium (1/2 MS medium
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with 1% sucrose and 1.2% agar), the roots of the als3-3 mutant did not differ
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morphologically from those of the WT (Figure 1A). In contrast, when grown on a P-
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medium, the length of als3-3 primary root was about 50% shorter than that of the WT.
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Accordingly, the length of root epidermal cells in the maturation zone were half as
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long in als3-3 as in the WT (Supplemental Figure 1A). At 11 DAG (days after
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germination), the root apical meristem (RAM) of the WT was 50% smaller on P-
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medium than on P+ medium, with the cellular organization of the RAM remaining
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well maintained (Figure 1B). At that time, the root meristematic cells of Pi-deficient
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als3-3 had lost their identity, becoming enlarged and highly vacuolated, and had
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prematurely differentiated into root hairs. Under Pi deficiency, lateral roots appeared
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at 5 DAG for als3-3 but at 7 DAG for the WT. By 8 DAG, lateral root density was two
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times greater for als3-3 than for the WT (Supplemental Figure 1B). On P- medium,
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although the primary root of als3-3 stopped growing by 4 DAG, its lateral roots
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continued to elongate. Under Pi deficiency, als3-3 also formed more and longer root
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hairs than the WT (Supplemental Figure 1C). A Pi concentration-dependent analysis
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indicated that Pi levels < 100 µM were required to distinguish root phenotypic
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differences between als3-3 and the WT (Supplemental Figure 2).
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and F2 progeny derived from selfed F1 plants segregated into mutant and WT
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phenotypes in a ratio of 1:3 (52:169), indicating that the als3-3 mutant phenotype was
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caused by a single recessive mutation. als3-3 was backcrossed to the WT four times
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before further characterization.
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als3-3 Over-accumulates Fe3+ in Roots Under Pi Deficiency
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We then examined the level of Fe in the roots of 8-day-old seedlings using the
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Perls staining method, which mainly stains labile (non-heme) Fe3+ (Merguro et al., 5
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mainly localized in the SCN, which includes the quiescent center (QC) and its
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surrounding initials, as well as in the cortex of the root apex (Figure 2A). This was
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consistent with the observations of Müller et al (2015). On P- medium, Fe3+
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accumulation in the SCN and cortex of WT roots was dramatically reduced and was
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undetectable in most samples; instead, a light-blue staining was evident proximal to
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the root apex, mostly in the maturation zone. For als3-3 on P- medium, however,
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there was a light-blue staining in the elongation zone and a dark-blue staining in the
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maturation zone. A similar Fe accumulation pattern was observed when a more
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sensitive Perls/DAB (diaminobenzidine) staining method was used (Figure 2B). In
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this method, the Perls staining was intensified with the addition of DAB, which stains
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both Fe2+ and Fe3+ (Roschzttardtz et al., 2009). With the Perls/DAB staining method,
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a low level of Fe accumulation was detected in the SCN and in all root cell layers in
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the root apex except the epidermis. Quantitative analysis indicated that the total Fe
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content was about four times higher in P- roots than in P+ roots of the WT (Figure 3).
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The Fe content in the roots of P- als3-3 was, however, only 25% higher than that in
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the roots of P- WT, indicating that the overstaining of Fe in P- als3-3 was mainly due
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to the conversion of Fe 2+ to Fe3+ rather than to an increase in total Fe content.
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When grown on P- medium in the absence of Fe, the root morphology of als3-3
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did not differ from that of the WT (Supplemental Figure 3), and Fe staining was not
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detected in either als3-3 or WT roots by the Perls method (Figure 2A). A very weak
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staining pattern was observed for roots of als3-3 and WT using the Perls/DAB
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method (Figure 2B). These results suggested that the enhanced inhibition of primary
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root growth of Pi-deficient als3-3 might be due to the over-accumulation of Fe3+ in its
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roots.
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Mutation in ALS3 Causes A Hypersensitive Root Response to Pi Deficiency
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als3-3 carries a T-DNA insertion near the end of the second exon of the gene
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AT2G37330 (Figure 4A). No transcript of AT2G37330 was detected in this line by
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RT-PCR analysis, indicating that als3-3 is a strong allele (Supplemental Figure 4A). 6
ACCEPTED MANUSCRIPT The AT2G37330 gene is 1,050 bp long and contains three exons and two introns.
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AT2G37330 was previously identified as ALS3 (ALUMINUM SENSITIVE 3), because
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its mutation resulted in plants being hypersensitive to Al toxicity (Larsen et al., 2005).
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ALS3 encodes a half-ABC (ATP-binding cassette) transporter-like protein that has
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seven predicted transmembrane domains (TMD) but lacks nucleotide-binding
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domains (NBDs). We then examined the Pi deficiency responses of als3-1 and als3-2
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(Larsen et al., 2005) and another T-DNA insertion allele (SALK_061074, designated
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als3-4). als3-1 carries a substitution of T for C at nucleotide 335, resulting in the
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conversion of a serine to a leucine (Larsen et al., 2005), and als3-2 and als3-4 contain
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T-DNA insertions in the 5’ UTR and the third exon of the AT2G37330 gene,
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respectively (Figure 4A). Like als3-3, all other als3 alleles were morphologically
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normal under Pi sufficiency but exhibited enhanced inhibition of primary root growth
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under Pi deficiency (Figure 4C). The root growth inhibition was less for als3-2 than
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for the other three als3 alleles. This was probably due to a residual expression of the
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ALS3 gene in this SALK line (Supplemental Figure 4A). To confirm that the T-DNA
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insertion in the AT2G37330 gene was responsible for the mutant phenotypes of
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Pi-deficient als3-3, we introduced the genomic sequence of the WT AT2G37330 gene
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into als3-3 under the control of the CaMV 35S promoter or its own promoter. Both
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gene constructs could fully complement the mutant root phenotypes (Figure 4D),
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demonstrating that the T-DNA insertion in the AT2G37330 gene was responsible for
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the Pi-hypersensitive phenotype of als3-3.
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(N), potassium (K), and Fe deficiencies, as well as under low pH (4.2). None of these
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stress treatments resulted in a growth difference between als3-3 and WT
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(Supplemental Figure 5), suggesting ALS3 specificity in the processes underlying Pi
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deficiency and Al toxicity responses.
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AtSTAR1 and ALS3 Function Similarly in Plant Responses to Pi Deficiency
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ABC transporters represent a large family in Arabidopsis; they are localized in
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different organelles, transport a diverse array of substrates, and thereby function in a 7
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transporter is composed of at least one TMD and one NBD. Given that ALS3 lacks an
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NBD, it must act with a partner protein to transport its substrate(s). OsSTAR1 and
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OsSTAR2 were previously reported to form a functional ABC transporter that is
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required for Al tolerance in rice (Huang et al., 2009). OsSTAR1 contains only an
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NBD, while OsSTAR2, the counterpart of ALS3 in rice, contains only a TMD.
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Knockout of AtSTAR1 (ABCI17, AT1G67940), the counterpart of OsSTAR1 in
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Arabidopsis, also resulted in plants that were hypersensitive to Al toxicity (Huang et
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al., 2010). These observations, in conjunction with the phenotypes described above
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for als3-3, suggested that AtSTAR1 and ALS3 might form a protein complex that
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functions as an ABC transporter in Arabidopsis. Consequently, if AtSTAR1 and
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ALS3 form a functional protein complex, the knockout of AtSTAR1 should also result
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in plants showing a hypersensitive phenotype to Pi deficiency. The strong allele of the
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AtSTAR1 gene, atstar1 (ABRC stock no. CS384144) contains a T-DNA insertion in
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its second exon that strongly decreases the transcription of the AtSTAR1 gene (Figure
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4B and Supplemental Figure 4B). We then examined the atstar1 phenotype in
17
response to Pi deficiency. The root growth inhibition in P- medium was significantly
18
greater for atstar1 than for the WT (Figure 4E) and was similar in degree to that
19
exhibited by als3-3. Like als3-3, atstar1 also over-accumulated Fe3+ in its roots on P-
20
medium (Figure 5). These phenotypic similarities indicated that AtSTAR1 and ALS3
21
components of common physiological mechanism involved in plant responses to
22
both Pi deficiency and Al toxicity. Furthermore, on our standard P- medium (1/2 MS
23
medium with 1% sucrose and 1.2% agar without Pi), the growth of plants
24
overexpressing either ALS3 or AtSTAR1 alone was similar to that of plants
25
concurrently overexpressing AtSTAR1 and ALS3. This was potentially due to residual
26
Pi (about 10 µM Pi) remaining in the agar, which was not stressful enough to
27
distinguish the Pi deficiency responses among the plants with different genotypes. We
28
then replaced agar in our standard P- medium with agarose so that the plants grown
29
on this modified medium would be more Pi starved. On this agarose-containing
30
medium, the transgenic plants concurrently overexpressing AtSTAR1 and ALS3 grew
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significantly better than the plants overexpressing either ALS3 or AtSTAR1 alone
2
(Supplemental Figure 6), thereby providing further evidence for ALS3 and AtSTAR1
3
interactions.
4
ALS3 and AtSTAR1 Directly Interact at the Tonoplasts
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Several approaches were taken to validate the inferred ALS3 and AtSTAR1
7
interactions. First, a yeast split-ubiquitin two-hybrid approach (Johnsson and
8
Varshavsky, 1994) was used, where ALS3 was fused to the C-terminal half of the
9
ubiquitin gene (Cub), and AtSTAR1 was fused to the mutated N-terminal half of the
10
ubiquitin gene (NubG). As the negative controls, the co-transformation of ALS3-Cub
11
with the NubG empty vector or co-transformation of the Cub empty vector and
12
NubG-AtSTAR1 did not enable the yeast cells to grow on the selective medium. In
13
contrast, the yeast cells co-transformed with ALS3-Cub and NubG-AtSTAR1
14
constructs were able to grow on the selective medium, indicating that ALS3 and
15
AtSTAR1 interacted to activate the expression of the reporter gene for growth
16
selection (Figure 6A).
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The ALS3 and AtSTAR1 interaction in planta was then validated in the leaves
17
of
Nicotiana
benthamiana
via
two
independent
approaches:
luciferase
19
complementation imaging (LCI) assays (Chen et al., 2008) and bimolecular
20
fluorescence complementation (BiFC) assays. For LCI, the coding sequences (CDSs)
21
of ALS3 and AtSTAR1 were fused to the N- and C-terminal half of the luciferase
22
(LUC) gene, respectively, resulting in the gene constructs ALS3-nLUC and
23
cLUC-AtSTAR1. Transient co-expression of these chimeras under the control of the
24
CaMV 35S promoter resulted in strong LUC activity (Figure 6B). In contrast,
25
co-expression of nLUC and cLUC, nLUC and cLUC-AtSTAR1, or ALS3-nLUC and
26
cLUC did not result in LUC activity. For BiFC analysis of the ALS3-AtSTAR1
27
interaction, the CDSs of ALS3 and AtSTAR1 were fused to the N- and C-terminal
28
halves, respectively, of the yellow fluorescence protein (YFP) gene. Transient
29
co-expression of ALS3-nYFP with cYFP-AtSTAR1 under the control of the CaMV 35S
30
promoter restored the YFP fluorescence (Figure 6C). The fluorescence signal in intact
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ACCEPTED MANUSCRIPT leaf tissue was observed predominantly in the periphery of leaf cells, with an
2
additional band surrounding the nucleus. Released vacuoles from the isolated
3
mesophyll protoplasts from transformed leaves exhibited the YFP signal, which was
4
associated with the tonoplast (Figure 6D). The vacuolar localization of the
5
ALS3-AtSTAR1 interaction was further confirmed by transient expression of the
6
BiFC constructs in protoplasts isolated from mesophyll cells of Arabidopsis, which
7
resulted in a YFP signal that did not co-localize with the plasma membrane-specific
8
dye, FM4-64 (Figure 6E). The absence of the fluorescence signal in specific regions
9
of the cell periphery (usually near the places where the chloroplasts were located)
10
provided additional evidence that ALS3 and AtSTAR1 interact at the tonoplast.
11
Co-expression of either ALS3-nYFP or cYFP-AtSTAR1 with the corresponding half of
12
the YFP empty vector did not result in a fluorescence signal in any of the above BiFC
13
assays.
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To further confirm the tonoplast localization of the ALS3/AtSTAR1 protein complex, the HA-tagged ALS3 (ALS3-HA) and FLAG-tagged
16
(AtSTAR1-FLAG) were transiently co-expressed in the leaves of N. benthamiana by
17
Agrobacterium infection. After 48 h, the total proteins were isolated from the infected
18
leaves and subjected to Western blot using HA- and FLAG-specific antibodies. The
19
results indicated that both proteins were successfully expressed in the leaves of N.
20
benthamiana (Figure 6F). Next, the total proteins were separated into soluble and
21
membrane fractions. The ALS3-HA was exclusively detected in the membrane
22
fraction whereas AtSTAR1-FLAG was found in both soluble and membrane fractions
23
(Figure 6G). Finally, we performed sucrose density-gradient centrifugation using
24
tonoplast- and plasma membrane-specific markers to determine the subcellular
25
localization of the ALS3/AtSTAR1 complex. The results showed that the distributions
26
of ALS3-HA and AtSTAR1-FLAG completely overlapped with the distribution of the
27
tonoplast-specific marker, V-ATPase, but not with distribution of the plasma
28
membrane-specific marker, H+-ATPase (Figure 6H).
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ACCEPTED MANUSCRIPT To determine whether the expression of ALS3 is affected by Pi availability, we
2
first performed quantitative real-time PCR (qPCR) assays using mRNA extracted
3
from 8-day-old WT seedlings. Pi deficiency induced upregulation of ALS3 in both
4
shoots and roots (Supplemental Figure 7A). To identify the tissues where ALS3
5
expression is induced, we fused a DNA fragment upstream of its transcription start
6
site and its genomic sequence to a GUS reporter gene (ALS3::ALS3-GUS) according
7
to Larsen et al. (2010) and transformed this gene construct into WT plants. On P+
8
medium, GUS activity was only detected in the vascular tissues of roots and
9
cotyledons of 8-day-old seedlings (Supplemental Figure 7B). In the root tips of P-
10
seedlings, the GUS expression domain extended from vascular tissues to all layers of
11
roots cells, including root hairs. No GUS expression was detected in the root
12
meristem under either P+ or P- conditions.
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The expression of AtSTAR1 was also slightly induced by Pi deficiency as
14
determined by qPCR (Supplemental Figure 7C). To determine its tissue-specific
15
expression patterns, we constructed an AtSTAR1::AtSTAR1-GUS gene chimera
16
according to Huang et al. (2010) and transformed it into WT plants. In the 8-day-old
17
AtSTAR1::AtSTAR1-GUS seedlings, the GUS gene was expressed in all parts of the
18
root except for the meristem (Supplemental Figure 7D). GUS staining was further
19
enhanced by Pi deficiency in the roots.
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previously investigated in Xenopus laevis oocytes, and the research indicated that the
24
protein complex transports UDP-glucose (UDP-Glc) but not UDP-glucuronic acid
25
(UDP-GlcA) or UDP-galactose (UDP-Gal) (Huang et al., 2009). Because
26
exogenously applied UDP-Glc alleviated the toxic effect of Al on the root growth of
27
rice star1 seedlings (Huang et al., 2009), we determined whether UDP-Glc and its
28
related compounds had similar effects on als3-3. The results showed that inclusion of
29
500 µM UDP-Glc or UDP-GlcA, but not UDP, glucose, or UDP-Gal, completely
30
rescued the short-root phenotype of als3-3 on P- medium (Figure 7). The lowest 11
ACCEPTED MANUSCRIPT 1
effective concentration of UDP-Glc was 100 µM (Supplemental Figure 8). UDP-Glc
2
could also rescue the root phenotypes of atstar1 grown on P- medium (Figure 7).
3
AtSTAR1 and ALS3 Interact to Mediate a Highly Electrogenic Transport in
5
Xenopus Oocytes
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To characterize the transport function of the ALS3/AtSTAR1 protein complex,
7
we investigated their electrophysiological properties when expressed in Xenopus
8
oocytes. The ALS3-AtSTAR1 interaction was first validated via BiFC, by coinjection
9
of cRNA chimeras. YFP signal was detected upon co-injection of ALS3::nYFP and
10
cYFP::AtSTAR1 (Figure 8A), while no background YFP signal was detected in cells
11
co-injected with ALS3::nYFP and the cRNA encoding the complementary cYFP.
12
Having validated the ALS3-AtSTAR1 structural interactions, we then determined the
13
functional interactions by examining the transport characteristics of ALS3, AtSTAR1,
14
and the protein complex via two-electrode voltage clamp (TEVC) of Xenopus oocytes.
15
Cells injected only with ALS3 or AtSTAR1 cRNA had resting membrane potentials
16
(RMPs) resembling the values in control cells (i.e., cells injected with water). In
17
contrast, cells co-injected with ALS3 and AtSTAR1 cRNA had significantly less
18
negative RMPs than control cells (Figure 8B). Under voltage clamp conditions, cells
19
expressing only ALS3 or AtSTAR1 did not show a significant difference in
20
electrogenic transport compared to that recorded in control cells. In contrast, cells
21
co-expressing ALS3 and AtSTAR1 showed large, slowly activating inward (negative)
22
currents (Figure 8C and 8D), which reversed (i.e., change in current sign) at a
23
potential significantly less negative than those recorded in cells expressing only ALS3
24
or AtSTAR1, or in control cells. By convention, the ALS3/AtSTAR1-mediated inward
25
(negative) currents are the product of net positive charge influx (e.g., Na+ or H+) or
26
net negative charge efflux (e.g., organic or inorganic anions), This inference is
27
consistent with the depolarization state (i.e., reduction of the net internal negative
28
charge) recorded in ALS3+AtSTAR1, relative to that found in control cells or in cells
29
expressing only ALS3 or AtSTAR1.
30
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We also determined whether the ALS3/AtSTAR1 complex could mediate Pi or 12
ACCEPTED MANUSCRIPT UDP-Glc transport; however, the magnitude of the ALS3/AtSTAR1-mediated currents
2
was insensitive to increases in intracellular Pi concentrations (i.e., loading up to 3
3
days) or UDP-Glc (i.e., microinjected) (Supplemental Figure 9). Overall, the above
4
results validated a functional interaction between ALS3 and AtSTAR1, and indicated
5
that this protein complex mediates significant transport across the membrane;
6
nonetheless, the nature of the endogenous (in oocytes) and/or in planta substrate(s)
7
for the ALS3/AtSTAR1 transporter remains unknown.
8
ALS3 and LPR1/2 Act in the Same Regulatory Pathway
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In Arabidopsis, LPR1 and its homolog LPR2 play important roles in regulating Pi
11
deficiency-induced inhibition of primary root growth (Svistoonoff et al., 2007). These
12
two proteins possess ferroxidase activity (Müller et al., 2015). lpr1 and lpr2 mutants
13
accumulated less Fe3+ in their roots than the WT and were insensitive to Pi
14
deficiency-induced inhibition of primary root growth. To determine whether ALS3
15
and LPR1/2 act in the same regulatory pathway, we constructed the triple mutant
16
als3-3lpr1lpr2. In our standard agar-containing P- medium, the primary root growth
17
of lpr1lpr2 was similar to that of the WT. Therefore, we used a modified
18
agarose-containing P- medium as mentioned earlier to test plant growth phenotypes.
19
In this modified medium, lpr1lpr2 was less sensitive to Pi deficiency than the WT, as
20
previously reported by Svistoonoff et al. (2007). The root growth phenotypes of
21
als3-3lpr1lpr2 resembled those of lpr1lpr2 (Figure 9A). In addition, we identified a
22
genetic suppressor of als3-3 (from EMS-mutagenized als3-3 plants) whose root
23
phenotype was similar to that of lpr1 (Figure 9B). Sequencing of the LPR1 gene in
24
this suppressor indicated a single point mutation in its second exon at position 1157,
25
with a G to A substitution resulting in a conversion of a glycine to an arginine (Figure
26
9C). We refer to this suppressor as als3-3/lpr1. Together, these results suggest that
27
ALS3 and LPR1/2 act in the same signalling pathway that regulates Pi
28
deficiency-mediated inhibition of primary root growth, with LPR1/2 being epistatic to
29
ALS3.
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Additional Evidence That the Over-accumulation of Fe3+ Is Responsible for
2
als3-3 Mutant Phenotypes As noted earlier, our results suggested that the over-accumulation of Fe3+ in roots
4
is directly linked to the als3-3 root phenotypes (Figure 2). To further test this
5
hypothesis, we examined Fe3+ accumulation in the roots of als3-3 grown on P-
6
medium supplemented with UDP-Glc. UDP-Glc treatment completely reversed the
7
over-accumulation of Fe3+ in als3-3 (Figure 10 and Supplemental Figure 10). Also, in
8
the als3-3lpr1hpr2 triple mutant and als3-3 suppressor als3-3/lpr1, the levels of Fe3+
9
accumulation were similar to that of lpr1hpr2, both being lower than that of the WT
10
(Figure 10 and Supplemental Figure 10). These results provided additional evidence
11
that the over-accumulation of Fe3+ in roots is responsible for the root phenotypes of
12
als3-3.
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DISCUSSION
The remodeling of RSA is a major developmental response of plants to Pi
16
deficiency and is believed to enhance a plant’s ability to acquire Pi. Although this
17
phenomenon has been well documented in a variety of plant species, the underlying
18
molecular mechanism(s) remain unclear. In this work, we identified the Arabidopsis
19
mutant hps10 as being hypersensitive to inhibition of primary root growth and
20
stimulation of lateral root production induced by Pi deficiency (Figure 1). hps10 was
21
further found to be a previously identified allele (als3-3) of ALS3, which is a key
22
determinant of plant tolerance to Al toxicity (Larsen et al., 2005). A similar result was
23
reported by Belal et al. (2015) during the course of this research. In their brief report,
24
the latter authors also found that als3-3 was hypersensitive to Pi deficiency-induced
25
remodeling of root architecture, and that the sensitivity of als3-3 to Pi deficiency was
26
sucrose-dependent. Our work corroborated their results, and together the results
27
suggest that the mechanism used to sense external Pi levels is impaired in als3-3. To
28
date, all of the reported Arabidopsis mutants with altered root responses to Pi
29
deficiency, except for lpr1 and lpr2, showed developmental abnormalities under
30
normal growth conditions (for examples, see Miura et al., 2005; Nacry et al., 2005;
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Jiang et al., 2007; Mayzlish-Gati et al., 2012; Singh et al., 2014). This indicates that
2
the effects of these mutations are not specific to Pi sensing and signaling. In contrast,
3
the root phenotype of als3-3 does not
4
growth conditions or in response to other types of nutrient deficiency (such as N, K,
5
and Fe deficiency) or to low pH conditions (Supplemental Figure 5). These results
6
suggest that ALS3 is not involved in the regulation of intrinsic developmental
7
programs but is an important component of specific pathways
8
responses to Pi deficiency. These results also suggest that als3-3 will be particularly
9
useful for studying Pi sensing mechanisms in higher plants.
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differ from that of the WT under normal
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controlling plant
ALS3 encodes a TMD of a bacterial-type ABC transporter. In rice, OsSTAR1
11
(which contains only an NBD) interacts with OsSTAR2 (which contains only a TMD),
12
forming a transport complex in cytosolic vesicle membranes of root cells (Huang et
13
al., 2009). AtSTAR1 and ALS3 are the Arabidopsis counterparts of OsSTAR1 and
14
OsSTAR2 (Huang et al., 2009, 2010). Our work and that of Belal et al (2015) showed
15
that like als3-3, the atstar1 mutant displays hypersensitive root responses to Pi
16
deficiency, suggesting that AtSTAR1 and ALS3 might interact to form a common
17
transporter complex (Figure 4E). The similarity in the over-accumulation of Fe3+ in
18
roots of atstar1 and als3-3 (Figure 5) also indicated that AtSTAR1 and ALS3 may
19
co-exist in the same protein complex. Before the current study, however, experimental
20
evidence for the direct interaction between ALS3 and AtSTAR1 in Arabidopsis was
21
lacking. In this study, we provided compelling evidence for the interaction between
22
ALS3 and AtSTAR1. Based on yeast two-hybrid, LCI, and BiFC assays and
23
membrane fractionation experiments, the current study demonstrated that ALS3 and
24
AtSTAR1 physically interact in the tonoplasts (Figure 6). This is consistent with the
25
finding that AtSTAR1 has been detected in the proteome of tonoplasts (Jaquinod et al.,
26
2007). The detection of AtSTAR1-FLAG in the soluble fraction in our experiments
27
was probably due to the overexpression of AtSTAR1-FLAG proteins, some of which
28
were not associated with ALS3 in the tonoplasts. Expression in Xenopus oocytes
29
further demonstrated that the AtSTAR1 and ALS3 interaction leads to the formation
30
of a functional transport complex, i.e., the co-expression of ALS3 and AtSTAR1 but
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not the expression of either protein alone resulted in a significant increase in
2
electrogenic transport activity (Figure 8). Arabidopsis lpr1 and lpr2 mutants are insensitive to Pi deficiency-induced
4
inhibition of primary root growth (Svistoonoff et al., 2007). Like als3-3, these two
5
mutants are also morphologically normal under Pi sufficiency. Through genetic
6
analysis, we showed that ALS3 and LPR1/LPR2 act in the same pathway in
7
regulating Pi deficiency-induced inhibition of primary root growth and that
8
LPR1/LPR2 mutations are epistatic to ALS3 (Figure 9). LPR1 is localized in both the
9
ER and cell walls and has ferroxidase activity that oxidizes Fe2+ to Fe3+ in the root
10
apoplast (Müller et al., 2015). In the WT under P+ conditions, Fe3+ mainly
11
accumulates in the SCN and cortex of the root apex, as revealed by histochemical
12
staining (Figure 2 and 5). This Fe3+ accumulation pattern agrees with the
13
tissue-specific expression pattern of the LPR1 gene as reported by Müller et al (2015).
14
Under P- conditions, the level of Fe3+ in the SCN and cortex of the root apex is
15
dramatically reduced, and the accumulation of Fe3+ is shifted to the upper part of the
16
root. In lpr1, the accumulation of Fe3+ was barely detected by Perls staining under
17
either P+ or P- conditions (Figure 10 and Supplemental Figure 10). For als3-3 on P+
18
medium, the Fe3+ accumulation pattern is similar to that of the WT; for als3-3 on P-
19
medium, however, Fe3+ accumulation is greatly increased behind the root apex
20
(Figure 2). This change in the Fe3+ accumulation pattern correlates with the root
21
phenotypes of als3-3 under both P+ and P- conditions and suggests that the
22
over-accumulation of Fe3+ is the cause of the enhanced inhibition of primary root
23
growth in Pi-deficient als3-3. This inference was further supported by the following
24
experimental evidence: 1) the hypersensitive root phenotype of als3-3 is no longer
25
evident on P-Fe- medium, and over-accumulation of Fe3+ in the root is also
26
diminished (Supplemental Figure 3 and Figure 2); 2) in the als3-3lpr1lpr1 triple
27
mutant and in a genetic suppressor of als3-3 (als3-3/lpr1), neither the Pi
28
hypersensitive phenotype nor the over-accumulation of Fe3+ is observed (Figure 9 and
29
10, Supplemental Figure 10); and 3) in the UDP-Glc-treated als3-3, whose
30
root-growth phenotypes are restored to those of the WT on P- medium, the level of
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ACCEPTED MANUSCRIPT Fe3+ accumulation is similar to that of the WT (Figure 7 and 10, Supplemental Figure
2
10). Taken together, these observations lead us to hypothesize that the
3
ALS3/AtSTAR1 protein complex mediates Pi deficiency-induced remodeling of RSA
4
by modulating Fe homeostasis in roots. The hypothesis could explain why
5
LPR1/LPR2 is epistatic to ALS3 in regulating Pi deficiency-induced inhibition of
6
primary root growth, i.e., the over-accumulation of Fe3+ in the root apoplast of als3-3
7
may be overcome by the loss of ferroxidase activity encoded by LPR1.
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The mutant phenotypes of both als3-3 and atstar1 are restored by exogenously
9
applied UDP-Glc or UDP-GlcA (Figure 7). LPR1 is associated with cell walls, and
10
the oxidation of Fe2+ to Fe3+ by LPR1 apparently occurs in the apoplast of roots
11
(Müller et al., 2015). The rescue effect of UDP-Glc and UDP-GlcA might result from
12
the chelation of Fe3+ by the Pi released from the UDP moiety as catalyzed by secreted
13
acid phosphatases. However, this does not seem to be the case because UDP and
14
UDP-Gal did not show the rescue effect. UDP-Glc is the active form of Glc, which is
15
the substrate for the biosynthesis of cellulose, the major structural element of cell
16
walls. UDP-Glc can also be converted to UDP-GlcA by a UDP-Glc dehydrogenase,
17
which will further produce UDP-xylose (UDP-Xyl) by a UDP-GlcA carboxylase.
18
UDP-Xyl is the active form of Xyl, which is the substrate for the biosynthesis of
19
xyloglucan. Another major component of the cell wall, pectin, is also rich in GlcA. In
20
plants, cellulose and xyloglucans together form a carbohydrate network that is
21
embedded in a matrix consisting of pectins, hemicellulose, and cell wall proteins.
22
Therefore, the rescue effect of UDP-Glc and UDP-GlcA on als3-3 root phenotypes
23
suggests that the root cells of als3-3 may have defects in the formation of xyloglucan
24
or pectins and that these defects alter cell wall architecture. Cell wall architecture
25
profoundly affects the function of cell wall-associated enzymes by restricting the
26
location of these enzymes in cell walls or by altering their enzymatic activity through
27
the electrostatic interactions between the enzyme molecules and pectins, which carry
28
a large number of negative charges. Using comparative transcriptome and proteome
29
analyses, a recent study found that the genes involved in cell wall modification and
30
ROS formation were overrepresented in the list of the genes whose expression was
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ACCEPTED MANUSCRIPT deregulated in pdr2 and lpr1lpr2 mutants (Hoehenwarter et al., 2016). This study also
2
reported that roots of pdr2 had increased pectin content and that the Arabidopsis
3
mutant qua1-2, which had reduced pectin content, was hypersensitive to Pi starvation.
4
Therefore, the over-accumulation of Fe3+ in als3-3 might result from the elevated
5
LPR1 activity in the root apoplast due to reduced pectin content. And, the
6
exogenously supplied UDP-Glc or UDP-GlcA could compensate the reduced pectin
7
biosynthesis in als3-3 and atstar1, which alleviates their hypersensitive root
8
phenotypes in responding to Pi deficiency. This notion was further supported by our
9
quantitative analysis of Fe content, which showed that the over-accumulation of Fe3+
10
in P- als3-3 might result from over-conversion of Fe2+ to Fe3+ due to the elevated
11
LPR1/LPR2 activities rather than from the overload of Fe from the growth medium
12
(Figure 3). Alternatively, the mutation in the ALS3/AtSTAR1 complex may elevate
13
the enzymatic activity of LPR1/LPR2 through an unknown mechanism that results in
14
the over-accumulation of Fe3+. The increased Fe3+ content, in turn, as proposed by
15
Müller (2015), generates more ROS, which greatly enhances the crosslinking of the
16
cell walls, leading to the hypersensitive root phenotypes of als3-3 and atstar1.
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The rice OsSTAR1/OsSTAR2 complex is localized in cytoplasmic membrane
18
vesicles and transports UDP-Glc but not UDP-GlcA in Xenopus oocytes (Huang et al.,
19
2009). The latter study suggested this was the underlying reason as to why
20
exogenously applied UDP-Glc but not UDP-GlcA alleviated the toxic effect of Al on
21
the growth of rice seedling roots. Huang et al. (2009) proposed that in the WT, the
22
Golgi-synthesized UDP-Glc is delivered to the cell surface to modify the cell walls,
23
thereby masking sites of Al3+ binding, resulting in increased Al tolerance in rice. In
24
rice osstar1 or osstar2 mutant, such delivery is impaired, making the rice seedlings
25
more sensitive to Al toxicity. This explanation does not seem to apply to Arabidopsis
26
for the following reasons: 1) OsSTAR1/OsSTAR2 is located in cytoplasmic vesicle
27
membranes whereas ALS3/AtSTAR1 is located in tonoplasts (Figure 6); 2) OsSTAR1
28
and OsSTAR2 are mainly expressed in roots while ALS3 and AtSTAR1 are expressed
29
in roots as well as in the vascular tissues of other organs (Supplemental Figure 7); 3)
30
the als3-3 mutant phenotypes can be rescued by both UDP-Glc and UDP-GlcA
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2
(Huang et al., 2009); and 4) ALS3/AtSTAR1 does not transport UDP-Glc in oocytes
3
(Supplemental Figure 9). Thus, ALS3/AtSTAR1 most likely uses a different
4
mechanism from that of OsSTAR1/OsSTAR2 to regulate root response to Pi
5
deficiency. Because both OsSTAR1 and AtSTAR1 show sequence similarity to the
6
bacterial Pi transporter pstB (Huang et al., 2009; Chan and Torriani, 1996), we also
7
attempted to determine whether ALS3/AtSTAR1 can transport Pi. Functional analysis
8
in oocytes demonstrated that Pi is not a substrate for the ALS3/AtSTAR1 transporter,
9
at least under the experimental ionic conditions in our studies (Supplemental Figure 9).
10
A future challenge is to understand how the defect in the tonoplast-localized
11
ALS3/AtSTAR1 transporter complex affects cell wall integrity, which in turn
12
interferes with the Fe homeostasis in root cells.
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Identification of HPS10 as ALS3 also suggested that ALS3 might play a critical
14
role in the cross-talk between P and Al. It has long been known that P interacts with
15
Al in regulating plant growth and metabolism (Hartwell and Pember, 1918; Clarkson,
16
1967), and that the inhibitory effect of Al on root growth can be ameliorated by
17
increasing the supply of Pi (Tan and Keltjens, 1990; Liao et al., 2006; Sun et al.,
18
2008). A high supply of Pi alters Al-induced exudation of organic acids and the
19
transcription of some Al-responsive genes (Liang et al., 2013). Such interactions have
20
been thought to occur through chemical or biochemical reactions because Pi can
21
deplete Al3+ levels by forming an Al(PO4)3 complex. However, by maintaining the
22
same level of free Al3+ activity in nutrient solutions, Liao et al. (2006) found that
23
increasing the Pi supply still suppressed Al-induced exudation of citrate. Thus, the
24
antagonistic effect of high Pi on Al-induced citrate exudation might not simply be due
25
to the chelation of Al by Pi. Instead, Pi and Al may use some common signaling
26
mechanisms to elicit plant responses to both Pi deficiency and Al toxicity. Perhaps
27
ALS3 is a key component that is shared by Pi and Al for their cross-talk. It follows
28
that als3-3 could be useful for studying the molecular mechanism of P and Al
29
interactions.
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METHODS
3
Plant Materials All Arabidopsis thaliana plants used in this study were in the Columbia-0
5
background. als3-1 (CS3848), the three T-DNA insertion lines for ALS3, i.e.,
6
SALK_011435 (als3-2), SALK_004094 (als3-3), and SALK_061074 (als3-4), and a
7
T-DNA insertion line for AtSTAR1, i.e., CS384144 (atstar1), were obtained from
8
ABRC. Genomic DNA was extracted from each T-DNA line and was analyzed via
9
PCR using the primers specific for each T-DNA insertion. The mRNA expression of
10
the corresponding gene in the T-DNA insertion lines was analyzed by a
11
semi-quantitative RT-PCR method. The lpr1lpr2 mutant was a kind gift from Dr.
12
Thierry Desnos (CEA Cadarach, France). The als3-3lpr1lpr2 triple mutant was
13
generated by genetic cross between als3-3 and lpr1lpr2. The sequences of all primers
14
used are listed in Supplemental Table S1.
15 16
Plant Growth Conditions
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Half-strength MS medium with 1% (w/v) sucrose, 0.1% MES (w/v), and 1.2%
18
(w/v) agar (Sigma Cat. No. A1296) or 0.8% agarose (Biowest Regular Agarose G-10)
19
was used as the standard Pi-sufficient (P+) medium. The Pi-deficient medium (P-)
20
was prepared by replacing KH2PO4 with K2SO4 in the P+ medium. For P+Fe- and
21
P-Fe- media, the 50 µM Fe-EDTA was omitted from P+ and P- media. For Pi
22
dose-response analysis, various amounts of KH2PO4 were added to the P- medium.
23
The N-, and K- media were prepared as described (Jung et al., 2009; Yong et al.,
24
2010).
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In some cases (the experiments shown in Figure 9 and Supplemental Figure 7),
26
the agar in the standard media was replaced with agarose to further reduce the amount
27
of residual Pi in the growth medium.
28
Seeds were surface-sterilized in 20% bleach for 10 min and washed three times
29
with sterile-distilled water before they were placed on plates containing media. After
30
the seeds were stratified at 4 °C in the dark for 2 days, the plates were placed 20
ACCEPTED MANUSCRIPT 1
vertically in a growth room with a photoperiod of 16-h light/8-h dark at 21 to 23°C.
2
The light intensity was 100 µmol m–2 s–1.
3 4
Analyses of Root Growth Phenotypes Seedlings grown on the plates with media were photographed with a digital
6
camera (Sony DSC-W50). The length of primary roots was measured with Digimize
7
software. The lateral roots were counted with the aid of an SZ61 stereomicroscope. To
8
examine the cellular organization of the root meristem, the root tips were excised and
9
mounted in 30 µl of HCG clearing solution (chloroacetaldehyde : water : glycerol
10
=8:3:1) under a coverslip. The root tips were then visualized and imaged with a
11
differential interference contrast (DIC) microscope (Olympus BX51) equipped with a
12
camera (Olympus DP71). The length of root cells was determined by measuring the
13
length of root epidermal cells in the maturation zone. For observation of root hair
14
patterns, the first 5-mm segment of the root tip of 8-day-old seedlings was
15
photographed with a stereomicroscope (Olympus SZ61) equipped with a digital
16
camera (Olympus DP72).
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Quantitative Real-time RT-PCR (qPCR)
qPCR analyses of the expression of ALS3 and AtSTAR1 were performed as
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Vector Construction and Plant Transformation To generate plant expression vectors for genetic complementation, a DNA
24
fragment containing the genomic sequence of the ALS3 gene and its 1.2-kb promoter
25
region was PCR amplified from the genomic DNA isolated from WT Arabidopsis
26
seedlings. The amplified DNA fragment was cloned into EcoR I and Kpn I sites of the
27
binary vector pCAMBIA1300, resulting in the construct ALS3::ALS3 gDNA. To
28
generate the ALS3 overexpression vector, the genomic sequence of ALS3 was cloned
29
into the plant expression vector pZH01 using the Gibson assembly cloning method
30
(Gibson et al., 2009), resulting in the construct 35S::ALS3 gDNA. To generate the 21
ACCEPTED MANUSCRIPT AtSTAR1 overexpression vector, the genomic sequence of AtSTAR1 was PCR
2
amplified from genomic DNA and used to replace the GUS gene on the plant vector
3
pBI121, resulting in the construct 35S::AtSTAR1 gDNA. For analyses of
4
tissue-specific expression patterns of ALS3 and AtSTAR1, the translational fusion
5
constructs ALS3::ALS3-GUS and AtSTAR1::AtSTAR1-GUS were generated according
6
to Larsen et al. (2005) and Huang et al. (2010), respectively. The sequences of the
7
primers used for PCR amplification of the ALS3 and AtSTAR1 genes are listed in
8
Supplemental Table S1.
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The above constructs were transferred into Agrobacterium tumefaciens strain
10
GV3101 and transformed into als3-3, atstar1, or WT plants using the floral dip
11
method (Clough and Bent, 1998).
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Histochemical Staining for GUS Activity
The histochemical analyses of GUS activity were performed as described by Jefferson et al. (1987).
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Yeast Two-hybrid (Y2H) Assay
The Y2H assay was performed using the DUALmembrane System (Biotech
19
Dualsystems). The coding sequence (CDS) of HPS10 was PCR amplified from cDNA
20
of WT Arabidopsis plants and fused with the C-terminal half of the ubiquitin gene
21
(Cub) to generate the construct pDHB-ALS3-Cub. The CDS of AtSTAR1 was PCR
22
amplified and was fused with the mutated N-terminal half of ubiquitin (NubG),
23
resulting in the construct pPR3-NubG-AtSTAR1. The sequences of the primers used
24
for Y2H assay are listed in Supplemental Table S1. Various combinations of plasmids
25
were then co-transformed into NMY51 yeast cells according to the manufacturer’s
26
instructions. The transformants were selected on SD medium without Leu and Trp.
27
The yeast cells were spotted on the indicated plates for growth assay as described
28
previously (Huang et al., 2009).
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Luciferase Complementation Imaging (LCI) Assays 22
ACCEPTED MANUSCRIPT For vector construction for LCI assays, the CDSs of the ALS3 and AtSTAR1
2
genes were PCR amplified from Arabidopsis cDNA and inserted into the vectors
3
pCAMBIA1300-nLUC and pCAMBIA1300-cLUC (Chen et al., 2008), respectively,
4
to generate the constructs ALS3-nLUC and cLUC-AtSTAR1. The sequences of the
5
primers used to amplify the CDSs of ALS3 and AtSTAR1 are listed in Supplemental
6
Table S1. The LCI assays were performed in the leaves of N. benthamiana according
7
to Sun et al. (2016).
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BiFC Assays
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For BiFC assays in the leaves of N. benthamiana, the CDSs of ALS3 and
11
AtSTAR1 were cloned into the plant binary expression vectors pCAMBIA1300-nYFP
12
and pCAMBIA1300-cYFP, respectively, to generate constructs ALS3-nYFP and
13
cYFP-AtSTAR1. The BiFC assays in N. benthamiana were carried out according to
14
Sun et al. (2016). The vacuoles were isolated from mesophyll protoplasts of the leaves
15
of N. benthamiana transformed with the BiFC vectors as described (Robert et al.,
16
2007).
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For BiFC assays in Arabidopsis mesophyll protoplasts, the CDSs of ALS3 and
18
AtSTAR1 were fused to the C-terminal and N-terminal fragments of YFP gene in the
19
plasmids pVYNE and pVYCER, respectively, resulting in the constructs ALS3-nYFP
20
and cYFP-AtSTAR1. Various combinations of BiFC constructs were co-transformed
21
into Arabidopsis mesophyll protoplasts via a PEG-mediated method as described
22
(Yoo et al., 2007). Transformed protoplasts were stained with a culture medium
23
supplemented with 5 µM FM4-64 (Invitrogen) for 15 min, and were observed with
24
confocal microscopy.
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Sucrose Density-Gradient Centrifugation
27
For the 35S::ALS3-HA and 35S::AtSTAR1-FLAG constructs, the CDSs of ALS3
28
and AtSTAR1 were PCR amplified and cloned into the modified plant binary
29
expression
30
pCAMBIA1300-3×Flag (Lu et al., 2016), respectively. The constructs were
vectors
pCAMBIA1300-3×HA 23
(Ma
et
al.,
2013)
and
ACCEPTED MANUSCRIPT 1
co-transformed into leaves of N. benthamiana by Agrobacterium-mediated infiltration.
2
After 48 h, three grams of fresh leaves was harvested. Total, soluble, and microsomal protein fractions were extracted from the leaves
4
as described (Michael Weaver et al., 2006), with minor modification. In brief, the
5
leaves were ground in the pre-chilled homogenization buffer [50 mM Tris.HCl (pH
6
7.5), 20% glycerol, 5 mM DTT, 2 mM EDTA, 7 mM MgCl2 and protease inhibitors
7
(Roche) ] on ice, and centrifuged at 10,000 × g for 15 min at 4°C. The supernatant
8
was then collected and centrifuged at 100,000 × g for 1 h at 4°C to obtain soluble (S)
9
and microsomal membrane (M) fractions. The microsomal pellet was suspended in
10
the suspension buffer [25 mM Tris.HCl (pH 7.5), 10% (w/w) sucrose, 1 mM DTT, 2
11
mM EDTA, 7 mM MgCl2, and protease inhibitors (Roche)]. For sucrose
12
density-gradient centrifugation, the suspended samples were then layered on a
13
20-50% (w/w) sucrose gradient formed by the Gradient Master 108 (Biocomp),
14
followed by centrifugation at 210, 000 × g for 16 h using a SW41Ti rotor (Beckman).
15
The 600-µl density gradient fractions were collected for Western blot analysis.
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Western Blot Analyses
Proteins were separated on 8% SDS-polyacrylamide gels, transferred to a PVDF
19
membrane, and then subjected to Western blot analyses with the indicated antibodies.
20
Rabbit polyclonal antibodies against subunit E of plant V-ATPase (AS07 213,
21
Agrisera) or plant H+-ATPase (AS07 260-100, Agrisera) were used at a dilution of
22
1:3000. Mouse monoclonal antibodies against HA (CWBIO) and FLAG (Abmart)
23
were used at a dilution of 1:2000. Secondary goat anti-rabbit antibody (CWBIO) or
24
goat anti-mouse antibody (Abmart) conjugated with horseradish peroxidase was used
25
at a dilution of 1:5000. The signal was detected by the chemiluminescence reaction
26
using the eECL Western Blot Kit (CWBIO).
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Confocal Microscopy
29
The fluorescence signals generated in BiFC assays were observed with an
30
LSM710 META laser scanning microscope (Zeiss). The wavelengths of 24
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excitation/emission were 514 nm/520-550 nm for YFP protein and 543 nm/575-630
2
nm for FM4-64. Images were analyzed with ZEN 2009 Light Edition software.
3 4
Fe Histochemical Staining Assay The accumulation of Fe in roots was detected using the Perls staining method as
6
described (Roschzttardtz et al., 2009). In brief, roots were excised from 8-day-old
7
seedlings and vacuum infiltrated with Perls staining solution containing equal
8
volumes of 4% (v/v) HCl and 4% (w/v) K-ferrocyanide for 15 min. After infiltration,
9
the samples were kept in the same solution for another 30 min at room temperature
10
before they were rinsed with ultrapure water to stop the reaction. Stained samples
11
were cleared in the HCG clearing solution (1 g/ml chloral hydrate in 15% glycerol)
12
and examined with a DIC microscope.
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For the DAB intensification, Perls-stained samples were incubated for 60 min in
14
methanol containing 0.01 M NaN3 and 0.3% H2O2 (30%). Then, the samples were
15
washed with 0.1 M Na-phosphate buffer (pH7.2) and incubated for 5 min in the same
16
buffer containing 0.025% (w/v) DAB (Sigma-Aldrich) and 0.005% H2O2. To stop the
17
reaction, the samples were washed several times with phosphate buffer. Stained
18
samples were cleared by mounting them in HCG clearing solution on glass slides and
19
were examined with DIC microscopy.
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Quantification of Fe Content in Roots Fe content of root tissues was determined by the spectrophotometric BPS method
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according to Tamarit et al. (2006).
Genetic Screening for als3-3 Suppressors A 2.5-g
quantity of
als3-3
seeds
was
mutagenized
with
a
0.4%
27
ethylmethanesulfonate (EMS) solution as described (Kim et al., 2006). The
28
mutagenized M1 seeds were grown in soil and self-pollinated to yield M2 seeds. The
29
M2 seeds were then sown on 1.2% agar-solidified P- medium to screen for mutants
30
with the same primary root length as the WT. The phenotypes of the putative hps10 25
ACCEPTED MANUSCRIPT 1
suppressors were reconfirmed in the M3 generation.
2 3
Protein-protein Interaction and Electrophysiological Assays in Xenopus Oocytes The CDSs of ALS3 and AtSTAR1 were cloned into the T7TS vector (Pineros et al.,
5
2008) and into BiFC expression vectors suitable for electrophysiological and
6
protein-protein interaction analysis, respectively (Nour-Eldin et al., 2006). The BiFC
7
chimeras consisted of i) ALS3::nYFP, with ALS3 fused to the N-terminal half of a
8
split YFP; and ii) cYFP::AtSTAR1, with the C-terminal half of a split YFP fused to
9
AtSTAR1. After the respective cDNAs were linearized, cRNA was synthesized using
10
an mMessage mMachine in vitro transcription kit (Ambion) according to the
11
manufacturer’s recommendations.
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For electrophysiological experiments, stage V-VI Xenopus laevis oocytes were
13
harvested and microinjected with 50 nl of H2O for control cells, 50 nl of 250 ng/µl of
14
either AtSTAR1 or ALS3 cRNA, or 50 nl of 250 ng/µl of a 1:1 ALS3/AtSTAR1
15
mixture (500 ng/µl total cRNA). Oocytes used for BiFC were injected with a 1:1
16
ALS3::nYFP+cYFP::AtSTAR1 cRNA mixture or ALS3::nYFP+cYFP mixture (250
17
ng/µl total cRNA). Microinjected oocytes were incubated in ND88 at 18°C for 2 days
18
before they were examined with confocal microscopy or were subjected to
19
electrophysiological analysis. For experiments evaluating the effect of increased
20
intracellular P concentration on ALS3-AtSTAR1 activity, cRNA-microinjected
21
oocytes were incubated for an additional 48 to 96 h in a modified ND88 solution with
22
or without 5 mM NaH2PO4 as described (Wang et al., 2015). YFP signals in Xenopus
23
oocytes were visualized using a TCS SP5 confocal laser-scanning microscope (Leica)
24
at excitation and emission wavelengths of 514 and 525–550 nm, respectively. The
25
standard ND96 bath solution used for electrophysiological recordings contained 96
26
mM NaCl, 1 mM KCl, and 1.8 mM CaCl2, with the pH adjusted to 7.5 or 5.5 with 5
27
mM HEPES-NaOH or HCl.
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In experiments concerning permeability to UDP-Glc, cells were microinjected
29
with 50 nl of 5 M HEPES buffer (pH 7.5, control) or with 50 nl of a 50 mM UDP-Glc
30
stock solution prepared in the latter buffer; microelectodes were inserted into cells 2 h 26
ACCEPTED MANUSCRIPT after microinjection. The 50-nl microinjection results in an approximately 4 mM
2
intracellular increase in UDP-Glc, assuming a 1.2-mm cell diameter (see Piñeros et al.,
3
[2008] for intracellular determinations). All electrophysiological measurements were
4
performed under two-electrode voltage-clamp (TEVC) using a GeneClamp 500
5
amplifier (Axon Instruments; www.moleculardevices.com) as described in Piñeros et
6
al. (2008). Cells were allowed to recover at least 10 minutes following microelectrode
7
impalement. Currents were elicited by voltage pulses stepped between values
8
indicated in the figures with a 10-sec rest at 0 or –20 mV between each pulse.
9
Steady-state current-voltage (I/V) relationships were constructed by measuring the
10
current amplitude at the end (i.e., at 2 sec) of the test pulse. Liquid junction potentials
11
were measured and corrected accordingly. Mean current values represent the average
12
of at least n (indicated in each figure legend) oocytes and two to three donor frogs.
13
Error bars denote SEM.
14
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ACCESSION NUMBERS
16
Sequence data from this article can be found in the TAIR database under the
17
following accession numbers: ALS3 (AT2G37330), AtSTAR1 (AT1G67940), LPR1
18
(AT1G23010), and LPR2 (AT1G71040).
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SUPPLEMENTAL INFORMATION
21
Supplemental information is available at Molecular Plant Online.
22
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FUNDING
24
This work was supported by funds from the Ministry of Science and Technology of
25
China (grant no. 2016YFD0100700 )
26
China (grant no. 31170238), and the Ministry of Agriculture of China (grant no.
27
2014ZX0800932B).
the National Natural Science Foundation of
28 29
AUTHOR CONTRIBUTIONS 27
ACCEPTED MANUSCRIPT 1
D.L., J.D., M.A.P., H.Y., A.S.M., and L.V. K. designed the research. J.D., M.A.P.,
2
X.L., H.Y., Y.L., and A.S.M. performed the research. D.L., J.D., M.A.P., H.Y., and
3
A.S.M., analyzed the data. D.L., J.D., M.A.P., and L.V. K. wrote the article.
4
ACKNOWLEDGEMENTS
6
We thank the Arabidopsis Biological Resource Center for providing the seed stocks of
7
the mutant lines, and Dr. Thierry Desnos for providing the lpr1lpr2 mutant.
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Roschzttardtz, H., Conejero, G., Curie, C., and Mari, S. (2009). Identification of the endodermal vacuole as the iron storage compartment in the Arabidopsis embryo. Plant Physiol. 151:1329-1338. Singh, A.P., Fridman, Y., Friedlander-Shani, L., Tarkowska, D., Strnad, M., and Savaldi-Goldstein, S. (2014). Activity of the brassinosteroid transcription factors BRASSINAZOLE RESISTANT1 and BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE -SUPPRESSOR1/BRASSINAZOLE RESISTANT2 blocks developmental reprogramming in response to low phosphate availability. Plant Physiol. 166: 678-688. Sun, L., Song, L., Zhang, Y., Zheng, Z., and Liu, D. (2016). Arabidopsis PHL2 and PHR1 act redundantly as the key components of the central regulatory system controlling transcriptional responses to phosphate starvation. Plant Physiol. 170:499-514. Sun, Q.B., Shen, R.F., Zhao, X.Q., Chen, R.F., and Dong, X.Y. (2008). Phosphorus enhances Al resistance in Al-resistance Lespedeza bicolor but not in Al-sensitive L. cuneata under relative high stress. Ann. Bot. 102:795-804. Svistoonoff, S., Creff, A., Reymond, M., Sigoillot-Claude, C., Ricaud, L., Blanchet, A., Nussaume, L., and Desnos, T. (2007). Root tip contact with low-phosphate media reprograms plant root architecture. Nat. Genet. 39: 792-796. Tamarit, J., Irazusta, V., Moreno-Cermeno, A., and Ros, J. (2006). Colorimetric assay for the quantitation of iron in yeast. Anal Biochem 351: 149-151. Tan, K., and Keltjens, W.G. (1990). Interaction between aluminum and phosphorus in sorghum plants. I. Studies with the aluminum sensitive sorghum genotypes TAM428. Plant Soil 124:15–23. Thibaud, M.C., Arrighi, J.F., Bayle, V., Chiarenza, S., Creff, A., Bustos, R., Paz-Ares, J., Poirier, Y., and Nussaume, L. (2010). Dissection of local and systemic transcriptional responses to phosphate starvation in Arabidopsis. Plant J. 64:775-789. Ticconi, C.A., Delatorre, C.A., Lahner, B., Salt, D.E., and Abel, S. (2004). Arabidopsis pdr2 reveals a phosphate-sensitive checkpoint in root development. Plant J. 37:801-814. Ticconi, C.A., Lucero, R.D., Sakhonwasee, S., Adamson, A.W., Creff, A., Nussaume, L., Desnos, T., and Abel, S. (2009). ER-resident proteins PDR2 and LPR1 mediate the developmental response of root meristems to phosphate availability. Proc. Nat. Acad. Sci. USA 106:14174-14179. Vance, C.P., Uhde-Stone, C., and Allan, D.L. (2003). Phosphorus acquisition and use: critical adaptations by plants for securing a nonrenewable resource. New Phytol. 157:423-447. Wang, C., Yue, W., Ying, Y., Wang, S., Secco, D., Liu, Y., Whelan, J., Tyerman, S.D., and Shou, H. (2015). Rice SPX-major facility superfamily3, a vacuolar phosphate efflux transporter, is involved in maintaining phosphate homeostasis in rice. Plant Physiol. 169:2822-2231. Wang, L., Li, Z., Qian, W., Guo, W., Gao, X., Huang, L., Wang, H., Zhu, H., Wu,
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ACCEPTED MANUSCRIPT J.W., Wang, D. et al. (2011). The Arabidopsis purple acid phosphatase AtPAP10 is predominantly associated with the root surface and plays an important role in plant tolerance to phosphate limitation. Plant Physiol. 157:1283-1299. Ward, J.T., Lahner, B., Yakubova, E., Salt, D.E., and Raghothama, K.G. (2008). The effect of iron on the primary root elongation of Arabidopsis during phosphate deficiency. Plant Physiol. 147:1181-1191. Yong, Z., Kotur, Z., and Glass, A.D. (2010). Characterization of an intact two-component high-affinity nitrate transporter from Arabidopsis roots. Plant J. 63:739-748. Yoo, S.D., Cho, Y.H., and Sheen, J. (2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat. Protoc. 2:1565-1572. Zheng, L., Huang, F., Narsai, R., Wu, J., Giraud, E., He, F., Cheng, L., Wang, F., Wu, P., Whelan, J., et al. (2009). Physiological and transcriptome analysis of iron and phosphorus interaction in rice seedlings. Plant Physiol. 151:262-274.
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Figure 1. Effect of Pi availability on the root phenotype of WT and als3-3 seedlings.
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(A) Morphology of WT and als3-3 seedlings grown on a Pi-sufficient (P+) or
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Pi-deficient (P-) medium at 8 and 11 DAG. Inset: A magnified view of the root tip of
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a P- als3-3 seedling. In the inset, the red arrow indicates the tip of the primary root. (B)
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Close view of the root tips of 11-day-old seedlings shown in (A). The arrows indicate
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the boundary between meristematic and elongation zones.
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Figure 2. Fe accumulation patterns as indicated by Perls (A) and Perls/DAB (B)
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staining in the roots of 8-day-old WT and als3-3 seedlings grown on P+Fe+ (P+),
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P-Fe+ (P-), and P-Fe- media.
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Figure 3. Fe contents in the roots of 8-day-old WT and als3-3 seedlings grown on P+
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and P- media. The values are means±SD. The asterisk indicates a significant
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difference from the WT (t-test, P<0.05).
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Figure 4. Molecular characterization of ALS3 and AtSTAR1 genes and root
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phenotypes of als3 and atstar1. (A) and (B) Diagrams showing the structure of the 32
ACCEPTED MANUSCRIPT ALS3 and AtSTAR1 genes, the positions of the point mutation in als3-1, and the
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position of the T-DNA insertions (the empty triangles) in als3-2, als3-3, als3-4, and
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atstar1. The black box, grey box, and the horizontal line represent the coding regions,
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untranslated regions, and introns, respectively. (C) Root phenotypes of the WT, als3-1,
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als3-2, als3-3, and als3-4 on P- medium. (D) Root phenotypes of the WT, als3-3, and
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two complementation lines on P- medium. (E) Root phenotypes of WT, als3-3, and
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atstar1 seedlings on P+ and P- media. In (C) to (E), the photographed seedlings were
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8 days old.
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Figure 5. Fe accumulation patterns as indicated by Perls (A) and Perls/DAB (B)
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staining in the roots of the 8-day-old WT and atstar1 seedlings grown on P+ and P-
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media.
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Figure 6. Yeast two-hybrid (A), LCI (B), and BiFC (C-E) assays indicated physical
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interaction between ALS3 and AtSTAR1. (A) Yeast cells co-transformed with various
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combinations of the plasmids were grown on synthetic dropout (SD) medium without
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Leu or Trp (left panel) or SD medium without Leu, Trp, His, or Ade (right panel) for 3
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days at 30ºC. The activation of the reporter genes by the SV40 large T antigen-Cub
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(large T-Cub) and NubG-the N-terminally truncated tumor suppressor protein p53
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(NubG-∆p53) or ALS3-Cub and NubI was used as a positive control. (B) The
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ALS3-nLUC and cLUC-AtSTAR1 fusion genes were co-expressed in the leaves of N.
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benthamiana, and bioluminescence was captured with a CCD camera. (C) The
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ALS3-nYFP and cYFP-AtSTAR1 fusion genes were co-expressed in the leaves of N.
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benthamiana, and fluorescence signals in the pavement cells were analyzed by
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confocal microscopy. (D) The fluorescence signals in the vacuoles isolated from the
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mesophyll protoplasts of the transformed leaves of N. benthamiana as shown in (C).
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(E) The ALS3-nYFP and cYFP-AtSTAR1 fusion genes were co-expressed in
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Arabidopsis protoplasts. Plasma membranes are stained red by FM4-64. The signal
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from the interaction between ALS3 and AtSTAR1 is shown in green. Inset: A close
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view of part of the cell periphery showing the non-overlapping of plasma membrane
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in tobacco leaves by Western blot using HA and FLAG antibodies. 1 and 2: total
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proteins extracted from uninfiltrated and Agrobacterium-infiltrated leaves of N.
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benthamiana. Rubsico was used as the protein loading control. (G) Detection of
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ALS3-HA and AtSTAR1-FLAG proteins in different cellular fractions. T: total
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proteins; S: soluble proteins; M: microsomal membrane fractions. V-ATPase: a
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tonoplast-specific marker. Rubisco was used as a soluble protein marker. (H) Sucrose
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density-gradient centrifugation. Microsomal membranes were fractionated over a
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20-50% (w/w) sucrose gradient, and samples of each fraction (20 µl each) were
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analyzed by Western blot using specific antibodies. H+-ATPase: a plasma
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membrane-specific marker.
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Figure 7. UDP-Glc and UDP-GlcA rescue the mutant phenotype of als3-3 and atstar1.
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The growth characteristics of 7-day-old als3-3 and atstar1 seedlings grown on P+, P-,
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or P- media supplemented with various compounds (500 µM).
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Figure 8. AtSTAR1 and ALS3 interact to form a functional transporter. (A) BiFC
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assays in Xenopus oocytes showing fluorescence (left panel) and bright field-merged
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(right panel) images resulting from the protein-protein interaction between AtSTAR1
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and ALS3 (top row). No BiFC fluorescence was observed in the controls in which
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oocytes were co-injected with ALS3::nYFP and the complementary cYFP (bottom
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row). (B) Resting membrane potentials (measured in a standard ND96 bath solution)
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of cells injected with water (control), ALS3, or AtSTAR1, or co-injected with both
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AtSTAR1 and ALS3 (n = 12 cells in each case). (C) Electrogenic transport in control
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cells, cells injected with either AtSTAR1 or ALS3, or cells co-injected with AtSTAR1
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and ALS3. Example of currents elicited in response to holding potentials ranging from
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0 to -130 mV (in 10-mV increments with an inter-episode holding potential of -20 mV
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for 10 sec) in AtSTAR1+ALS3 co-injected cells (right panel), or cells injected only
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with water, AtSTAR1, or ALS3 (left panel). Symbols of each injection correspond to
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those shown in (B). Cells were bathed in standard ND96 (pH 7.5) solution. (D) Mean
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current/voltage (I/V) relationships from recordings like those shown in (C). The
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straight and dotted arrows above the X-axis indicate the reversal potential (Erev)
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recorded in cells co-injected with AtSTAR1 and ALS3 (full circles) and all other
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control and single injections. Symbols correspond to those in (B and C) (n = 10 cells).
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Figure 9. Root phenotypes of the WT and various mutants grown on
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agarose-containing P+ and P- media. (A) 8-day-old seedlings of the WT, als3-3,
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lpr1lpr2, and als3-3lpr1lpr2. (B) 8-day-old seedlings of the WT, als3-3, lpr1, and a
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genetic suppressor of als3-3, als3-3/lpr1. (C) Diagram illustrating the position of the
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point mutation in the LPR1 gene in als3-3/lpr1.
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Figure 10. Fe accumulation patterns as indicated by Perls (A) and Perls/DAB (B)
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staining in the maturation zones of the roots of 8-day-old seedlings of the WT,
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UDP-Glc-treated als3-3, and various mutants grown on P- medium.
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Figure 1. Effect of Pi Availability on the Root Phenotype of WT and als3-3 Seedlings.
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(A) Morphology of WT and als3-3 seedlings grown on a Pi-sufficient (P+) or Pi-deficient (P-) medium at 8 and 11 DAG. Inset: A magnified view of the root tip of a P- als3-3 seedling. In the inset, the red arrow
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Figure 2. Fe Accumulation Patterns as Indicated by Perls (A) and Perls/DAB (B) Staining in the Roots
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of 8-Day-Old WT and als3-3 Seedlings Grown on P+Fe+ (P+), P-Fe+ (P-), and P-Fe- Media.
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Figure 3. Fe Contents in the Roots of 8-Day-Old WT and als3-3 Seedlings Grown on P+ and P- Media. The values are means±SD. The asterisk indicates a significant difference from
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Figure 4. Molecular Characterization of ALS3 and AtSTAR1 Genes and Root Phenotypes of als3
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and atstar1.
(A) and (B) Diagrams showing the structure of the ALS3 and AtSTAR1 genes, the position of the point
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mutation in als3-1, and the positions of the T-DNA insertions (the empty triangles) in als3-2, als3-3, als3-4, and atstar1. The black box, grey box, and the horizontal line represent the coding regions, untranslated regions, and introns, respectively. (C) Root phenotypes of the WT, als3-1, als3-2, als3-3, and als3-4 on Pmedium. (D) Root phenotypes of the WT, als3-3, and two complementation lines on P- medium. (E) Root phenotypes of WT, als3-3, and atstar1 seedlings on P+ and P- media. In (C) to (E), the photographed seedlings were 8 days old.
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Figure 5. Fe Accumulation Patterns as Indicated by Perls (A) and Perls/DAB (B) Staining in the
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Roots of the 8-Day-Old WT and atstar1 Seedlings Grown on P+ and P- Media.
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Figure 6. Yeast Two-Hybrid (A), LCI (B), and BiFC (C-E) Assays Indicated Physical Interaction between ALS3 and AtSTAR1.
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(A) Yeast cells co-transformed with various combinations of the plasmids were grown on synthetic dropout (SD) medium without Leu or Trp (left panel) or SD medium without Leu, Trp, His, or Ade (right panel) for 3
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days at 30ºC. The activation of the reporter genes by the SV40 large T antigen-Cub (large T-Cub) and NubGthe N-terminally truncated tumor suppressor protein p53 (NubG-Δp53) or ALS3-Cub and NubI was used as a positive control. (B) The ALS3-nLUC and cLUC-AtSTAR1 fusion genes were co-expressed in the leaves of N. benthamiana, and bioluminescence was captured with a CCD camera. (C) The ALS3-nYFP and cYFPAtSTAR1 fusion genes were co-expressed in the leaves of N. benthamiana, and fluorescence signals in the pavement cells were analyzed by confocal microscopy. (D) The fluorescence signals in the vacuoles isolated from the mesophyll protoplasts of the transformed leaves of N. benthamiana as shown in (C). (E) The ALS3nYFP and cYFP-AtSTAR1 fusion genes were co-expressed in Arabidopsis protoplasts. Plasma membranes are stained red by FM4-64. The signal from the interaction between ALS3 and AtSTAR1 is shown in green. Inset: A close view of part of the cell periphery showing the non-overlapping of plasma membrane and tonoplast. (F) Detection of expression of ALS3-HA and AtSTAR1-FLAG proteins in tobacco leaves by Western
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blot using HA and FLAG antibodies. 1 and 2: total proteins extracted from uninfiltrated and Agrobacteriuminfiltrated leaves of N. benthamiana. Rubsico was used as the protein loading control. (G) Detection of ALS3HA and AtSTAR1-FLAG proteins in different cellular fractions. T: total proteins; S: soluble proteins; M: microsomal membrane fractions. V-ATPase: a tonoplast-specific marker. Rubisco was used as a soluble protein marker. (H) Sucrose density-gradient centrifugation. Microsomal membranes were fractionated over a 20-50% (w/w) sucrose gradient, and samples of each fraction (20 μl each) were analyzed by Western blot
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Figure 7. UDP-Glc and UDP-GlcA Rescue the Mutant Phenotype of als3-3 and atstar1.
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The growth characteristics of 7-day-old als3-3 and atstar1 seedlings grown on P+, P-, or P- media
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supplemented with various compounds (500 μM).
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Figure 8. AtSTAR1 and ALS3 Interact to Form a Functional Transporter.
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(A) BiFC assays in Xenopus oocytes showing fluorescence (left panel) and bright field-merged (right panel) images resulting from the protein-protein interaction between AtSTAR1 and ALS3 (top row). No BiFC fluorescence was observed in the controls in which oocytes were co-injected with ALS3::nYFP and the complementary cYFP (bottom row). (B) Resting membrane potentials (measured in a standard ND96 bath
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solution) of cells injected with water (control), ALS3, or AtSTAR1, or co-injected with both AtSTAR1 and ALS3 (n = 12 cells in each case). (C) Electrogenic transport in control cells, cells injected with either AtSTAR1 or
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ALS3, or cells co-injected with AtSTAR1 and ALS3. Example of currents elicited in response to holding potentials ranging from 0 to -130 mV (in 10-mV increments with an inter-episode holding potential of -20 mV for 10 sec) in AtSTAR1+ALS3 co-injected cells (right panel), or cells injected only with water, AtSTAR1, or ALS3 (left panel). Symbols of each injection correspond to those shown in (B). Cells were bathed in standard ND96 (pH 7.5) solution. (D) Mean current/voltage (I/V) relationships from recordings like those shown in (C). The straight and dotted arrows above the X-axis indicate the reversal potential (Erev) recorded in cells coinjected with AtSTAR1 and ALS3 (full circles) and all other control and single injections. Symbols correspond to those in (B and C) (n = 10 cells).
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Media.
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Figure 9. Root Phenotypes of the WT and Various Mutants Grown on Agarose-Containing P+ and P-
(A) 8-day-old seedlings of the WT, als3-3, lpr1lpr2, and als3-3lpr1lpr2. (B) 8-day-old seedlings of the WT, als3-3, lpr1, and a genetic suppressor of als3-3, als3-3/lpr1. (C) Diagram illustrating the position of the point mutation in the LPR1 gene in als3-3/lpr1.
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Figure 10. Fe Accumulation Patterns as Indicated by Perls (A) and Perls/DAB (B) Staining in the Maturation Zones of the Roots of 8-Day-Old Seedlings of the WT, UDP-Glc-treated als3-3, and
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Various Mutants Grown on P- Medium.