Perturbation of Auxin Homeostasis Caused by Mitochondrial FtSH4 Gene-Mediated Peroxidase Accumulation Regulates Arabidopsis Architecture

Molecular Plant  •  Volume 7  •  Number 5  •  Pages 856–873  •  May 2014

RESEARCH ARTICLE

Perturbation of Auxin Homeostasis Caused by Mitochondrial FtSH4 Gene-Mediated Peroxidase Accumulation Regulates Arabidopsis Architecture a Guangdong Key Lab of Biotechnology for Plant Development, College of Life Sciences, South China Normal University, Guangzhou 510631, China b Hunan Provincial Key Laboratory of Phytohormones and Growth Development, Hunan Agricultural University, Changsha 410128, China

ABSTRACT  Reactive oxygen species and auxin play important roles in the networks that regulate plant development and morphogenetic changes. However, the molecular mechanisms underlying the interactions between them are poorly understood. This study isolated a mas (More Axillary Shoots) mutant, which was identified as an allele of the mitochondrial AAA-protease AtFtSH4, and characterized the function of the FtSH4 gene in regulating plant development by mediating the peroxidase-dependent interplay between hydrogen peroxide (H2O2) and auxin homeostasis. The phenotypes of dwarfism and increased axillary branches observed in the mas (renamed as ftsh4-4) mutant result from a decrease in the IAA concentration. The expression levels of several auxin signaling genes, including IAA1, IAA2, and IAA3, as well as several auxin binding and transport genes, decreased significantly in ftsh4-4 plants. However, the H2O2 and peroxidases levels, which also have IAA oxidase activity, were significantly elevated in ftsh4-4 plants. The ftsh4-4 phenotypes could be reversed by expressing the iaaM gene or by knocking down the peroxidase genes PRX34 and PRX33. Both approaches can increase auxin levels in the ftsh4-4 mutant. Taken together, these results provided direct molecular and genetic evidence for the interaction between mitochondrial ATP-dependent protease, H2O2, and auxin homeostasis to regulate plant growth and development. Key words:  Arabidopsis thaliana; auxin; development; FtSH4; hydrogen peroxide; IAA oxidation; peroxidase.

Introduction A group of membrane-localized ATP-dependent proteases has been named as AAA-protease (ATPase associated with diverse cellular activities protease, AAA-protease), and found in all eubacteria and eukaryotes. These proteases act on areas of the mitochondria and chloroplasts where there is molecular injury or incorrect assembly of membrane proteins (Nolden et al., 2005). AAA-protease achieves its proteolytic activity and molecular chaperone-like functions using two domains: the AAA-domain and the peptide domain in the same single peptide. The proteolytic domain is located at the back of the AAA-domain, and its enzymatic activity is metal-dependent (Kolodziejczak et al., 2002). There are three main types of the ATP-dependent proteases in mitochondria: Lon, Clp, and FtSH (filamentation temperature-sensitive). To date, four FtSH genes have been identified in Arabidopsis mitochondria. FtSH3 and FtSH10 are considered to be m-AAA proteases (reviewed by Janska et al., 2010), while FtSH4 and

FtSH11 showed i-AAA-protease characteristics (Urantowka et al., 2005). The function of plant mitochondrial AAA-protease is still very unclear at present but complementation experiments show that the important functions of m-AAA-protease in fungi and plants are conserved (Kolodziejczak et  al., 2002). With the exception of AtFtSH11, the Arabidopsis mitochondrial AAA proteases are related to the plant oxidative phosphorylation system (Kolodziejczak et  al., 2007). AtFtSH4 influences the vegetative growth of Arabidopsis late rosette

1 To whom correspondence should be addressed. E-mail Yangchw@scnu. edu.cn, tel./fax 86-20-85210855

© The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi:10.1093/mp/ssu006, Advance Access publication 30 January 2014 Received 17 November 2013; accepted 14 January 2014

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Shengchun Zhanga, Juelin Wua, Dongke Yuana, Daowei Zhanga, Zhigang Huangb, Langtao Xiaob, and Chengwei Yanga,1

Zhang et al.  •  The Roles of FtSH4 Gene in Auxin Homeostasis   

of H2O2 and peroxidase levels. The dwarfism and increased in axillary branching of the ftsh4-4 mutant could be reversed by expressing the iaaM gene or by knocking down the peroxidase genes PRX34 and PRX33, both of which elevated auxin level in ftsh4-4 mutant. Collectively, our data indicate that the mitochondrial ATP-dependent protease, FtSH4, is a modulator between H2O2 and auxin homeostasis to regulate plant growth and development.

RESULTS The mas Mutation Shows Dwarfism and Increased Axillary Branching Previously, a dwarf Arabidopsis mutant showing increased axillary shoots was generated, in the process of researching the AtMMS21-interacting AtSCE1a gene, by using the empty pCanG vector-mediated transformation of wild-type plants (Huang et al., 2009). A homozygous mutant was created and named as mas (More Axillary Shoots) according to its phenotype. The developmental phenotypes of the mas mutants were not detectable in young seedlings (Figure 1A). However, in the mas mutant plants, the rosette leaves stopped growing, and began to become curly after 3 weeks (Figure 1B). The phenotypic defects in the mas mutant plants became more pronounced after bolting at the 4-week-old stage. Commonly, the 6-week-old mas mutant plants had five to eight rosette axillary shoots compared to one to two for wild-type plants (Figure 1C). Interestingly, more than two branches were initiated from the same cauline leaf axil in the mas mutant plants and up to five axillary branches (Figure  1F). Because more fresh shoots originated from the axillary buds, the mas mutant plants displayed a stay-green phenotype after the wild-type plants had died (Figure 1D). The axillary meristems continued to form and subsequently grew to inflorescence branches from the axils of both rosette leaves (up to 12) and cauline leaves (up to 44 branches per rosette inflorescence) in the mas mutant plants. All mas mutant plants had quaternary branching whereas only 93.75% of the wild-type plants had tertiary branching (Supplemental Table 1). After ceasing growth, there was a mean of six axillary shoots from the base rosette of the mas mutant, but only two from wild-type plants (Figure 1H), and there was a mean of three axillary shoots produced by cauline leaves of the mas mutants compared to one for wild-type plants (Figure  1I). However, the main inflorescence shoot apex and the axillary shoot apex of mas mutant plants showed a loss of vitality (Figure  1E), which results in a severe dwarfing phenotype. The ultimate height of mas mutants was only around 6 cm compared to >20 cm for wild-type plants (Figure 1G).

Isolation of the MAS Gene Genetic analysis indicated that the mas mutation co-segregates with a T-DNA insertion, and MAS is a haploinsufficient gene (Supplemental Tables 2 and 3). To identify the relevant mas mutant gene, an adaptor ligation-mediated PCR revealed

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leaves formation under short-day conditions, depending on preventing the accumulation of oxidized proteins (Gibala et al., 2009). Numerous studies on the chloroplast FtSH protease have found that chloroplast FtSH proteases act mainly through reactive oxygen species (ROS) to control the leaf morphological changes (Kato et  al., 2009). There has, however, been little study into the function of plant mitochondrial FtSH protease. The integrity of the mitochondrial inner membrane depends on a number of membrane-localized FtSH/AAA proteases, and these proteases can very specifically degrade badly folded or unassembled inner membrane protein. ROS are key signaling molecules that regulate growth and development and coordinate responses to biotic and abiotic stresses in plants (reviewed by Apel and Hirt, 2004). During biotic and abiotic stress events, there is a close interplay between ROS and other plant signaling molecules and hormones, such as calcium, salicylic acid (SA), abscisic acid (ABA), jasmonic acid, ethylene, nitric oxide, and gibberellins (Bright et al., 2006; Gudesblat et al., 2007; Desikan et al., 2008; Galvez-Valdivieso et  al., 2009). Transcriptomic data revealed that various aspects of auxin homeostasis and signaling were modified by apoplastic ROS (Blomster et al., 2011). The best-known case where ROS directly influences the action of auxin is a hydrogen peroxide (H2O2)-dependent MAPK cascade that negatively affects auxin sensitivity by downregulation of auxin-inducible gene expression (Nakagami et al., 2006). Interaction between ROS and auxin is also suggested by altered auxin homeostasis through increased H2O2 levels and by observed changes in plant architecture provoked by ROS impingement on auxin signaling (reviewed by Potters et al., 2007, 2009). Mitochondrial ROS production in Arabidopsis can mediate crosstalk between abscisic acid and auxin signaling (He et al., 2012). Auxin homeostasis could be altered by H2O2-induced changes to PINOID gene expression, which affects polar auxin transport (Pasternak et  al., 2005). In addition to the influence on auxin homeostasis through the regulation of enzymes involved in auxin biosynthesis and conjugation (reviewed by Ljung et al., 2002; Woodward and Bartel, 2005), oxidative degradation of auxin through H2O2dependent peroxidases occurs as well (Gazarian et al., 1998; Ljung et al., 2002). Though several phenotypic analyses suggest that ROS can interact with auxin to regulate the plant growth and development (Sagi et  al., 2004; Bashandy et  al., 2010; Tognetti et  al., 2010), deeper and more comprehensive evidence of molecular biology and genetics, especially the mediators of ROS–auxin interaction, is required to clarify. This study reports the identification and characterization of one mutant, mas, related to interaction between H2O2 and auxin. The MAS is a FtSH protease gene and identified as FtSH4, previously shown to be involved in late rosette leaf development under short-day conditions (Gibala et al., 2009). The ftsh4-4 mutation caused the decrease of free IAA concentration, the perturbation of auxin signaling, and the elevation

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that the T-DNA insertion was located in the promoter region of the gene AtFtSH4 (At2g26140), which encodes for a mitochondrial AAA-protease protein and is involved in late rosette leaf development under short-day conditions (Gibala et al., 2009). The precise position of the T-DNA insertion was 224 base pairs upstream of the ATG (Figure 2A), and verified using the three primers PCR (Figure  2B). Western blot and real-time PCR were performed to assess FtSH4 expression in the homozygous T-DNA lines and no expression was detected in the mas mutant plants (Figure 2C and 2D). Thus, MAS was renamed as FtSH4, and the T-DNA-inserted loss-of-function mas mutant was renamed as ftsh4-4 according to the nomenclature of ftsh4 mutants (Gibala et  al., 2009). We further

compared the phenotypes of both ftsh4-4 and ftsh4-1 under short-day conditions. Both of them had the same phenotype with abnormal late rosette leave development and increased H2O2 level (Supplemental Figure  1), indicating that ftsh4-4 was an allele of ftsh4-1. To clarify whether the dwarfism and the increased axillary branching phenotypes were indeed caused by the loss of the FtSH4 function, the cDNA of FtSH4 gene was constructed into a complementation vector under the control of the FtSH4 promoter, and transformed into ftsh4-4 mutant plants. The homozygous progeny plants were screened and verified by real-time PCR (Figure 2D). In the ftsh4-4 mutant phenotypes, characteristics such as plant height, axillary branch number,

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Figure 1.  The Phenotype of mas Mutant Plants. (A) Two-week-old seedlings. Bar = 1 cm. (B) Four-week-old plants. (C) Six-week-old plants. Bar = 2 cm. (D) Eleven-week-old plants. Left, wild-type plant; right, mas mutant plants. Bar = 2 cm. (E) Axillary branches (tillers) from the rosette leaves of 11-week-old mas plants. Main stem, the main inflorescence stems with axillary shoots from cauline leaves. (F) Cauline leaves show more than one axillary bud in mas plants. (G) The average terminal height of mas mutant plants. (H) The average axillary branch number of the mas plants from the rosette leaves. (I) The number of secondary axillary branches present on mas cauline leaves. (G–I), 32 11-week-old plants were analyzed for each of three biological replicates.

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Figure 2.  Identification of mas Mutant Plants. (A) The localization of the T-DNA inserted in the genomic DNA. The light gray line and black line before the ATG is the promoter region, the black box is the exon, and the dark gray line is the intron. The left primers (LP) and right primers (RP) are the genomic primers used for verification. (B) Genomic DNA verification by PCR with three primers. Lanes 1 and 4: left border (LB) and genomic LP; lanes 2 and 5: LB and genomic RP; lanes 3 and 6: LP and RP. The positions of LP and RP are shown in (A). (C) FtSH4 gene completely reverses the ftsh4-4 mutant plant phenotypes. Top, photographs of 7-week-old plants. 1#, 2#, and 3# indicate ftsh4-4 plants transformed with pFtSH4:FtSH4 constructs. Bottom, real-time PCR verification of the FtSH4 gene expression in different genotype plants in the top. For real-time PCR, three technical replicates were performed for each of three biological replicates. (D) Protein expression verification of the ftsh4-4 mutant and complementation plants by Western blot. C1 indicates the 1# line complementation plants. (E) AAA-domain is necessary for FtSH4 function in plant growth and development. Photograph of the 4-week-old different ftsh4-4 mutant plants. (F) Axillary branch number of the different ftsh4-4 mutant plants.

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FtSH4 Gene Expression Pattern In order to elucidate the function of FtSH4, its expression pattern was investigated by the real-time PCR and the FtSH4

promoter–β-glucuronidase (GUS) fusion expression analysis. The FtSH4 transcript could be detected at different growth stages (Figure  3A) and in all tested tissues (Figure  3B). To study the FtSH4 expression pattern in detail, the homozygous single copy insertion pFtSH4:GUS plants were used for GUS staining. In the 5-day-old seedlings, GUS expression was more intense and located in the vascular tissue of both the hypocotyl and the cotyledons, especially at the shoot apex and the branching zone of the vascular tissue that lies at the base of the shoot apical meristem (Figure 3C). In the 15-dayold seedlings, the GUS activity was mainly observed in the vascular system of rosette leaves and petioles, as well as the shoot apex (Figure 3D). Within the inflorescence, FtSH4 was expressed in the inflorescence stem and the vascular tissue of the cauline leaves, with the strongest intensity found at the base of the cauline leaves where axillary meristems are initiated (Figure  3E). Clearly, the FtSH4 gene is expressed strongly in the new axillary branch outgrowths from the base of rosette leaves and the cauline leaves (Figure  3F).

Figure 3.  Expression Patterns of FtSH4 Gene. (A) FtSH4 expressed stably in different growth stages detected by real-time PCR. The numbers 1–5 indicate 1–5-week-old wild-type plants. (B) FtSH4 expressed in different tissues detected by real-time PCR. Seedling and root are from 15-day-old plants, and the other tissues were from 35-day-old plants. For real-time PCR in (A) and (B), three technical replicates were performed for each of three biological replicates. (C) pFtSH4:GUS expressed in the 5-day-old seedling cotyledons, shoot apex, and hypocotyls. (D) pFtSH4:GUS expressed in the 15-day-old seedling rosette leaves. (E) pFtSH4:GUS expressed in the branching sites and branches of the 25-day-old seedlings on MS plates. (F) pFtSH4:GUS expressed in the branching sites and branches from the rosette axillary buds of 35-day-old seedlings. Red arrows in (E) and (F) indicate the branching sites and branches.

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and rosette leaf shape were rescued by the expression of FtSH4 in different transgenic ftsh4-4 mutant lines (Figure 2D), demonstrating that the ftsh4-4 mutant phenotypes were due to the non-functioning of FtSH4. FtSH4 protein has two functional domains, including the AAA-domain and the peptide domain, in the same single peptide. In order to test which domain takes charge for the ftsh44 phenotype, two homozygous lines of the altered FtSH4 variants were generated in the ftsh4-4 mutant (Supplemental Figure  2). FtSH4m1 (the AAA-domain deletion) could not reverse the dwarfing or the increased branching in ftsh4-4 mutant plants. However, FtSH4m2 (the protease-domain deletion) could reverse dwarfing and decrease branching of the ftsh4-4 mutant (Figure 2E and 2F). These results demonstrated that the AAA-domain of FtSH4 was necessary for its function.

Zhang et al.  •  The Roles of FtSH4 Gene in Auxin Homeostasis   

The expression pattern of FtSH4, as shown by GUS staining, correlated spatially with the morphological abnormalities of ftsh4-4 mutants.

Level of IAA Declines in ftsh4-4 Mutant Plants

in 7-day-old ftsh4-4 seedlings was similar to that of wild-type plants (Figure  4B). To illustrate the effect of IAA deficiency in ftsh4-4 mutant plants, a tryptophan-2-monooxygenase gene called iaaM from Agrobacterium to convert tryptophan to indole-3-acetamide (IAM), which is subsequently hydrolyzed into IAA by the hydrolase iaaH (Camilleri and Jouanin, 1991; Romano et al., 1995), was transformed into and overexpressed in ftsh4-4 mutants. The iaaM transgenic ftsh4-4 mutation showed a wild-type phenotype, taller than the other ftsh4-4 mutant plants when grown in the light for 5 weeks (Figure 4C), and the extent of recovery was proportional to the level of iaaM expression (Supplemental Figure 4). These results suggest that increasing the levels of endogenous IAA could reverse the defects seen in typical untransformed ftsh44 phenotypes. To further investigate the reduced auxin levels, the mutant plants were crossed with the auxin response marker line ProDR5:GUS (Ulmasov et  al., 1997; Vieten et  al., 2005). GUS staining (Figure 5A) and GUS activity (Figure 5D) were lower in the inflorescence stem of ftsh4-4 compared with wild-type

Figure 4.  Excessive Branch Production Is Inhibited by Exogenous Auxin. (A) Six-week-old plants sprayed with 5 μM NAA every 4 d from 3 weeks old for a further 3 weeks. (B) IAA levels were determined in the 7-day-old and 4-week-old wild-type plants, homozygous ftsh4-4 mutant plants, and pFtSH4:FtSH4-ftsh4-4 plants. Data are means of three biological repetitions ± SE. (C) Auxin synthesis gene iaaM overexpression completely reverses the ftsh4-4 mutant phenotypes. The iaaM-ftsh4-4–5 indicates the fifth-line plants that overexpressed iaaM in the ftsh4-4 mutant. All plants are 35 d old. Bar = 2 cm.

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According to the features of the ftsh4-4 mutant phenotypes, it was predicted that auxin content had been blocked in the ftsh4-4 mutant plants. To verify this hypothesis, exogenous auxin, NAA, was sprayed on to 3-week-old seedlings every 4 d for 3 weeks. As expected, the ftsh4-4 plant’s tendency to dwarfism and multiple branching was reversed by application of the exogenous NAA (Figure  4A), which implied that the FtSH4 gene played an important role in auxin-mediated plant development. However, no change was observed in FtSH4 gene transcript level after treatment with exogenous IAA, NAA, or 2,4-D (Supplemental Figure 3). These results suggest that FtSH4 may regulate plant height and branching by regulating the endogenous auxin level. As we speculated, the auxin (IAA) concentration was almost two-fold lower in the 4-week-old ftsh4-4 mutant plants, but

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ProDR5:GUS plants, and GUS staining was also reduced significantly in the axillary meristems of ftsh4-4 ProDR5:GUS plants (Figure 5A), supporting the finding that auxin responses were repressed in ftsh4-4 mutants. The change in auxin levels in the ftsh4-4 mutant shoots suggests that the mutation may also change callus regeneration capacity and/or shoot regeneration from calli. The capacity of ftsh4-4 mutant plants to dedifferentiate and form calli was first evaluated on callus-inducing medium plates. Callus regeneration was not modified in the ftsh4-4 mutant, indicating that cell division was not affected (Figure  5B). When calli were transferred onto shoot-inducing medium plates containing fixed concentrations of synthetic cytokinin 6-BA without IAA, wild-type calli dedifferentiated to form adventitious shoots after 15 d (Figure  5C). Leaf primordia could be observed after 7 d and the number of calli with adventitious shoots increased gradually. However, ftsh4-4 mutant calli were unable to dedifferentiate in the absence of exogenous auxin, suggesting that auxin was the limiting factor for shoot regeneration in the ftsh4-4 mutant.

Furthermore, IAA biosynthesis genes were detected in ftsh4-4 mutant plants. TAA1 (Stepanova et al., 2008; Tao et al., 2008), YUCCA or YUCCA1 (Zhao et  al., 2001), and CYP79B2 (Zhao et al., 2002), three key genes that play important roles in the Trp-dependent IAA biosynthesis pathway, were chosen for transcript detection. Interestingly, there were no changes in these important IAA biosynthesis gene transcripts in ftsh44 mutant plants compared to wild-type plants at different growth stages (Figure 6A).

Alterations in Auxin Transport and Signaling Gene Expression in the ftsh4-4 Mutant Plants To elucidate how the FtSH4 genes regulate the auxin pathway, a genome-wide expression analysis was performed using samples from 4-week-old wild-type and ftsh4-4 plants grown under normal growth conditions. No IAA biosynthesis gene showed significant expression change in the ftsh4-4 mutant (Supplemental Table 4). However, expression of several important auxin binding, transport, and response genes, including the AFB2, ABCB19, PIN4, PIN7, LAX2, IAA3, and NDPK2 decreased significantly in the ftsh4-4 mutant plants (Table 1

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Figure 5.  Auxin Reporter ProDR5:GUS Gene Expression Changed in the ftsh4-4 Inflorescence Stem. (A) Localization of the ProDR5:GUS activity in the 7-day-old wild-type Col-0 and in ftsh4-4 cotyledon, hypocotyl, root, root apex, and the 6-weekold inflorescence stems and whole plants. (B, C) The ftsh4-4 mutant plants are less susceptible to the de novo shoot induction system. Explants or callus from the wild-type or ftsh4-4 mutant plants in the callus-inducing medium (B) or the shoot-inducing medium (C). (D) GUS activity of ProDR5:GUS plants inflorescence stems measured by a MUG assay. MUG, 4-methylumbelliferyl β-D-glucuronide; three technical replicates were performed for each of three biological replicates.

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(A) IAA biosynthesis gene transcript levels did not significantly change in ftsh4-4 compared to wild-type plants. (B) Aux/IAA gene transcript levels were down-regulated in ftsh4-4 mutant plants. (C) GH3 gene transcript levels did not change in ftsh4-4 mutants. Three technical replicates were performed for each of three biological replicates.

and Supplemental Table  4). In addition to these important genes, many SAUR-like auxin-responsive genes changed significantly in the ftsh4-4 mutant; and a series of Aux/IAA genes, including IAA1 and IAA2, appeared in the microarray data, although their decreased fold in the ftsh4-4 mutant is not significant (Supplemental Table 5). Auxin is detected by the TIR1 receptor, which then initiates transcription of the responsive genes (Lau et al., 2008). There are three major classes (Aux/IAAs, SAURs, and GH3s) of early or primary auxin response genes, which play important roles in the auxin signaling pathway (reviewed by Hagen and Guilfoyle, 2002). To further elucidate the expression of the auxin response genes in ftsh4-4 mutants, three Aux/IAA protein family genes (IAA1, IAA2, and IAA3) that play important regulatory roles in the auxin signaling pathway (Tian et  al., 2002; Yang et al., 2004), and six Group II GH3s genes that may function in auxin homeostasis by conjugating amino acids to IAA and reducing the availability of free auxin (Staswick et  al., 2005) were also chosen for transcript detection. The expression of all three IAAs genes significantly decreased in ftsh4-4 mutant plants (Figure 6B), indicating that auxin signaling in ftsh4-4 mutant plants was blocked. However, the expression levels of all six GH3 genes, including GH3.1, GH3.2, GH3.3, GH3.5, GH3.6, and GH3.17, had no significant changes in ftsh4-4 mutants (Figure 6C).

FtSH4 Mutation Increases the H2O2 Level and Exogenous Antioxidants Partially Rescue the ftsh4-4 Phenotypes It has been demonstrated that ROS content in the ftsh4-1 mutant increased (Gibala et al., 2009). Consistently with that, elevated H2O2 levels were detected in the ftsh4-4 mutant plants from seedling to adult stages, particularly at the 3-week-old growth stage (Figure 7C). Interestingly, the high H2O2 level in the ftsh4-4 mutants could be reduced by FtSH4 complementation (Figure 7D). We speculated that the phenotype defect of ftsh4-4 could be rescued to a certain extent by decreasing the H2O2 level, if the FtSH4 gene functions through ROS. As expected, its dwarf and multi-branched phenotypes were all partially rescued by exogenously supplying antioxidants, ascorbic acid (AsA) (Figure 7A). The exogenous AsA (1 mM) increased the ftsh44 height to 12 cm and decreased the numbers of primary branches to three (Supplemental Figure 5B). From the results above, it was concluded that the phenotypic changes in the ftsh4-4 mutant plants were mainly caused by the accumulation of H2O2. However, FtSH4 expression was not affected by exogenous antioxidants, such as AsA, reduced glutathione (GSH), or H2O2 (Figure 7B), indicating that FtSH4 may control the H2O2 production or the signaling connected with plant growth and development.

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Figure 6.  Auxin Synthesis and Signaling Gene Transcript Levels in ftsh4-4 Mutants Compared to Wild-Type Plants.

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Table 1.  Changed Expression of the Auxin Signaling and Hydrogen Peroxide Catabolism Genes in ftsh4-4 Mutant.

Gene

Protein

Function

Fold change

P-value (%)

Auxin signaling genes At3g28860

ABCB19

ATPase, auxin efflux transporter

0.36280

0.13445

At2g21050

LAX2

A member of the AUX1 LAX family of auxin influx carriers

0.44217

0.19049

At2g01420

PIN4

Auxin hydrogen symporter/transporter

0.45573

0.49087

At1g23080

PIN7

Auxin hydrogen symporter/transporter

0.45810

0.33063

At1g04240

SHY2/IAA3

Regulates multiple auxin responses in roots

0.46097

0.66483

At5g63310

NDPK2

ATP binding/nucleoside diphosphate kinase

0.33633

0.48203

At3g26810

AFB2

Auxin binding/ubiquitin–protein ligase

0.49773

0.42575

At4g08770

PER37

Peroxidase, putative

At3g49120

PER34

ATPCB/ATPERX34/PERX34/PRXCB (PEROXIDASE 34); peroxidase

3.5764

0

At3g49110

PER33

ATPCA/ATPRX33/PRX33/PRXCA (PEROXIDASE 33); peroxidase

2.8182

0

At4g37530

PER51

Peroxidase, putative

2.8165

0

At2g37130

PER21

Peroxidase 21 (PER21) (P21) (PRXR5)

2.6962

0

At3g21770

PER30

Peroxidase 30 (PER30) (P30) (PRXR9)

2.3799

0

At4g37520

PER50

Peroxidase 50 (PER50) (P50) (PRXR2)

2.03

0.0887

At2g43350

GPX3

Glutathione peroxidase 3

2.2353

0

FtSH4 Mutation Increases the Peroxidase Levels Plant cells have evolved effective mechanisms to modulate steady-state levels of ROS inside and outside cells (reviewed by Mittler et  al., 2004). The increase in H2O2 levels in the ftsh4-4 mutant plants could be caused either by an increase in production enzymes or by a decrease in scavenger enzymes. However, the expression of the important H2O2 scavenger enzyme genes, CAT1, CSD1, and FDS1, showed no significant change at different growth stages of the ftsh4-4 mutant plants (Supplemental Figure  6). Interestingly, microarray data showed that the expression of a series of peroxidase genes, such as PRX37, PRX33, and PRX34, increased significantly in ftsh4-4 mutants compared with wild-type plants (Table  1 and Figure  8A). Furthermore, benzidine staining showed that the peroxidase isozyme content and activity increased significantly in the ftsh4-4 mutant plants (Figure 8B and Supplemental Figure  7). PRX33 and PRX34 play significant roles during the H2O2 generation process in Arabidopsis (Daudi et  al., 2012). In the present study, PRX33 or PRX34 knockdown not only clearly reduced the peroxidase isozyme level in ftsh4-4 mutants (Figure 8C), but also increased their final height (Figure  8C) and reduced the final number of axillary branches (Figure  8D). These data, combined with the increased expression of numerous peroxidase genes increased in ftsh4-4 mutants, implied that the peroxidases

10.5387

0.0648

play important roles in the FtSH4-dependent plant growth and development. Plants use various mechanisms to spatially and temporally regulate IAA concentration and gradients (auxin homeostasis). Besides de novo biosynthesis, degradation, and transport, another functional IAA catabolism type is IAA oxidation (Meudt and Gaines, 1967). In Arabidopsis, oxidation of IAA to 2-oxindole-3-acetic acid (oxIAA) is regarded as an important pathway for IAA degradation (Östin et al., 1998; Kowalczyk and Sandberg, 2001; Kai et al., 2007; Peer et al., 2013; Pencík et  al., 2013). Although the dioxygenase to auxin oxidation (DAO) gene has so far been identified to perform oxidation of IAA to oxIAA in rice (Zhao et al., 2013), the oxidative decarboxylation of IAA by plant peroxidases is thought to be a major degradation reaction involved in controlling the in vivo levels of IAA (Gazarian et al., 1996). The peroxidases also have an IAA oxidase activity (Savitsky et al., 1999) and so may control endogenous levels of IAA. Interestingly, IAA content increased significantly in the ftsh4-4 mutant plants by PRX34 and PRX33 knockdown expression (Figure 8E), indicating that the increased peroxidase level resulted in declined IAA content in the ftsh4-4 mutant plants. We further detected the content changes of exogenous stable deuterium labeled-IAA in the wild-type, ftsh4-4, FtSH4-ftsh4-4, prx33/prx34, and prx33/prx34/ftsh4-4 plants. The stable deuterium labeled-IAA

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Hydrogen peroxide catabolism genes

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(A) AsA can rescue the ftsh4-4 mutant plant branching phenotype. Eight-week-old plants were photographed, and these plants were sprayed by 1 mM AsA solution every 3 d from 3  weeks old for a further 2 weeks. There were 32 plants analyzed for each of three biological replicates. (B) The abundance of the FtSH4 gene transcripts was not affected by H2O2 or antioxidants. The 15-day-old seedlings treated by water with or without 1 mM H2O2, 1 mM AsA, or 1 mM GSH respectively for 3 h. AsA, ascorbic acid; GSH, reduced glutathione. (C) H2O2 concentration increased significantly in mas mutant plants from the third week onwards compared to wild-type plants. (D) H2O2 concentration increased significantly in the ftsh4-4 mutant plants, and restored to normal levels by FtSH4 expression. The 21-day-old plants were stained by DAB solution. Bar = 2 cm. Three technical replicates were performed for each of three biological replicates.

was degraded fastest in ftsh4-4 mutants, and slowest in prx33/prx34/ftsh4-4 mutants, compared to that in wild-type plants. Furthermore, the deuterium labeled-IAA degradation speed in the ftsh4-4 mutant would also be recovered by FtSH4 expression and delayed by PRX34 and PRX33 knockdown expression (Table 2). Based on these results, the catabolism of both endogenous and exogenous IAA could be regulated by peroxidases in the ftsh4-4 mutant.

IAA Restores the H2O2-Caused Auxin Deficiency in the ftsh4-4 Phenotype From the results above, we speculated that the lack of phenotypic variation in the young ftsh4-4 mutant plants may be due to low H2O2 levels. In order to verify this hypothesis, 2-dayold seedlings were transplanted into the H2O2 (0.1 mM) plates to grow for another 5 d. Shorter hypocotyls and hyponastic cotyledons in the ftsh4-4 mutant plants were observed under a low H2O2 concentration (0.1 mM) (Figure 9A and 9B), which is opposite to the characteristic auxin overproduction phenotypes observed in iaaM overexpression lines (Romano et al., 1995). Interestingly, these shortages could be reversed by a low concentration IAA (0.01  μM) treatment (Figure  9A and 9B). However, although the exogenous H2O2 could induce a high peroxidase level in wild-type plants, exogenous IAA did not decrease the high peroxidase level in the ftsh4-4 mutant

(Figure 9C). These results indicated that exogenous IAA could reverse the phenotypes of ftsh4-4 mutants by restoring the H2O2-caused auxin deficiency.

Discussion Auxin plays important roles in regulating plant apical dominance and shoot branching (reviewed by Shimizu-Sato et  al., 2009). The FtSH4 gene was expressed mainly at sites of auxin synthesis, transport, and accumulation, such as shoot apexes, vascular tissues, and branch outgrowth sites but weakly in the root (Figure 3) and corresponded with the dwarfism and multiple auxiliary branched phenotypes of ftsh4-4 mutants (Figure 1). Thus, we speculate that the FtSH4 gene may mediate auxin metabolism, transport, or signaling. The following observations support this hypothesis. First, the ftsh4-4 mutant plants had a reduced free IAA content (Figure 4B) and their dwarfism and increased axillary branching phenotypic characteristics could be reversed by endogenous IAA (Figure 4C) or exogenous NAA (Figure 4A and Supplemental Figure 5A). Second, ProDR5:GUS displayed a decreased expression level in the ftsh4-4 mutant inflorescence stems (Figure 5A). Third, the ftsh4-4 mutant calli were unable to regenerate shoots in the absence of exogenous auxin. Fourth, several auxin polar transport genes, including LAX2, an auxin influx carrier (Petrasek et al., 2006); PIN4 and

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Figure 7.  Exogenous Antioxidants Restore the Branching Phenotypes of ftsh4-4 Mutant Plants.

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Table 2.  Deuterium Labeled-IAA Content Changes in the ftsh4-4 Mutant Plants.

Plant genotype

[2H5]-IAA content (ng g–1 FW) 24 h after feeding

48 h after feeding

72 h after feeding

Col

2461.45 ± 193.59

1423.81 ± 183.50

625.70 ± 77.43

ftsh4-4

2530.83 ± 161.95

1142.15 ± 120.02

b

387.06 ± 26.50b

FtSH4-ftsh4-4

2513.13 ± 214.01

1486.22 ± 212.10

658.39 ± 144.77

prx33/prx34

2587.38 ± 188.84

1638.65 ± 175.37a

884.76 ± 64.83b

ftsh4-4/prx33/prx34

2557.45 ± 173.37

1297.47 ± 165.47

536.55 ± 84.32

a Means significantly different at P < 0.05, compared to Col. b Means significantly different at P < 0.01, compared to Col. Three technical replicates were performed for each of three biological replicates.

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Figure 8.  FtSH4 Regulates the Plant Development through Peroxidase Metabolism. (A)Real-time PCR for determining expression of the four peroxidase genes in wild-type and ftsh4-4 mutant plants. Four-week-old plants were analyzed, and three technical replicates were performed for each of three biological replicates. (B) Peroxidase isozyme content increased significantly in the ftsh4-4 mutant plants, but was reduced by PRX34 and PRX33 knockdown. Top: phenotypes of plants used for peroxidase assays; middle: PAGE assay and benzidine staining for peroxidase content of each plant shown in the top panel; bottom: Coomassie blue-stained gel for the peroxidase isozyme PAGE assay of the wild-type and mutant plants is shown. (C) PRX34 and PRX33 knockdown reduced the ftsh4-4 dwarfism. (D) PRX34 and PRX33 knockdown reduced the number of ftsh4-4 axillary branches. The 11-week-old plants were analyzed, and 32 plants measured for each of three biological replicates in (C) and (D). (E) IAA content increased significantly in the ftsh4-4 mutant plants by PRX34 and PRX33 knockdown expression. Four-week-old plants were analyzed, and three technical replicates were performed for each of three biological replicates.

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(A) A low concentration of H2O2 (0.1 mM) shortened the ftsh4-4 mutant plant hypocotyls (middle), and the low concentrations of IAA (0.01 μM) can reverse phenotype changes (bottom). The asterisk indicates significant differences compared with control plants (P < 0.005). (B) Hypocotyls length statistics of the seedlings in (A). (C) Exogenous IAA did not decrease the peroxidase level in the ftsh4-4 mutant. Five-week-old plants, sprayed with 1 mM H2O2 or 5 μM IAA every 3 d from 3 weeks old for a further 2 weeks, were analyzed.

PIN7, two auxin efflux carriers (Bainbridge et  al., 2008); and ABCB19/MDR1, an ATPase, also involved in auxin efflux transport (Lewis et  al., 2009), were all significantly reduced in the ftsh4-4 mutant plants (Table 1 and Supplemental Figure 8). The decline in expression of these genes may cause the lower IAA content in the ftsh4-4 mutant. Finally, a family of early response genes were significantly reduced in ftsh4-4 mutants (Figure 6, Table 1, Supplemental Tables 5, and Supplemental Figure 8, and Supplemental Figure 9A and 9B). Recent studies have shown that oxidative stress induces a broad spectrum of auxin-like effects, referred to as stressinduced morphological responses, in Arabidopsis seedlings with alterations in auxin levels and/or distribution (Pasternak et  al., 2005; Blomster et  al., 2011; reviewed in Leyser, 2005; Potters et al., 2009; Tognetti et al., 2012). The H2O2 content increased gradually with age in ftsh4-4 mutant plants except at the transition phase, where it increased rapidly, which coincided with a noticeable change in visible characteristics (Figure  1B). These results indicated that the H2O2 may play important roles in regulating the ftsh4-4 development. Redox-signaling pathways play important roles in modulating the plant development to adapt to their growth environment (Foyer and Noctor, 2005). In the GO-biological-process analysis of the microarray data, the obviously changed gene numbers that were related to oxidation reduction were counted: 35 were down-regulated and 50 were up-regulated (Supplemental Figure  9C), indicating that FtSH4 mutation affected the redox system significantly. Considering that the dwarfism and increased branching in ftsh4-4 mutants caused by auxin blocking could be partially reversed by the exogenous antioxidants AsA (Figure 7A), we proposed the existence

of a link between the redox system and auxin homeostasis in ftsh4-4 mutants. The expression of key IAA biosynthesis genes was not affected, but free IAA content decreased significantly, indicating that there should be another pathway to maintain the low IAA level in ftsh4-4 mutants. Plants use various mechanisms to spatially and temporally regulate IAA concentration and gradients (auxin homeostasis). Besides de novo biosynthesis, degradation, transport, and the synthesis and hydrolysis of two main groups of IAA conjugates (reviewed by Ljung et  al., 2002; Woodward and Bartel, 2005), another functional IAA catabolism type is IAA oxidation (Meudt and Gaines, 1967). The expression of IAA conjugate-related GH3 genes was not changed in ftsh4-4 mutants (Figure 6C), and the IAA-Asp and IAA-Ala content of ftsh4-4 mutants was similar to that of wild-type plants (Supplemental Table  6), implying that the IAA catabolism style in the ftsh4-4 mutant may be oxidation. Peroxidases have an indole-3-acetic acid (IAA) oxidase activity to play important roles in IAA oxidation (Savitsky et al., 1999) and IAA-Ile oxidative degraded (Park and Park, 1987). Hence, the oxidative decarboxylation of IAA by plant peroxidases is thought to be a major degradation reaction involved in controlling the in vivo level of IAA (Gazarian et al., 1996). Our results showed that peroxidase gene expressions were up-regulated and peroxidase isozyme levels increased significantly, which is consistent with the decline in free IAA levels found in the ftsh4-4 mutant plants (Figure  8), and IAA-Ile level was reduced obviously in the ftsh4-4 mutant (Supplemental Table  6), suggesting that IAA content may be regulated by peroxidase.

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Figure 9.  Exogenous IAA Reverses the H2O2 Effects on the ftsh4-4 Mutant Plants.

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arranged on the surface of the solid medium and were given a cold treatment at 4°C for 72 h. Seeds were germinated and seedlings were grown on a light shelf under a 16-h/8-h light/ dark cycle for 12 d, then the seedlings were transferred into well-watered potting mix (FAERDIGBLANDING SUBSTRATE, Pindstrup Inc., Denmark). Light was supplied by cool- and warm-white fluorescent bulbs, reaching an intensity approximately 100 μmol m–2 s–1 on the surface of the shelf. The double mutant lines in this study were created by cross-pollination between the relevant mutants. The mutant line ftsh4-1 (SALK_035107) was obtained for the Arabidopsis Biological Resource Center. Their genotypes were verified by resistance and PCR.

METHODS

Figure 10.  Simplified Model of FtSH4 Function.

Plant Materials and Growth Conditions Seeds of A. thaliana (Columbia 0 ecotype background) were sowed in a Petri dish containing sterile solid medium consisting of 1 MS salt, 1.5% sucrose, and 0.8% Phytagel (Sigma, St. Louis, MO) at pH 5.7. Seeds were first surface-sterilized and

Plant Treatments For the exogenous auxin treatment, 3-week-old plants spayed by 5  μM NAA every 4 d for a further 3 weeks, then the branching phenotype pictures of ftsh4-4 mutant were taken. For the exogenous AsA treatment, 1 mM AsA solution sprayed every 3 d onto the 3-week-old plant for a further 2 weeks, and the phenotype pictures of the 8-week-old ftsh4-4 mutant were taken. For the exogenous H2O2 treatment, 0.1 or 1 mM H2O2 with or without 0.01 μM IAA was added to the

Loss of FtSH4 function resulted in a reduction in protein stability in the mitochondria, and led to a H2O2 rise. High levels of H2O2 induced high levels of peroxidase production, which may function as an IAA oxidase. Increased peroxidase resulted in free IAA degradation and auxin transport and signaling perturbation, which promote the production of excessive axillary branches and dwarfism phenotypes. IM, inner membrane; OM, outer membrane; Prxs, peroxidases.

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In Arabidopsis thaliana, 44 out of 73 sequences encoding class III peroxidases have been reported to be putatively involved in the regulation of the cell elongation process through the catabolism of auxin in the cell wall (reviewed by Kawano, 2003). It is also generally accepted that the oxidation of IAA by peroxidases catalyzes the generation of ROS (Schopfer et  al., 2002). Our results show that PRX37, PRX33, and PRX34 were the most highly expressed peroxidase genes in the ftsh4-4 mutant plants (Table  1) and that peroxidase isozyme content also increased (Figure 8B). It has been shown that two highly homologous Arabidopsis peroxidases, PRX33 and PRX34, are involved in the reactions that promote cell elongation or regulate growth through auxin oxidase activity (Passardi et  al., 2006); and overexpression of PRX37 in Arabidopsis produced a dwarf phenotype with delayed development (Pedreira et  al., 2010). Knockdown of the expression of PER34 and PER33 restored the IAA level partially (Figure  8E), delayed the exogenous IAA degradation (Table 2), and reduced the peroxidase isozyme content, the number of axillary branches, and the extent of dwarfism of ftsh4-4 mutants (Figure 8C and 8D). These results indicate that ftsh4-4 growth and developmental defects may be due to the elevated peroxidase gene expression and peroxidase isozyme content which, in turn, would affect auxin-mediated growth. Overall, the work described here provides strong evidence that FtSH4 plays an important role in mediating the H2O2– auxin interaction. We propose a simplified model (Figure 10) showing that FtSH4 acts in the auxin pathway to regulate plant axillary branching by affecting auxin homeostasis. In this model, FtSH4-dependent protein assembly and/or stability inhibits H2O2 production, which in turn regulates IAA metabolism, transport, and auxin signaling. The increase in peroxidase enzyme concentration and peroxidase activity caused by the FtSH4 mutation may play a role in this regulation according to previous research, and the peroxidases may function as the direct mediators between ROS and auxin signaling interaction. To determine the precise mechanism of FtSH4 regulating IAA metabolism, transport, and signaling during plant development, it will be important to identify the direct target of FtSH4 protease and to understand how they are regulated. The targets of FtSH4 may be very complicated due to its mitochondrial membrane localization. In addition, the IAA oxidase activity of peroxidase (e.g. PRX33, PRX34, and PRX37) needs more detailed analysis and to be identified in Arabidopsis.

Zhang et al.  •  The Roles of FtSH4 Gene in Auxin Homeostasis   

Plasmid Constructs and Plant Transformation The promoter (2.0 kb before ATG) and the coding sequence (CDS) of FtSH4 were PCR-amplified from Arabidopsis genomic DNA and cDNA, respectively, and cloned into the T Cloning vector (Takara). All PCR amplifications were carried out with high-fidelity DNA polymerase (Pfu Ultra DNA polymerase). The sequence of the cloned promoter or CDS or promoter with CDS was verified by DNA sequencing and subcloned into pCAMBIA1303 and modified pBA002 binary vectors. For promoter analysis, the 2000-bp FtSH4 promoter was subcloned into pCAMBIA1303 and added to the front of the GUS gene to drive the expression of GUS gene (pFtSH4:GUS). For the complementation experiment (pFtSH4: FtSH4), the promoter of FtSH4 was used to drive the expression of FtSH4 CDS. Plants were transformed with Agrobacterium tumefaciens using the floral dipping method (Clough and Bent, 1998). The transformants were selected on agar plates containing 25 μg ml–1 hygomycin or 37.5 μg ml–1 Basta, and verified using PCR with construct-specific primers. All the transformed plants were selected for two more generations and homozygous transgenic plants (T3) were used for further characterization.

Genomic DNA Extraction and T-DNA Insertional Mutant Screening For isolation to the homozygote mutants, seeds were selected on MS medium supplemented with 50 mg l–1 kanamycin to select homozygous ftsh4-4 mutants. Homozygous T-DNA insertion mutants were identified using a PCR method. Genomic DNA was extracted using a quick CTAB method (Rogers and Bendich, 1988) and used for PCR reactions with the primers recommended in the SALK (Rosso et al., 2003) protocols.

Gene Expression Analysis Real-time PCR was performed as previously described (Zhang et al., 2013) and transcript data were normalized using UBQ10 gene as an internal control. Error bars were presented to indicate the standard error of the mean. All experiments were performed with three replicates. The primers used in this paper for quantitative RT–PCR are listed in Supplemental Table 7.

Histochemical Analyses The histochemical staining of H2O2 was performed as previously described (Jabs et  al., 1996) with minor modifications. In the case of H2O2, leaf were vacuum infiltrated with 1 mg ml–1 DAB in 50 mM Tris-acetate (pH 3.8) and incubated at 25ºC in dark for 24 h. Leaf discs were rinsed in 80% (v/v) ethanol for 10 min at 70ºC, mounted in lactic acid/phenol/water (1:1:1; v/v), and photographed. Histochemical detection of GUS activity was performed as previously described (Zhang et al., 2009). Fluorometric determination of GUS activity was done using 4-MUG (Sigma-Aldrich) as substrate (Jefferson et al., 1987).

Determination of IAA Concentrations Col-0 and ftsh4-4 plants were grown on MS agar plates under long-day conditions. Two weeks after germination, seedlings were transplanted to grown in soil for a further 2 weeks, then shoot tissue were pooled and frozen in liquid nitrogen for quantification of auxin content. Three replicates were analyzed for each sample under the same conditions. IAA was extracted and purified as described (Andersen et  al., 2008). After methylation, the samples were trimethyl-silylated and analyzed by gas chromatography–selected reaction monitoring–mass spectrometry (Ljung et al., 2005). For the [2H5]-IAA measurement, 200 mg of fresh plant tissue were homogenized in 80% methanol/H2O (1 ml), centrifuged at 15 200 g for 10 min, and the supernatant was evaporated in a Jouan RCT-60. The [2H5]-IAA fraction was re[-dissolved in 0.1 mol l–1 sodium phosphate solution (pH 7.8, 200 μl) and subjected to a Sep-Pak C18 column (Waters). The column was washed with H2O (2 ml). [2H5]-IAA was eluted with 80% methanol (1.5 ml) and evaporated to dryness again. 2 H5 -IAA was analyzed by LC–ESI–MS/MS (LC20AD-MS8030 plus system, Shimadzu). The chromatography was performed on an Acquity UPLC BEH C18 column (2.1 × 100 mm id, 1.7  μm, Waters). The [2H5]-IAA fraction was re-dissolved in 10% methanol/H2O (50 μl) and injected with 5 μl to HPLC; the column over temperature was set at 40°C. Elution of the samples was carried out with 0.05% aqueous acetic acid (solvent A) and methanol (solvent B), and a gradient mode ((min/%/%) for 0/90/10, 3/20/80; 5.0/20/80, 6.0/90/10) at a flow rate of 0.25 ml min–1. MS/MS conditions were as follows: collision energy –18.0 eV, mass-to-charge ratio 181/134.1. The linear dynamic range was determined by injecting a standard fraction of [2H5]-IAA in a wide concentration range.

Measurement of Hydrogen Peroxide Content H2O2 contents were determined by the POD-coupled assay protocols. About 0.1 g Arabidopsis leaves were ground in liquid N2 and the powder was extracted in 2 ml 1 M HClO4 in the presence of insoluble polyvinylpyrrolidone (5%). The homogenate was centrifuged at 12 000 g for 10 min, and the supernatant was neutralized with 5 M K2CO3 to pH 5.6 in the presence of 100 μl 0.3 M phosphate buffer, pH 5.6. The solution was centrifuged at 12 000 g for 1 min, and the sample was

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MS medium plates, then the seedlings, which germinated for 2 d on MS medium, were transplanted into the H2O2 plates to grow for a further 5 d, then the phenotypes photos were taken. For the effects of exogenous phytohormones on the FtSH4 expression, the 15-day-old seedlings treated by water with or without 10 μM NAA, 6-BA, or 2,4-D, respectively, for 4 h. For the effects of the H2O2 or antioxidants on the FtSH4 expression, the 15-day-old seedlings treated by water with or without 1 mM H2O2, 1 mM AsA, or 1 mM GSH, respectively, for 3 h. For the [2H5]-IAA feeding experiment, the 18-day-old seedlings were fed with 100 μM [2H5]-IAA (CAS 76937–78–5, Olchemim LTD, Czech Republic) by root absorption for 6 h. Then the seedlings were transferred from the medium containing [2H5]-IAA, and the aboveground part were used for [2H5]-IAA detection.

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incubated for 10 min with 1 unit ascorbate oxidase to oxidize ascorbate prior to assay. The reaction mixture was composed of 0.1 M phosphate buffer, pH 6.5, 3.3 mM 3-(dimethylamino) benzoic acid, 0.07 mM 3-methyl-2-benzothiazoline hydrazone, and 0.3 units peroxidase. The reaction was initiated by addition of 200 μl sample. The absorbance change at 590 nm was monitored at 25ºC.

Peroxidase Isozyme Native-PAGE Analyses

Peroxidase Activity Assay Peroxidase activity was assayed in a reaction mixture consisting of 100 mM potassium phosphate buffer (pH 6.5), 15 mM guaiacol, 0.05% (v/v) H2O2, and 60 μl enzyme extract diluted between 1:40 and 1:80 (v/v) with assay buffer. The reaction was initiated by adding H2O2, and the oxidation of guaiacol was determined based on the increase in A470. One peroxidase unit is defined as the amount of enzyme that produces 1 μmol min–1 oxidized guaiacol under the above assay conditions.

Analysis of FtSH4 Protein Total protein of 3-week-old plants were extracted and separated by SDS–PAGE. The gel blot was probed with the FtSH4 antibody (Agrisera, www.agrisera.com) and detected using ECL plus (Amersham Pharmacia, www.gelifesciences.com).

Callus Induction and Plant Regeneration For callus formation and plant regeneration, different inflorescence stem part explants were harvested from 6-week-old wild-type or ftsh4-4 mutant plants were used for callus induction for 3 weeks on MS medium supplemented with 2.2 μM 2,4-D. Then, calli were transferred to the MS-modified regeneration media supplemented with 10 μM 6-BA, but without IAA and incubated under the same light conditions as previously described. After two additional weeks, regenerated shoot meristems were observed.

Accession Numbers Sequence data from this article can be found in the Arabidopsis Information Resource database under the following accession numbers: FtSH4 (At2g26140), PRX33 (At3g49110), PRX34 (At3g49120). Gene IDs for genes used for real-time quantitative PCR can be found in Supplemental Table 7.

Microarray Analyses Total RNA of the experimental (4-week-old ftsh4-4 mutant) and reference (wild-type) was extracted with the TRIzol reagent (Invitrogen) and purified with a NucleoSpin® RNA

SUPPLEMENTARY DATA Supplementary Data are available at Molecular Plant Online.

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One gram of 5-week-old Arabidopsis seedlings were ground in liquid N2 and the powder was extracted in 5 ml 0.02 M KH2PO4 in the presence of insoluble polyvinylpyrrolidone (10%) at 4ºC for 1 h. The homogenate was filtered, and then centrifuged at 3000 g for 10 min, and the supernatant was the peroxidase isozymes solution and collected for analysis. About 20 μl peroxidase isozymes sample was separated by the native-PAGE under reducing conditions. The gel was stained by the ascorbic acidbenzidine solution for 5 min, and then washed using water four times. The 50-ml staining mixture solution was composed of 0.45 ml acetic acid, 0.056 g benzidine, 1 ml 3% hydrogen peroxide, and the remaining was water. Two blue peroxidase bands would be appeared by this stained method in Arabidopsis, and the larger band was chosen as the comparable marker.

clean-up Kit (MACHEREY-NAGEL, Germany) following the manufacturer’s instructions. The Arabidopsis genome-wide long oligonucleotide microarray was constructed in-house at CapitalBio Corporation (Beijing, China). Briefly, 5′-aminomodified 70-mer probes representing 26 173 A.  thaliana genes from the Arabidopsis Genome Oligo Set Version 3.0 (Operon), and internal and external controls were printed on amino silaned glass slides using a SmartArray microarrayer (CapitalBio Corp.). Fluorescent-labeled DNA (Cy3 and Cy5-dCTP) was produced through Eberwine’s linear RNA amplification method and subsequent enzymatic reaction. Briefly, double-stranded cDNA containing T7 RNA polymerase promoter sequence was synthesized with 1  μg of total RNA using Reverse Transcription System, RNase H, DNA polymerase I, and T4 DNA polymerase, according to the manufacturer’s recommended protocol (CapitalBio). The resulting labeled DNA (labeled control and test samples) was quantitatively adjusted based on the efficiency of Cy-dye incorporation and mixed into 80 μl hybridization solution (3 SSC, 0.2% SDS, 25% formamide, and 5 Denhart’s solution). Individually labeled cRNAs were not pooled before hybridization. DNA in hybridization solution was denatured at 95ºC for 3 min prior to loading onto a microarray. Arrays were hybridized was performed in a CapitalBio BioMixerTM II Hybridization Station overnight at a rotation speed of 8 r.p.m. and a temperature of 42ºC and washed with two consecutive solutions (0.2% SDS, 2 SSC at 42ºC for 5 min, and 0.2 SSC for 5 min at room temperature). Arrays were scanned with a confocal LuxScan 10K-A scanner and the images obtained were then analyzed using LuxScanTM 3.0 software (both from CapitalBio). For individual channel data extracts, faint spots were removed for which intensities were below 400 units after background subtraction in both channels (Cy3 and Cy5). A  space- and intensity-dependent normalization based on a LOWESS program was employed. To determine the significant differentially expressed genes, Significance Analysis of Microarrays were performed using one class comparison in the Significant Analysis of Microarray software (SAM, version 3.02). Genes were determined to be significantly differentially expressed with a selection threshold of false discovery rate, FDR <5%, and fold change >2.0 in the SAM output result (N (biological replicates) = 3). Significant differentially expressed genes are shown in Supplemental Table 5.

Zhang et al.  •  The Roles of FtSH4 Gene in Auxin Homeostasis   

FUNDING This work is supported by the National Natural Science Foundation of China (31370350, 31271471, 91317312), the Guangzhou Pearl River new star project for science and technology (2012J2200033), the Education Department of Guangdong Province (2012CXZD0019), and the Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (2010).

Acknowledgments

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We thank Jie Le for providing the iaaM vector and Zhigang Huang and Langtao Xiao for their help on IAA measurement. We also thank Professor Frederick M. Ausubel from the Harvard Medical School for providing the prx34 and prx33 prx34 mutant seeds. No conflict of interest declared.

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