Ubiquitin-Specific Protease 14 (UBP14) Is Involved in Root Responses to Phosphate Deficiency in Arabidopsis

Molecular Plant

•

Volume 3

•

Number 1

•

Pages 212–223

•

January 2010

RESEARCH ARTICLE

Ubiquitin-Specific Protease 14 (UBP14) Is Involved in Root Responses to Phosphate Deficiency in Arabidopsis Wen-Feng Li, Paula J. Perry, Nulu N. Prafulla and Wolfgang Schmidt1 Institute of Plant and Microbial Biology, Academia Sinica, 115 Taipei

ABSTRACT A mutant isolated from a screen of EMS-mutagenized Arabidopsis lines, per1, showed normal root hair development under control conditions but displayed an inhibited root hair elongation phenotype upon Pi deficiency. Additionally, the per1 mutant exhibited a pleiotropic phenotype under control conditions, resembling Pi-deficient plants in several aspects. Inhibition of root hair elongation upon growth on low Pi media was reverted by treatment with the Pi analog phosphite, suggesting that the mutant phenotype is not caused by a lack of Pi. Reciprocal grafting experiments revealed that the mutant rootstock is sufficient to cause the phenotype. Complementation analyses showed that the PER1 gene encodes an ubiquitin-specific protease, UBP14. The mutation caused a synonymous substitution in the 12th exon of this gene, resulting in a lower abundance of the UBP14 protein, probably as a consequence of reduced translation efficiency. Transcriptional profiling of per1 and wild-type plants subjected to short-term Pi starvation revealed genes that may be important for the signaling of Pi deficiency. We conclude that UBP14 function is crucial for adapting root development to the prevailing local availability of phosphate. Key words:

Phosphate deficiency; root hairs; ubiquitin-specific protease; root development.

INTRODUCTION Root hairs are tip-emergent tubular outgrowths of rhizodermal cells that mediate the uptake of water and provide access to immobile nutrients such as phosphate (Pi). In Arabidopsis, root epidermal cells are arranged in a file pattern, composed of alternating root hair (trichoblasts) and non-root hair cells (atrichoblasts). The differentiation of epidermal cells into either cell type is dependent on the position with regard to the underlying cortical cells. Cell fate acquisition is controlled by a regulatory circuit of transcription factors, involving feedback loops between both cell types (Bernhardt et al., 2005; Kwak and Schiefelbein, 2007; Savage et al., 2008). In cells that adopt the non-hair cell fate, the homeodomain-leucine zipper transcription factor GLABRA2 (GL2) represses the initiation of root hair formation, reduces the number of cell divisions, and promotes longitudinal elongation. Trichoblasts develop from epidermal cells that are located over the clefts of two cortical cells (H-position). In these cells, GL2 expression is blocked by a signal that is presumably stronger over anticlinal cortical cell walls. The signal is thought to be perceived by the leucine-rich receptor-like kinase SCRAMBLED (SCM), which causes a reduced expression of the WEREWOLF (WER) gene (Kwak and Schiefelbein, 2008). WER encodes a Myb-type transcription factor that forms a complex with the bHLH dimer, GL3/EGL3,

and a WD40 repeat protein, TTG, thereby supporting the synthesis of GL2. The WER–GL3/EGL3–TTG complex also promotes the formation of CPC, a Myb transcription factor that moves, via plasmodesmata, from non-hair cells to hair cells, where it competes with WER for binding sites on the GL3/EGL2–TTG complex. The CPC–GL3/EGL3 complex does not support the formation of GL2, and the cell enters the default (root hair) pathway (reviews by Ishida et al., 2008; Schiefelbein et al., 2009). Following cell specification, root hair initiation commences in the trichoblasts with the formation of a cell wall bulge close to the basal end of the cell. The third stage of root hair development, the elongation of the hairs by tip growth, is associated with extensive cytoskeleton reorganization, deposition of wall materials and cell membranes to the growing tip, tip-focused influx of Ca2+, and migration of the nucleus toward the tip (Schiefelbein et al., 1992). The molecular

1 To whom correspondence should be addressed. E-mail [email protected]. edu.tw, tel. +886 2 2789 2997.

ª The Author 2009. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS. doi: 10.1093/mp/ssp086, Advance Access publication 10 November 2009 Received 16 July 2009; accepted 24 August 2009

Li et al.

machinery of tip growth is complex, involving GTP binding proteins (Jones et al., 2002; Carol et al., 2005), actin filaments, microtubules for vesicle transport (Grierson and Schiefelbein, 2002; Samaj et al., 2006), and regulatory mitogen-activated protein kinases (Samaj et al., 2002). Transcriptional profiling studies with a mutant that is defective in root hair elongation, rhd2, revealed some 605 genes with possible functions in polar growth of root epidermal cells, thus defining the ‘root-hair morphogenesis transcriptome’ (Jones et al., 2006). The root epidermal pattern becomes plastic during postembryonic development. In particular, the lack of immobile nutrients such as Pi and iron (Fe) has been shown to affect the patterning and characteristics of root hairs, leading to an increased absorptive surface of the root (reviewed by Schmidt, 2008). Upon Pi deficiency, root epidermal cell elongation is reduced, leading to a higher frequency of hairs per unit root length (Sa´nchez-Caldero´n et al., 2005; Ma et al., 2003). In addition, the number of root hairs per unit length is increased as a result of the formation of hairs in ectopic position and by an increase in rhizodermal cells in the H-position resulting from cell proliferation in the ground tissue (Ma et al., 2001; Mu¨ller and Schmidt, 2004). The Pi deficiency-specific root hair pattern has been postulated to be under the control of a mechanism that acts downstream of, but is biased by, the WER patterning cascade (Savage and Schmidt, 2008). Supported by computer simulations that were fitted to biological data, it has been hypothesized that an activator-inhibitor mechanism is superimposed onto the embryonic patterning controlled by WER, which confers plasticity to the system and acclimates the plants to changes in environmental conditions. Since the mobility of Pi is limited in many soil topographies, a higher surface area of the root conferred by increases in the number and length of root hairs improve Pi acquisition efficiency. Root hairs formed upon Pi starvation are considerably longer than those formed under control conditions, suggesting that not only cell specification, but also the elongation process is altered upon Pi starvation (Mu¨ller and Schmidt, 2004). How the Pi signal is perceived and translated into changes in root hair patterning is not known. Phosphate starvation responses have been shown to be regulated by a conserved Myb transcription factor, PHR1, which, in turn, is controlled by the SUMO E3 ligase SIZ1 (Rubio et al., 2001; Miura et al., 2005, see also Schachtman and Shin, 2007 for a review). Ectopic expression of PHR1 caused an increased abundance of the miR399d, a member of the microRNA family that regulates a subset of Pi starvation genes by affecting the abundance of the ubiquitin conjugating E2 enzyme 24, PHO2 (Chiou et al., 2006; Bari et al.; 2006; Nilsson et al., 2007). PHO2 critically controls Pi homeostasis by regulating a subset of Pi-starvation genes including transporters mediating the uptake and translocation of Pi (Delhaize and Randall, 1995; Dong et al., 1998; Aung et al., 2006; Bari et al., 2006). Mutants such as pho2 accumulate high levels of Pi in their shoots (Delhaize and Randall, 1995). Upon Pi starvation, miR399 is strongly induced and has been suggested to systemically suppress PHO2

d

Phosphate Deficiency Responses in Arabidopsis Roots

|

213

through transcript cleavage (Pant et al., 2008; Lin et al., 2008). Alterations in the root hair pattern have been described for mutants harboring defects in some of the genes with suggested function in Pi homeostasis; so far, however, genes specifically involved in the root hair phenotype typical of Pi-deficient plants have not been described. A mutant isolated in a genetic screen of an EMS-mutagenized population in the Col-0 background for individuals that failed to respond to Pi starvation by increasing the frequency and length of root hairs was named phosphate deficiency root hair defective1 (per1) and further characterized. We show here that the mutation caused a synonymous substitution in an exon of ubiquitin-specific protease (UBP) 14, an ortholog of yeast UBP14, Dictyostelium UbpA, and human IsoT. Homozygous mutations in Arabidopsis thaliana UBP14 were found to be embryo-lethal, indicating that UBP14 plays a critical role in early development (Doelling et al., 2001). AtUPB14 cleaves ubiquitin from polypeptides with broad substrate specificity and frees Lys48-linked multi-ubiquitin chains (Doelling et al., 2001). The per1 mutant shows a phosphate-specific defect in root hair elongation and several defects in radial root patterning and epidermal cell differentiation, suggesting that the balance between ubiquitin monomers and ubiquitin chains is critical for root development and the root responses to Pi.

RESULTS per1 Mutant Plants Are Defective in Pi Deficiency-Induced Root Hair Formation Growth of Arabidopsis plants on media deprived of Pi caused significant and nutrient-specific changes in the radial patterning of the roots. As reported previously for the Col-0 accession, the root hairs formed upon Pi deficiency were partly derived from atrichoblasts (Ma et al., 2003; Mu¨ller and Schmidt, 2004; Figure 1 and Table 1). Growth under Pi-deficient conditions was associated with cell proliferation in the ground tissue, leading to an increased number of cortical and epidermal cells (Figure 1 and Table 1). Reduced elongation of the trichoblasts and the surplus of epidermal cells in the H-position led to an approximately 35% increase in root hair frequency in the default (H-) position. As typically observed for this growth type, root hairs formed upon Pi deprivation were markedly longer than those formed under control conditions. To identify genes involved in the induction of the root hair phenotype associated with Pi deficiency, a forward genetic screen for the isolation of mutants that are affected quantitatively or qualitatively in the formation of root hairs under low Pi conditions has been undertaken using an EMS-mutagenized seed population. One of the mutants identified in this screen was further characterized and named perfect1 (per1, Pi deficient root hair defective1). The mutant showed Pi-specific defects in root hair elongation, but this phenotype was compromised in the presence of Pi. An initial characterization of the mutant has been carried out by Mu¨ller (2006). Segregation

214

|

Li et al.

d

Phosphate Deficiency Responses in Arabidopsis Roots

Figure 1. Effect of Pi Supply on Root Hair Formation of Wild-Type and per1 Mutant Plants. Confocal micrographs of wild-type (A, B) and per1 (C, D) roots grown under control (A, C) and low Pi conditions (B, D). In per1 roots, root hairs fail to elongate under low Pi conditions. (E–H) Cross-sections from wild-type (E, F) and per1 (G, H) roots of control (E, G) and low Pi plants (F, H). Under low Pi conditions, root hairs are formed in ectopic positions, namely over tangential wall of underlying cortical cells. Plants were analyzed 14 d after sowing. Scale bar = 75 lm. Table 1. The Number of Root Hairs in the H- and N-Positions and the Cortical and Epidermal Cell Numbers of the Col-0 Wild-Type and the per1 Mutant. Root hair number (root hairs/section) Genotype

H

Root hairs in N-position (%)

N a

a

0.8

Tissue cell number (cells/section) Cortex

Epidermis a

8.01 6 0.00

26.6 6 0.11a

Col-0 high Pi

1.26 6 0.05

0.01 6 0.01

Col-0 low Pi

1.70 6 0.07b

0.48 6 0.04b

22.0

8.72 6 0.04b

28.3 6 0.20b

per1 high Pi

1.41 6 0.07a

0.07 6 0.02c

4.7

9.74 6 0.08c

28.7 6 0.14b

b

b

17.0

d

28.8 6 0.10b

per1 low Pi

1.85 6 0.08

0.38 6 0.04

8.98 6 0.03

A total of 350 cross-sections from 10 roots (35 cross-sections per root) were analyzed for each growth type and genotype. Different letters in each column denote statistical difference (p , 0.001). H, root hairs in the H-position; N, root hairs in the N-position.

of the mutant phenotype observed in the F2 generation approached the 3:1 ratio expected for Mendelian segregation of a single, recessive mutation. Root hair elongation was normal when per1 plants were grown on control (2.5 mM Pi) media (Figure 1C). Root hairs of per1 plants initiated but failed to elongate when subjected to Pi starvation (Figure 1D). Inhibition of elongation of root hairs occurred early in root hair development, resulting in short and deformed hairs. In contrast to the wild-type, a substantial number of root hairs was formed in positions normally occupied by non-hair cells when plants were grown under control conditions (Table 1). The number of ectopic hairs was markedly increased under low

Pi conditions in the mutant with no significant differences between per1 and Col-0 plants. per1 and wild-type also showed a similar increase in root hair density upon growth on low Pi media with regard to hairs formed in the H-position (Table 1). In addition to alterations in root hair frequency and distribution, the number of cortical cells was increased in the mutant under control conditions while a higher cortical cell number in Col-0 was only associated with low Pi conditions (Table 1). In some per1 roots, more than twice as many cortical cells as typically found in the wild-type were present. Interestingly, these differences were not observed when per1 and Col-0 plants were grown under low Pi supply. The number of epidermal

Li et al.

cells was increased in per1 plants independent of the concentration of Pi in the media and was equivalent to wild-type plants grown on low Pi media (Table 1). Under control conditions, the irregular radial pattern of epidermal cells found in per1 plants coincided with a high variation in longitudinal cell length. Cell length varied within a single cell file, with clusters of short cells interspersed between the otherwise normally elongated cells. The variable cell length was probably caused by irregular cell divisions (Figure 2A). In addition, the identity of trichoblasts and atrichoblasts files was also affected in the mutant. Under control conditions, cell files changed identity along the longitudinal axis (Figure 2B), and individual trichoblasts were interspersed with atrichoblasts cell files (Figure 2C).

Identification of the per1 Locus Previously conducted genetic mapping placed the PER1 gene between nucleotides 7162174 and 7209300 of chromosome 3 (Mu¨ller, 2006). The open reading frames (ORFs) of all 19 genes in this region were amplified by PCR from a Col-0 and a per1

d

Phosphate Deficiency Responses in Arabidopsis Roots

|

215

plant and sequenced to identify point mutations. A synonymous substitution in the third position of a conserved proline four-fold redundancy genetic code (CCC to CCT) was found in the 12th exon of At3g20630, which encodes an ubiquitinspecific protease, UBP14. To investigate whether the protein profile is affected by the mutation, we compared total protein from shoots and roots of the wild-type and mutant. Similar to what has been reported for seeds of embryo-lethal Atubp14 mutants (Doelling et al., 2001), SDS–PAGE profiles were not significantly different between per1 and Col-0 plants, indicating that protein abundance was not severely affected by the mutation (Figure 3A). Immunoblot analysis with anti-UBP14 antibodies showed that the level of the UBP14 protein was markedly reduced in shoots. However, no significant differences were detectable between the roots of wild-type and per1 plants (Figure 3B).

Complementation of per1 with the Wild-Type Gene A 2645-bp fragment containing the wild-type PER1 (At3g20630) coding sequence, 87 bp upstream of the

Figure 2. Root Phenotype of the per1 Mutant and per1–UBP14 Plants. Confocal micrographs of per1 roots grown on control media (A–C), low Pi media (D), and on Pi-free media containing the phosphate analog phosphite (E). (A–C) Primary root of per1 plants grown on control media; arrows indicate a cluster of irregular, short epidermal cells (A) longitudinal cell division (B) and ectopic root hairs interspersed in non-hair cell files (C). (D) Compilation of confocal pictures of the primary root of a per1 plant grown on low Pi media. (F, G) Pi-sufficient (F) and Pi-deficient (G) per1 plants complemented with the wild-type sequence of UBP14 (per1–UBP14). Plants were analyzed 14 d after sowing. Note the wild-type-like marked difference in longitudinal cell length between atrichoblasts (at) and trichoblasts (t) in per1–UBP14 plants. Scale bars: I = 150 lm, others = 75 lm.

216

|

Li et al.

d

Phosphate Deficiency Responses in Arabidopsis Roots

translational start codon and 164 bp downstream of the stop codon, was amplified from Col-0 with engineered BamHI/KpnI sites and cloned into the vector pCR2.1TOPO to generate pTOPO–PER1. The construct was verified by sequencing. The cDNA fragment was cut with BamHI/KpnI and subcloned into BamHI/KpnI-digested pBIN–pROK2 to generate the pROK–

PER1 binary vector, which contains At3g20630 cDNA driven by the 35S CaMV promoter. This vector was introduced into Agrobacterium tumefaciens strain GV3101 and used to transform per1 plants via floral dipping. We obtained 20 independent kanamycin-resistant transgenic lines. Transformants with high transcript abundance were selected and tested for their ability to elongate root hairs when grown on low Pi media. per1–UBP14 plants displayed wild-type-like root hair elongation, thus demonstrating that a defect in PER1 gene causes the phenotype of the per1 mutant (Figure 2F and 2G). Quantification of the root hair length revealed no marked differences among wild-type, per1, and per1–UBP14 plants under control conditions (Figure 4A). When grown on low Pi media, root hair length was reduced by about 60% in per1 mutant plants, but was unaffected in per1–UBP14 plants. It should

A

300

Root hair length (µm)

250

wild-type per1 per1-UBP14

200

150

100

50

0

Control

B

Low Pi

500 Trichoblasts Atrichoblasts

Cell length (µm)

400

300

200

100

0

wt + Pi

wt low Pi

per1 +Pi

per1 low Pi

Figure 4. Root Hair Length and Longitudinal Cell Length of Root Epidermal Cells of Col-0 and per1 Plants. Figure 3. The PER1 Mutation Affects the Abundance of UBP14 Protein, But Not the Overall Protein Pattern. (A) SDS–PAGE of crude protein extracts from leaves and roots of the wild-type and of per1 plants. (B) Western analysis with anti-UBP14 antibodies. (C) Quantification of Western analysis based on two independent experiments. Error bars represent SE.

(A) Root hair length of wild-type, per1, and per1–UBP14 plants grown on control and low Pi media. Bars represent the average of 100 root hairs from five roots. (B) Longitudinal length of root epidermal cells. Bars indicate the average of 200 cells of primary roots, measured 2–6 mm from the quiescent center. Error bars indicate standard error of the means. Plants were analyzed 14 d after sowing.

Li et al.

d

Phosphate Deficiency Responses in Arabidopsis Roots

|

217

be noted that while root hair length under low Pi conditions varied with Pi concentrations between 0.5 and 25 lM Pi and duration of growth in the wild-type, the short root hair type of per1 plants was unaffected by these parameters. The longitudinal atrichoblast cell length in per1 roots was markedly shorter than those of the wild-type; hence, the difference between root hair bearing and hairless cells was less significant in the mutant (Figure 4). This lack of difference was reverted back to wild-type in per1–UBP14 plants (Figure 2F). In contrast to Col-0 plants, the longitudinal cell length of per1 roots was not significantly affected by the growth conditions.

The per1 Phenotype Is Rescued by Phosphate and Independent on the Light Intensity The effect of Pi presence locally can be mimicked by applying the Pi analog phosphite (Phi). Phi is readily taken up by plant roots but cannot be metabolized (Carswell et al., 1997). As a result, Pi-deficient plants fed with Phi have been shown to specifically suppress some Pi deficiency responses, such as anthocyanin accumulation and the expression of genes involved in Pi uptake, while the internal Pi concentration continues to decrease, resulting in growth inhibition (Carswell et al., 1997; Ticconi et al., 2001). In this study, mutant plants grown on Pi-free media supplemented with Phi showed reduced root growth, but the root hairs elongated in a similar way to those of the wild-type under Pi-sufficient conditions, indicating that the rescue of the phenotype by Phi is due to a local rather than by a systemic signal (Figure 2E). Further evidence that supports a hypothesis suggesting the existence of a root autonomous response also comes from reciprocal grafting experiments, in which the per1 phenotype was observed exclusively in grafts with per1 roots. Grafting of the wild-type shoot on the mutant rootstock does not affect the per1 phenotype (Figure 5). Previously, we demonstrated that root hair elongation under control conditions is sensitive to light. Root hair length was substantially reduced when the light intensity was increased from 50 to 70 lmol 1 s 1 (Yang et al., 2008). In the present study, growing plants at 70 lmol 1 s 1 on control media inhibited the elongation of the root hairs without affecting root hair initiation (Figure 4). However, light intensity had no effect on plants grown on low Pi media, resulting in similar phenotypes at both light conditions (data not shown). In contrast to wild-type plants, root hair elongation under control conditions was not markedly affected by light in per1 roots. Growing plants on media with low Pi induced the phenotype typical of Pi-deficient per1 roots, independent of the light intensity (Figure 6).

The per1 Phenotype Is Specific to Pi Deficiency To determine whether the phenotype of the per1 mutant is specific to Pi starvation, we subjected wild-type and per1 plants to manganese (Mn)- and Fe-deficient conditions. Low

Figure 5. Reciprocal Grafting between the Wild-Type and per1 Mutant Plants. Scion/rootstock combinations were wt/wt (A, D) wt/per1 (B, E), and per1/wt (C, F). Pictures show root tips (D–F) and the root hair zone between 2 and 6 mm from the tip (A–C). Plants were grafted after 9 d of growth on control media, then transferred to Pi-free media and analyzed 16 d after sowing.

Figure 6. Light-Induced Inhibition of Root Hair Elongation. Effect of 70 lmol 1 s 1 on root hair elongation in roots of control wt plants (A), control per1 plants (B), and low Pi per1 plants (C). Plants were analyzed 10 d after sowing. Scale bars = 150 lm.

availability of either mineral nutrient has been shown to induce extra root hairs, with characteristics that are specific to the respective growth type (Mu¨ller and Schmidt, 2004; Yang et al., 2008). When grown under Mn deficiency, wild-type

218

|

Li et al.

d

Phosphate Deficiency Responses in Arabidopsis Roots

plants react by forming long root hairs, some of which are branched at their base (Yang et al., 2008). A similar phenotype was observed in per1 plants (Figure 7A and 7B). Similar to Mn deficiency, low Fe availability was shown to increase the abundance of root hairs in Arabidopsis (Mu¨ller and Schmidt, 2004). The per1 mutant also behaved in a manner similar to wild-type plants under Fe deficiency (Figure 7C and 7D). Quantification of the root hair length revealed a slight decrease in root hair length in per1 plants under Mn deficiency and no change upon Fe deficiency, indicating that the short root hair phenotype of the per1 mutant was specifically induced under Pi deprivation (Figure 7E).

Figure 7. Specificity of the per1 Phenotype. Effect of Mn (A, B) and Fe (C, D) starvation on the wild-type (A, C) and per1 (C, D) root phenotype. (E) Quantification of root hair length under Mn and Fe-deficient conditions. Root hair length was normalized to that of the wildtype under the respective conditions. Mn-deficient plants were analyzed 7 d after sowing, Fe-deficient plants were transferred at day 8 to media deprived of iron and analyzed 6 d after transfer. Scale bars = 75 lm.

Short-Term Transcriptional Responses to Pi Starvation and Elemental Analysis To identify genes that were involved in Pi sensing and signaling, we subjected wild-type and per1 plants to a short-term Pi deficiency treatment and analyzed the transcriptional changes in roots by using the Arabidopsis ATH1 gene chip. An experimental period of 10 h was chosen to detect transiently expressed genes potentially involved in the relay of the Pi deficiency signal. Fifty-three genes were found to be differentially expressed upon Pi deficiency in the wild-type, the majority of which (37 genes) was up-regulated (Supplemental Table 1). Among this group, genes involved in Ca transport and binding, cell wall alterations, and in signal transduction were over-represented. No genes with known function in Pi uptake or distribution were detected as being responsive to Pi at this time-point. Acid phosphatase 5 (At3g17790), a Pi deficiency marker, was 2.8-fold up-regulated upon short-term Pi deficiency treatment. Two genes with developmental functions that are important for the Pi deficiency phenotype were found to be induced by Pi starvation, the exocyst gene AtEXO70A1 (At5g03540), and the organic cation transporter gene AtOCT1 (At1g73220). AtEXO70A1 has been shown to be required for polar growth of root hairs and was suggested to play a regulatory role in root hair development (Synek et al., 2006). AtOCT1 function was shown to be linked with lateral root formation (Lelandais-Brie`re et al., 2007), typical for Pi-deficient plants. In per1 plants, a smaller number (24) of genes was found to be differentially expressed upon Pi deficiency when compared to the wild-type with an overlap of seven genes, among them, the Pi deficiency marker AtACP5 (Supplemental Table 2). Two genes that are known to be involved in Fe homeostasis, encoding the oligopeptide transporter OPT3 (At4g16370) and the transcription factor BHLH039 were markedly down-regulated when the plants were subjected to Pi-deficient conditions, suggesting an anticipation of the Fe overload that is associated with growth under Pi-deplete conditions (Misson et al., 2005). When signals of per1 roots genes were compared to the wild-type under control conditions, the expression of 58 genes was found to be significantly different, the majority of which were present at a low abundance when compared to the wildtype (Supplemental Table 3). Under low Pi conditions, the expression of 72 genes was affected in the mutant (41 up-, 31 down-regulated; Supplemental Table 4). In tendency, genes that showed higher transcript abundance in per1 plants also appears to be down-regulated in Col-0 roots upon Pi starvation. Those genes that were less represented in per1 roots were generally up-regulated in the Col-0 background (WT) upon Pi deficiency. A total of 12 genes are part of the root hair elongation transcriptome (Jones et al., 2006), most of which were found to be less expressed in per1 roots when compared to Col-0 under low Pi conditions. To investigate whether the PER1 mutation affects the elemental composition, the ion concentration in roots and shoots

Li et al.

was measured by inductively coupled plasma optical emission spectroscopy (ICP–OES). As expected, both wild-type and mutant showed a dramatic reduction in P concentration in both leaves and roots when grown on low Pi media (Figure 8A). The concentration of P in per1 plants did not differ significantly from the wild-type. As reported previously (Misson et al., 2005), the concentration of Fe was markedly higher when plants were grown on low Pi media (Figure 8B). This increase was more pronounced in per1 mutants but not in per1–UBP14 complementation lines. This difference between wild-type and mutant plants was observed in four independent experiments. The concentration of other metals was not affected by the mutation.

DISCUSSION Considerable variation in Pi availability due to spatial-temporal heterogeneity in Pi distribution is counteracted by plants through implementation of a suite of reactions aimed at improving Pi acquisition and distribution (Vance et al., 2003; Ticconi and Abel, 2004; Doerner, 2008). Increasing the root surface area by altering root architecture and forming of extra root hairs are widespread responses among plant species and is thought to confer fitness by adjusting developmental programs to the prevailing conditions (Desnos, 2007; Lo´pez-

Figure 8. Analysis of Mineral Ion Concentration of Roots and Shoots from Control and Pi-Deficient Plants. (A) Macronutrients. (B) Micronutrients. Control plants were grown on media containing 2.5 mM Pi. Plants were analyzed 14 d after sowing. R, roots; S, shoots.

d

Phosphate Deficiency Responses in Arabidopsis Roots

|

219

Bucio et al., 2003). In support of this assumption, root hair length and density have been shown to vary among Arabidopsis accessions and to contribute to the efficiency of Pi acquisition (Narang et al., 2000). Despite a profound knowledge regarding rhizodermal cell fate determination and differentiation of hair-bearing cells (Lee and Schiefelbein, 2002; Dolan, 2006; Jones et al., 2006; Ishida et al., 2008), the mechanism underlying the formation of extra root hairs upon Pi starvation has not been addressed at the molecular level. We show here that in Arabidopsis, a ubiquitin-specific protease, UBP14, is crucial for the elongation of root hairs specifically under conditions of low Pi availability. In the per1 mutant, root hairs fail to elongate properly in Pideficient plants. The root hairs that are formed are wider than those of the wild-type, leading to a phenotype resembling the tip1 mutant (Hemsley et al., 2005). The per1 mutation is the result of a synonymous substitution in the 12th exon of UBP14, which presumably leads to reduced protein translation efficiency. Homozygous Atubp14 mutants were shown to be embryolethal, indicating an essential function of UBP14 in embryo development (Doelling et al., 2001). Complementation of the per1 mutant with the wild-type sequence rescues the effects of the mutation, thus providing evidence to suggest that the single base substitution is the cause of the per1 phenotype. Although the mutation did not affect the amino acid sequence of the protein, it appears that at least some of the UBP14mediated processes are compromised, probably as a consequence of reduced efficiency in protein synthesis. CCT and CCC are the major codons for high- and low-expression genes in the CCN codon group, respectively (Morton and Wright, 2007). Thus, the change from CCC to CCT might have an effect on translational efficiency. This assumption is supported by a reduced level of UBP14 protein in the per1 mutant. The reduction in protein abundance was more pronounced in leaves, but reciprocal grafting experiments and the effect of phi imply that the phenotype is not affected by the shoot. It thus appears that subtle differences in UBP14 may be sufficient to alter root development. Interestingly, although UBP14 is expressed throughout the plant, lower expression levels have been observed in pollen and root hair cells (www.genevestigator. ethz.ch/at/). Thus, a slight decrease in translation efficiency might have greater consequences in these tissues, in particular under stress conditions. From the function of UBP14, it is tempting to speculate that the phenotype of the per1 plants is the result of a reduced recycling of Lys48-linked ubiquitin chains. Although the elongation of root hairs was indistinguishable from the wild-type under control conditions, the overall root phenotype of per1 plants appeared to be more affected when Pi was present at sufficient amounts in the growth media. This was in particular relevant with regard to the longitudinal length of epidermal cells and to the radial pattern of the peripheral root tissues, which are irregular and highly variable in the mutant. It appears that the developmental program induced under

220

|

Li et al.

d

Phosphate Deficiency Responses in Arabidopsis Roots

Pi-deficient conditions is less affected by the mutation than under Pi-sufficient conditions. Several findings suggest that the per1 phenotype under control conditions is, at least in part, due to a disturbed adjustment to the prevailing Pi level, resulting in a phenotype that resembled Pi-deficient plants. First, the root hair pattern was less regular when compared to the wild-type, leading to a reduced effect of the position-dependent formation of root hairs, which is typically found in Pi-deficient plants. This was associated with the formation of considerably shorter atrichoblasts, suggesting a loss in cell identity. In addition, similar to what has been observed in Pi-deficient plants, the elongation of the root hairs in control per1 roots was not inhibited by increasing the light intensity from 50 to 70 lmol 1 s 1. Second, when grown under Pi-sufficient conditions, the number of epidermal cells was higher in the mutant, resembling one of the Pi-deficient phenotypes of the wild-type. While the uptake of Pi does not seem be greatly affected, the per1 mutant did accumulate more Fe than the wild-type—a phenomenon that has been well described but not yet clarified in terms of molecular mechanism in Pi-deficient plants (Mission et al., 2005; Ward et al., 2008). The shortened root hair phenotype of Pi-starved per1 mutant plants was rescued by the application of the phosphate analog phosphite, indicating that the phenotype is not caused by a lack of Pi, but is rather due to a disturbed Pi signaling. It thus appears that the cellular regulatory circuits are affected under both low and high Pi availability. The phenotype of per1 plants under control conditions does not appear to be caused by transcriptional regulation of the Piresponsive genes. Only a relatively small number of genes appear to be affected by this mutation at the transcriptional level. Genes with known function in Pi homeostasis such as SIZ1, BHLH32, PHO2, and the major Pi transporters Pht1;1 and Pht1;4 are not significantly affected by this mutation. Moreover, the P concentration in the leaves and roots of Pi-sufficient per1 plants do not deviate significantly from that of the wild-type, suggesting that the Pi-deficiency-like phenotype of Pi-sufficient per1 plants is not the result of an altered cellular Pi concentration. It seems more likely that the mutation affects Pi homeostasis at the post-transcriptional level. Regulation at the protein level has been ascribed to the transcription factor PHR1 via the SUMO E3 ligase SIZ1 (Miura et al., 2005). The E2 ubiquitin conjugase PHO2 (UBC24) is a central component in Pi homeostasis. PHO2 transcript abundance is regulated by miR399 in a systemic manner and antagonized by the ribo-regulators IPS/At4. The level of miR399 expression is regulated by PHR1, placing the SIZ1/PHR1 regulation at the top of the known cascade in Pi regulation (Aung et al., 2006; Bari et al., 2006). The multitude of regulatory sites makes it likely that a disturbed de-ubiquitination and/or recycling of ubiquitin chain affect the phenotype. Short-term treatment with Pi-free media induced a number of genes in Col-0 that may reflect components in the early signaling events associated with Pi deficiency. A subset of the induced genes are related to Ca binding and transport,

suggesting that Ca signaling is crucial for cellular Pi homeostasis. Only a small group of genes were affected by short-term Pi deficiency in both Col-0 and per1 mutant roots. Among this group was the Ca-binding protein ATCAL4, a member of the large family of EF hand, Ca-binding motif containing calmodulin-like (CML) proteins of unknown function. Together with another gene coding for an EF hand-containing protein (At3g47480), a putative Ca2+-ATPase (At3g22910) and PBP1, this group was the most responsive gene in the wild-type, suggesting that Ca is an important second messenger in early Pi signaling. Both ATCAL4 and PBP1 were shown to interact with the protein serine/threonine kinase PINOID, mediating auxin signaling (Benjamins et al., 2003). In both genotypes, SPX1 (At5g20150) was found to be among the most Pi-responsive genes. A role of AtSPX1 in Pi homeostasis, acting downstream of the MYB transcription factor PHR1, has recently been suggested (Duan et al., 2008). The early and robust up-regulation of AtSPX1 points to a potential key role in cellular Pi homeostasis. Genes that are affected transcriptionally in per1 roots under low Pi conditions appear to be involved in establishing lateral root formation and root hair elongation rather than in signaling. In conclusion, we show here that a mutation in the Arabidopsis UBP14 gene, resulting in a reduced abundance of a gene product, causes a variety of developmental defects that resemble the root phenotype of Pi-deficient plants under control conditions. Under Pi-deficient conditions, the point mutation in UBP14 led to a nutrient-specific defect in root hair elongation, which was partly reflected by a reduced expression of genes that have been associated with root hair elongation as revealed by microarray analysis (Jones et al., 2006). In addition to a crucial role in development, UBP14 may act as a regulator in the abundance of proteins involved in the adaptation to changing environmental conditions. Since homozygous Atubp14 mutants are embryo-lethal, the weak allele reported here provides a useful tool for follow-up studies on the function of UBP14 in Arabidopsis.

METHODS Plant Material and Growth Conditions Plants were grown in a growth chamber on agar medium as described by Estelle and Somerville (1987). Seeds of Arabidopsis (Arabidopsis thaliana), ecotype Col-0 were obtained from the Arabidopsis Biological Resource Center (Ohio State University) and surface-sterilized by immersing them in 5% (v/v) NaOCl for 5 min and 96% ethanol for 7 min, followed by four rinses in sterile water. The medium was composed of (mM): KNO3 (5), MgSO4 (2), Ca (NO3)2 (2), KH2PO4 (2.5), (lM): H3BO3 (70), MnCl2 (14), ZnSO4 (1), CuSO4 (0.5), NaCl (10), Na2MoO4 (0.2) and 40 lM FeEDTA, solidified with 0.3 % Phytagel (Sigma-Aldrich). Sucrose (43 mM) and Mes (4.7 mM) were included and the pH was adjusted to 5.5. Seeds were placed onto Petri dishes containing agar medium either with

Li et al.

high Pi (2.5 mM, control plants) or low Pi (0.025 mM, low Pi plants) and kept for 1 d at 4
C in the dark, before being transferred to a growth chamber and grown at 21
C under continuous illumination (50 lmol m 2 s 1, Phillips TL lamps). Light intensity was varied as indicated. The lower concentration of potassium due to the reduced KH2PO4 concentration was compensated for by addition of KCl. The plants were analyzed 14 d after sowing. Manganese deficiency was applied by sowing plants on media deprived of Mn. Manganese-deficient plants were analyzed 7 d after sowing. Fe deficiency was applied by transferring 8-day-old plants to media deprived of Fe containing 100 lM 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine sulfonate to scavenge free Fe resulting from impurities of the chemicals used to make up the media. Hypocotyl micrografting experiments were performed with wild-type and per1 plants grown for 9 d on media described above. Two days prior to grafting, plants were grown in the dark for 48 h at 27
C. Dark treatment was continued for 1 d after grafting. Plants were then transferred to either control or low Pi media and the phenotype was observed at day 16. The efficiency of the micrografting was ;25%. For gene expression analysis, roots were harvested and immediately frozen in liquid nitrogen.

Microarray Experiments and Data Analysis The Affymetrix GeneChip Arabidopsis ATH1 Genome Array was used for microarray analysis. Total RNA from roots of control and low Pi plants was isolated with the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions. Nucleic acid quantity was evaluated by using a NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, USA). All RNA samples were quality assessed by using the Agilent Bioanalyzer 2100 (Agilent). cRNA synthesis was performed by use of the GeneChip One-Cycle Target Labeling Kit (Affymetrix). GeneChips were hybridized with 15 lg of fragmented cRNA. Hybridization, washing, staining, and scanning procedures were performed as described in the Affymetrix technical manual. Data from the mircoarray experiments were imported directly into GeneSpring (version 7.0, Agilent). The software was used to normalize data per chip, to the 50th percentile and per gene to the control samples. The data were then filtered using the following: (1) by removing genes that were flagged as absent in one of the replicates of the control plants for genes down-regulated in low Pi conditions and in low Pi plants for genes that are up-regulated by low Pi; (2) by expression level to remove those genes that were deemed to be unchanging between values 0.5 and 2.0 (more than two-fold difference). Plants for microarray analysis were grown for 10 d on control media and then transferred to either fresh control or Pifree media for 10 h.

Light Microscopy Root samples were fixed, dehydrated, and then embedded in Technovit 7100 (Heraeus Kulzer, Wehrheim, Germany) resin in

d

Phosphate Deficiency Responses in Arabidopsis Roots

|

221

gelatine capsules, in accordance with the manufacturer’s instructions. Transverse sections (30 lm) were cut using a RM 2255 Leica microtome (Leica, Nussloch, Germany). Sections were dried and stained with toluidine blue (0.05%) on glass slides and examined using bright-field on an Imager Z1 (Zeiss, Jena, Germany) microscope. The number of cortical and epidermal cells and the rate of root hairs in the H- and Npositions root hairs were counted in 350 cross-sections from 10 roots of each genotype and growth type. Root hair length was determined by using a light microscope equipped with a scale in the eyepiece. One hundred root hairs from five roots were measured for each genotype and growth type.

Confocal Microscopy and Cell Length Measurements Plants were placed in 10 mg ml 1 propidium iodide solution (PI) for 1 min. The plant was gently rinsed with water for 2 min. The root was removed and mounted in fresh water. The roots where then observed using a Confocal Laser Scanning Microscope (Zeiss LSM510 Meta). The peak excitation k and emission k for PI was 536 and 620 nm, respectively. The cell length of trichoblasts and atrichoblasts was measured using ImageJ (http://rsb.info.nih.gov/ij/). The position of each cell was calculated from the cumulative length of all cells between the cell and the quiescent center. The datasets were then smoothed and interpolated into 25-mm-spaced data points using a kernel-smoothing routine (Beemster and Baskin, 1998); this was performed using a Microsoft Excel (version 97) macro, which enables the average calculation between replicate roots.

Ion Content Determination Elemental analysis was carried out by inductively coupled plasma (ICP) atomic absorption with a Perkin Elmer Optima 5300 DV optical emission spectrometer (OES) on roots of wild-type (Col-0) and per1 mutant plants. Samples were digested in nitric acid in a microwave digestion unit (CEM, Matthews, USA). Tomato leaves were used as standard reference material.

SDS–PAGE and Western Blot Analysis Total protein was prepared from roots of plants grown either on low Pi or control media. Extracts were obtained by grinding tissue on ice in extraction buffer as described by Connolly et al. (2002). Extracts were mixed in a 1:1 ratio with 8% lithium dodecyl sulfate (LDS) sample buffer (Invitrogen) containing 300 mM DTT, heated at 70
C for 10 min, then centrifuged for 10 min at 15 000 g. Total protein (about 15 lg) was separated by denaturing 7% SDS–PAGE (NuPAGE Novex Tris-acetate gels, Invitrogen), then stained with Coomassie Brilliant Blue for checking the amount of total protein, or electrotransferred to PVDF membrane filters (Immobilon-P, Millipore Co, Bedford, MA) overnight at 4
C for Western analysis. AtUBP14 antibodies raised against recombinant protein were described elsewhere (Doelling et al., 2001). Filters were blocked for 1 h in TBS-buffer (20 mM TRIS-HCl, pH 7.5, 150 mM NaCl)

222

|

Li et al.

d

Phosphate Deficiency Responses in Arabidopsis Roots

containing 5% (w/v) skimmed milk powder and incubated for 3 h with the antiserum. After a 1-h incubation with the secondary antibody (horseradish peroxydase-conjugate antirabbit IgG, Sigma-Aldrich Co., St Louis, MO), the band corresponding to AtUBP14 was detected by chemiluminescence (SuperSignal West Substrate, Pierce, Rockford, IL) on autoradiography film (Kodak X-Omat AR, Eastman Kodak Co., Rochester, NY). Quantification of the Western analysis was performed with the Quantity One (Biorad) software program.

Carol, R.J., Takeda, S., Linstead, P., Durrant, M.C., Kakesova, H., Derbyshire, P., Drea, S., Zarsky, V., and Dolan, L. (2005). A RhoGDP dissociation inhibitor spatially regulates growth in root hair cells. Nature. 438, 1013–1016.

Accessions

Chiou, T.J., Aung, K., Lin, S.I., Wu, C.C., Chiang, S.F., and Su, C.L. (2006). Regulation of phosphate homeostasis by MicroRNA in Arabidopsis. Plant Cell. 18, 412–421.

The microarray data from this study have been deposited in NCBI Gene Expression Omnibus (accession number GSE15649).

SUPPLEMENTARY DATA

Carswell, M.C., Grant, B.R., and Plaxton, W.C. (1997). Disruption of the phosphate-starvation response of oilseed rape suspension cells by the fungicide phosphonate. Planta. 203, 67–74. Connolly, E.L., Fett, J.P., and Guerinot, M.L. (2002). Expression of the IRT1 metal transporter is controlled by metals at the levels of transcript and protein accumulation. Plant Cell. 14, 1347–1357.

Delhaize, E., and Randall, P.J. (1995). Characterization of a phosphate-accumulator mutant of Arabidopsis thaliana. Plant Physiol. 107, 207–213.

Supplementary Data are available at Molecular Plant Online.

Desnos, T. (2008). Root branching responses to phosphate and nitrate. Curr. Opin. Plant Biol. 11, 82–87.

FUNDING

Doelling, J.H., Yan, N., Kurepa, J., Walker, J., and Vierstra, R.D. (2001). The ubiquitin-specific protease UBP14 is essential for early embryo development in Arabidopsis thaliana. Plant J. 27, 393–405.

This work was supported by the NSC Taiwan (NSC96-2311-B-001022-MY3 to W.S.).

ACKNOWLEDGMENTS UBP14 antibodies were kindly provided by R.D. Vierstra (University of Wisconsin-Madison, USA). We thank K.C. Yeh (ABRC, Taipei, Taiwan) for his help with ICP–OES analysis. We also thank T.W.J. Yang (IPMB Taipei, Taiwan), M. Mu¨ller (IPK Gatersleben, Germany), and T.J. Buckhout (HU Berlin, Germany) for critical comments on the manuscript. Affymetrix GeneChip assays were performed by the Affymetrix Gene Expression Service Lab (http://ipmb.sinica.edu. tw/affy/), supported by Academia Sinica. Experiments and data analysis were performed in part through the use of the inductively coupled plasma optical emission spectrometer at Agricultural Biotechnology Research Center of Academia Sinica and with the assistance of Yan-Chen Huang. No conflict of interest declared.

REFERENCES Aung, K., Lin, S.I., Wu, C.C., Huang, Y.T., Su, C.L., and Chiou, T.J. (2006). pho2, a phosphate overaccumulator, is caused by a nonsense mutation in a microRNA399 target gene. Plant Physiol. 141, 1000–1011.

Doerner, P. (2008). Phosphate starvation signaling: a threesome controls systemic P(i) homeostasis. Curr. Opin. Plant Biol. 11, 536–540. Dolan, L. (2006). Positional information and mobile transcriptional regulators determine cell pattern in the Arabidopsis root epidermis. J. Exp. Bot. 57, 51–54. Dong, B., Rengel, Z., and Delhaize, E. (1998). Uptake and translocation of phosphate by pho2 mutant and wild-type seedlings of Arabidopsis thaliana. Planta. 205, 251–256. Duan, K., Yi, K., Dang, L., Huang, H., Wu, W., and Wu, P. (2008). Characterization of a sub-family of Arabidopsis genes with the SPX domain reveals their diverse functions in plant tolerance to phosphorus starvation. Plant J. 54, 965–975. Estelle, M.A., and Somerville, C. (1987). Auxin-resistant mutants of Arabidopsis thaliana with an altered morphology. Mol. Gen. Genet. 206, 200–206. Grierson, C., and Schiefelbein, J. (2002). Roots hairs. In The Arabidopsis Book, Somerville C.R. and Meyerowitz E.M., eds (Rockville, MD: American Society of Plant Biologists), aspb.org/ publications/Arabidopsis/, doi: 10.1199/tab.0060, pp. 1–22. Hemsley, P.A., Kemp, A.C., and Grierson, C.S. (2005). The TIP GROWTH DEFECTIVE1 S-acyl transferase regulates plant cell growth in Arabidopsis. Plant Cell. 17, 2554–2563.

Bari, R., Datt Pant, B., Stitt, M., and Scheible, W.R. (2006). PHO2, microRNA399, and PHR1 define a phosphate-signaling pathway in plants. Plant Physiol. 141, 988–999.

Ishida, T., Kurata, T., Okada, K., and Wada, T. (2008). A genetic regulatory network in the development of trichomes and root hairs. Annu. Rev. Plant Biol. 59, 365–386.

Beemster, G.T.S., and Baskin, T.I. (1998). Analysis of cell division and elongation underlying the developmental acceleration of root growth in Arabidopsis thaliana. Plant Physiol. 116, 1515–1526.

Jones, M.A., Raymond, M.J., and Smirnoff, N. (2006). Analysis of the root-hair morphogenesis transcriptome reveals the molecular identity of six genes with roles in root-hair development in Arabidopsis. Plant J. 45, 83–100.

Benjamins, R., Ampudia, C.S., Hooykaas, P.J., and Offringa, R. (2003). PINOID-mediated signaling involves calcium-binding proteins. Plant Physiol. 32, 1623–1630. Bernhardt, C., Zhao, M., Gonzalez, A., Lloyd, A., and Schiefelbein, J. (2005). The bHLH genes GL3 and EGL3 participate in an intercellular regulatory circuit that controls cell patterning in the Arabidopsis root epidermis. Development. 132, 291–298.

Jones, M.A., Shen, J.J., Fu, Y., Li, H., Yang, Z., and Grierson, C.S. (2002). The Arabidopsis Rop2 GTPase is a positive regulator of both root hair initiation and tip growth. Plant Cell. 14, 763–776. Kwak, S.H., and Schiefelbein, J. (2007). The role of the SCRAMBLED receptor-like kinase in patterning the Arabidopsis root epidermis. Dev. Biol. 302, 118–131.

Li et al.

d

Phosphate Deficiency Responses in Arabidopsis Roots

|

223

Kwak, S.H., and Schiefelbein, J. (2008). A feedback mechanism controlling SCRAMBLED receptor accumulation and cell-type pattern in Arabidopsis. Curr. Biol. 18, 1949–1954.

factor involved in phosphate starvation signaling both in vascular plants and in unicellular algae. Genes Dev. 15, 2122–2133.

Lee, M.M., and Schiefelbein, J. (2002). Cell pattern in the Arabidopsis root epidermis determined by lateral inhibition with feedback. Plant Cell. 14, 611–618.

Samaj, J., et al. (2002). Involvement of the mitogen-activated protein kinase SIMK in regulation of root hair tip growth. EMBO J. 21, 3296–3306.

Lelandais-Brie`re, C., Jovanovic, M., Torres, G.A., Perrin, Y., Lemoine, R., Corre-Menguy, F., and Hartmann, C. (2007). Disruption of AtOCT1, an organic cation transporter gene, affects root development and carnitine-related responses in Arabidopsis. Plant J. 51, 154–164.

Samaj, J., Muller, J., Beck, M., Bohm, N., and Menzel, D. (2006). Vesicular trafficking, cytoskeleton and signalling in root hairs and pollen tubes. Trends Plant Sci. 11, 594–600.

Lin, S.I., Chiang, S.F., Lin, W.Y., Chen, J.W., Tseng, C.Y., Wu, P.C., and Chiou, T.J. (2008). Regulatory network of microRNA399 and PHO2 by systemic signaling. Plant Physiol. 147, 732–746. Lo´pez-Bucio, J., Cruz-Ramı´rez, A., and Herrera-Estrella, L. (2003). The role of nutrient availability in regulating root architecture. Curr. Opin. Plant Biol. 6, 280–287. Ma, J.F., Goto, S., Tamai, K., and Ichii, M. (2001). Role of root hairs and lateral roots in silicon uptake by rice. Plant Physiol. 127, 1773–1780. Ma, Z., Baskin, T.I., Brown, K.M., and Lynch, J.P. (2003). Regulation of root elongation under phosphorus stress involves changes in ethylene responsiveness. Plant Physiol. 131, 1381–1390. Misson, J., et al. (2005). A genome-wide transcriptional analysis using Arabidopsis thaliana Affymetrix gene chips determined plant responses to phosphate deprivation. Proc. Natl Acad. Sci. U S A. 102, 11934–11939. Miura, K., et al. (2005). The Arabidopsis SUMO E3 ligase SIZ1 controls phosphate deficiency responses. Proc. Natl Acad. Sci. U S A. 102, 7760–7765. Morton, B.R., and Wright, S.I. (2007). Selective constraints on codon usage of nuclear genes from Arabidopsis thaliana. Mol. Biol. Evol. 24, 122–129. Mu¨ller, M. (2006). Arabidopsis root hair development in adaptation to iron and phosphate supply Dissertation, Humboldt-University Berlin, Germany. Mu¨ller, M., and Schmidt, W. (2004). Environmentally induced plasticity of root hair development in Arabidopsis. Plant Physiol. 134, 409–419. Narang, R.A., Bruene, A., and Altmann, T. (2000). Analysis of phosphate acquisition efficiency in different Arabidopsis accessions. Plant Physiol. 124, 1786–1799. Nilsson, L., Mu¨ller, R., and Nielsen, T.H. (2007). Increased expression of the MYB-related transcription factor, PHR1, leads to enhanced phosphate uptake in Arabidopsis thaliana. Plant Cell Environ. 30, 1499–1512. Pant, B.D., Buhtz, A., Kehr, J., and Scheible, W.R. (2008). MicroRNA399 is a long-distance signal for the regulation of plant phosphate homeostasis. Plant J. 53, 731–738. Rubio, V., Linhares, F., Solano, R., Martı´n, A.C., Iglesias, J., Leyva, A., and Paz-Ares, J. (2001). A conserved MYB transcription

Sa´nchez-Caldero´n, L., Lo´pez-Bucio, J., Chaco´n-Lo´pez, A., CruzRamı´rez, A., Nieto-Jacobo, F., Dubrovsky, J.G., and HerreraEstrella, L. (2005). Phosphate starvation induces a determinate developmental program in the roots of Arabidopsis thaliana. Plant Cell Physiol. 46, 174–184. Savage, N.S., and Schmidt, W. (2008). From priming to plasticity: the changing fate of rhizodermic cells. Bioessays. 30, 75–81. Savage, N.S., Walker, T., Wieckowski, Y., Schiefelbein, J., Dolan, L., and Monk, N.A. (2008). A mutual support mechanism through intercellular movement of CAPRICE and GLABRA3 can pattern the Arabidopsis root epidermis. PLoS Biol. 6, e235. Schachtman, D.P., and Shin, R. (2007). Nutrient sensing and singaling: NPKS. Annu. Rev. Plant Biol. 58, 47–69. Schiefelbein, J., Kwak, S.H., Wieckowski, Y., Barron, C., and Bruex, A. (2009). The gene regulatory network for root epidermal cell-type pattern formation in Arabidopsis. J. Exp. Bot. 60, 1515–1521. Schiefelbein, J.W., Shipley, A., and Rowse, P. (1992). Calcium influx at the tip of growing root-hair cells of Arabidopsis thaliana. Planta. 187, 455–459. Schmidt, W. (2008). Inner voices meet outer signals: the plasticity of rhizodermic cells. Plant Science. 174, 239–245. Synek, L., Schlager, N., Elia´s, M., Quentin, M., Hauser, M.T., and Za´rsky´, V. (2006). AtEXO70A1, a member of a family of putative exocyst subunits specifically expanded in land plants, is important for polar growth and plant development. Plant J. 48, 54–72. Ticconi, C.A., and Abel, S. (2004). Short on phosphate: plant surveillance and countermeasures. Trends Plant Sci. 9, 548–555. Ticconi, C.A., Delatorre, C.A., and Abel, S. (2001). Attenuation of phosphate-starvation responses. by phosphite in Arabidopsis thaliana. Plant Physiol. 127, 963–972. Vance, C.P., Uhde-Stone, C., and Allan, D.L. (2003). Phosphorus acquisition and use: critical adaptations by plants for securing a non-renewable resource. New Phytol. 157, 423–447. 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. Yang, T.J., Perry, P.J., Ciani, S., Pandian, S., and Schmidt, W. (2008). Manganese deficiency alters the patterning and development of root hairs in Arabidopsis. J. Exp. Bot. 59, 3453–3464.

Copyright of Molecular Plant is the property of Oxford University Press / UK and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.