Rapid synthesis of auxin via a new tryptophan-dependent pathway is required for shade avoidance in plants.

Plants grown at high densities perceive a decrease in the red to far-red (R:FR) ratio of incoming light, resulting from absorption of red light by canopy leaves and reflection of far-red light from neighboring plants. These changes in light quality trigger a series of responses known collectively as the shade avoidance syndrome. During shade avoidance, stems elongate at the expense of leaf and storage organ expansion, branching is inhibited, and flowering is accelerated. We identified several loci in Arabidopsis, mutations in which lead to plants defective in multiple shade avoidance responses. Here we describe TAA1, an aminotransferase, and show that TAA1 catalyzes the formation of indole-3-pyruvic acid (IPA) from L-tryptophan (L-Trp), the first step in a previously proposed, but uncharacterized, auxin biosynthetic pathway. This pathway is rapidly deployed to synthesize auxin at the high levels required to initiate the multiple changes in body plan associated with shade avoidance.

Tip growth defective1 interacts with cellulose synthase A3 to regulate cellulose biosynthesis in Arabidopsis.

KEY MESSAGE: AtTIP1 physically and genetically interacts with AtCESA3. AtCESA3 undergoes S-acylation, possibly mediated by AtTIP1, suggesting a specific role of AtTIP1 in cellulose biosynthesis and plant development. S-acylation is a reversible post-translational lipid modification of proteins catalyzed by protein S-acyl transferases (PATs). S-acylation is important for various biological molecular mechanisms including cellulose biosynthesis. Cellulose is synthesized by the cellulose synthase A (CESA) complexes (CSCs) at the plasma membrane. However, specific PAT involving in cellulose biosynthesis has not been identified and the precise mechanism by which PAT regulates the CESAs is largely unknown. Here, we report isolation of tip1-5, an allele of Tip Growth Defective1 (AtTIP1/AtPAT24) with a premature stop codon. tip1-5 genetically interacts with ixr1-2, a point mutant of AtCESA3 which encodes a catalytic subunit of CSC synthesizing primary wall cellulose. We show that AtTIP1 physically interacts with AtCESA3. AtCESA3 undergoes S-acylation, which is possibly mediated by AtTIP1, suggesting a functional relationship between AtTIP1 and AtCESA3. Moreover, the interfascicular fiber cells in the primary inflorescence stems of tip1-5 ixr1-2 double mutant contain thinner cell walls and significantly less crystalline cellulose compared to the single mutants. These results highlight the positive regulation of AtTIP1 in cellulose biosynthesis, and a specific role of AtPAT in plant development.

The ESCRT-I components VPS28A and VPS28B are essential for auxin-mediated plant development.

The highly conserved endosomal sorting complex required for transport (ESCRT) pathway plays critical roles in endosomal sorting of ubiquitinated plasma membrane proteins for degradation. However, the functions of many components of the ESCRT machinery in plants remain unsolved. Here we show that the ESCRT-I subunits VPS28A and VPS28B are functionally redundant and required for embryonic development in Arabidopsis. We conducted a screen for genetic enhancers of pid, which is defective in auxin signaling and transport. We isolated a no--cotyledon in pid 104 (ncp104) mutant, which failed to develop cotyledons in a pid background. We discovered that ncp104 was a unique recessive gain-of-function allele of vps28a. VPS28A and VPS28B were expressed during embryogenesis and the proteins were localized to the trans-Golgi network/early endosome and post-Golgi/endosomal compartments, consistent with their functions in endosomal sorting and embryogenesis. The single vps28a and vps28b loss-of-function mutants did not display obvious developmental defects, but their double mutants showed abnormal cell division patterns and were arrested at the globular embryo stage. The vps28a vps28b double mutants showed altered auxin responses, disrupted PIN1-GFP expression patterns, and abnormal PIN1-GFP accumulation in small aberrant vacuoles. The ncp104 mutation may cause the VPS28A protein to become unstable and/or toxic. Taken together, our findings demonstrate that the ESCRT-I components VPS28A and VPS28B redundantly play essential roles in vacuole formation, endosomal sorting of plasma membrane proteins, and auxin-mediated plant development.

NCP2/RHD4/SAC7, SAC6 and SAC8 phosphoinositide phosphatases are required for PtdIns4P and PtdIns(4,5)P2 homeostasis and Arabidopsis development.

Phosphoinositides play important roles in plant growth and development. Several SAC domain phosphoinositide phosphatases have been reported to be important for plant development. Here, we show functional analysis of SUPPRESSOR OF ACTIN 6 (SAC6) to SAC8 in Arabidopsis, a subfamily of phosphoinositide phosphatases containing SAC-domain and two transmembrane motifs. We isolated an Arabidopsis mutant ncp2 that lacked cotyledons in seedling and embryo in pid, a background defective in auxin signaling and transport. NCP2 encodes RHD4/SAC7 phosphoinositide phosphatase. SAC6, SAC7 and SAC8 exhibit overlapping and specific expression patterns in seedling and embryo. The sac6 sac7 embryos either fail to develop into seeds, or have three or four cotyledons. The embryo development of sac7 sac8 and sac6 sac7 sac8 mutants is significantly delayed or lethal, and the seedlings are arrested at early stages. Auxin maxima are decreased in double and triple sac mutants. The contents of PtdIns4P and PtdIns(4,5)P2 in sac6 sac7 and sac7 sac8 mutants are dramatically increased. Protein trafficking of the plasma membrane (PM)-localized protein PIN1 and PIN2 from trans-Golgi network/early endosome back to PM is delayed in sac7 sac8 mutants. These results indicate that SAC6-SAC8 are essential for maintaining homeostasis of PtdIns4P and PtdIns(4,5)P2, and auxin-mediated development in Arabidopsis.

AtMOB1 Genes Regulate Jasmonate Accumulation and Plant Development.

The MOB1 proteins are highly conserved in yeasts, animals, and plants. Previously, we showed that the Arabidopsis (Arabidopsis thaliana) MOB1A gene (AtMOB1A/NCP1) plays critical roles in auxin-mediated plant development. Here, we report that AtMOB1A and AtMOB1B redundantly and negatively regulate jasmonate (JA) accumulation and function in Arabidopsis development. The two MOB1 genes exhibited similar expression patterns, and the MOB1 proteins displayed similar subcellular localizations and physically interacted in vivo. Furthermore, the atmob1a atmob1b (mob1a/1b) double mutant displayed severe developmental defects, which were much stronger than those of either single mutant. Interestingly, many JA-related genes were up-regulated in mob1a/1b, suggesting that AtMOB1A and AtMOB1B negatively regulate the JA pathways. mob1a/1b plants accumulated more JA and were hypersensitive to exogenous JA treatments. Disruption of MYC2, a key gene in JA signaling, in the mob1a/1b background partially alleviated the root defects and JA hypersensitivity observed in mob1a/1b. Moreover, the expression levels of the MYC2-repressed genes PLT1 and PLT2 were significantly decreased in the mob1a/1b double mutant. Our results showed that MOB1A/1B genetically interact with SIK1 and antagonistically modulate JA-related gene expression. Taken together, our findings indicate that AtMOB1A and AtMOB1B play important roles in regulating JA accumulation and Arabidopsis development.

Modulation of Auxin Signaling and Development by Polyadenylation Machinery.

Polyadenylation influences gene expression by affecting mRNA stability, transport, and translatability. Here, we report that Cleavage stimulation Factor 77 (AtCstF77), a component of the pre-mRNA 3'-end polyadenylation machinery, affects polyadenylation site (PAS) selection in transcripts of some auxin signaling genes in Arabidopsis (Arabidopsis thaliana). Disruption of AtCstF77 reduced auxin sensitivity and decreased the expression of the auxin reporter DR5-GFP Null mutations of cstf77 caused severe developmental defects, but were not lethal as previously reported. cstf77-2 genetically interacted with transport inhibitor response 1 auxin signaling f-box 2 auxin receptor double mutants, further supporting that polyadenylation affects auxin signaling. AtCstF77 was ubiquitously expressed in embryos, seedlings, and adult plants. The AtCstF77 protein was localized in the nucleus, which is consistent with its function in pre-mRNA processing. We observed that PASs in transcripts from approximately 2,400 genes were shifted in the cstf77-2 mutant. Moreover, most of the PAS shifts were from proximal to distal sites. Auxin treatment also caused PAS shifts in transcripts from a small number of genes. Several auxin signaling or homeostasis genes had different PASs in their transcripts in the cstf77-2 mutant. The expression levels of AUXIN RESISTANT 2/INDOLE-3-ACETIC ACID 7 were significantly increased in the cstf77-2 mutant, which can partially account for the auxin resistance phenotype of this mutant. Our results demonstrate that AtCstF77 plays pleiotropic and critical roles in Arabidopsis development. Moreover, disruption of AtCstF64, another component of the polyadenylation machinery, led to developmental defects and reduced auxin response, similar to those of the cstf77-2 mutant. We conclude that AtCstF77 affects auxin responses, likely by controlling PAS selection of transcripts of some auxin signaling components.

NCP1/AtMOB1A Plays Key Roles in Auxin-Mediated Arabidopsis Development.

MOB1 protein is a core component of the Hippo signaling pathway in animals where it is involved in controlling tissue growth and tumor suppression. Plant MOB1 proteins display high sequence homology to animal MOB1 proteins, but little is known regarding their role in plant growth and development. Herein we report the critical roles of Arabidopsis MOB1 (AtMOB1A) in auxin-mediated development in Arabidopsis. We found that loss-of-function mutations in AtMOB1A completely eliminated the formation of cotyledons when combined with mutations in PINOID (PID), which encodes a Ser/Thr protein kinase that participates in auxin signaling and transport. We showed that atmob1a was fully rescued by its Drosophila counterpart, suggesting functional conservation. The atmob1a pid double mutants phenocopied several well-characterized mutant combinations that are defective in auxin biosynthesis or transport. Moreover, we demonstrated that atmob1a greatly enhanced several other known auxin mutants, suggesting that AtMOB1A plays a key role in auxin-mediated plant development. The atmob1a single mutant displayed defects in early embryogenesis and had shorter root and smaller flowers than wild type plants. AtMOB1A is uniformly expressed in embryos and suspensor cells during embryogenesis, consistent with its role in embryo development. AtMOB1A protein is localized to nucleus, cytoplasm, and associated to plasma membrane, suggesting that it plays roles in these subcellular localizations. Furthermore, we showed that disruption of AtMOB1A led to a reduced sensitivity to exogenous auxin. Our results demonstrated that AtMOB1A plays an important role in Arabidopsis development by promoting auxin signaling.

The jasmonic acid signaling pathway is linked to auxin homeostasis through the modulation of YUCCA8 and YUCCA9 gene expression.

Interactions between phytohormones play important roles in the regulation of plant growth and development, but knowledge of the networks controlling hormonal relationships, such as between oxylipins and auxins, is just emerging. Here, we report the transcriptional regulation of two Arabidopsis YUCCA genes, YUC8 and YUC9, by oxylipins. Similar to previously characterized YUCCA family members, we show that both YUC8 and YUC9 are involved in auxin biosynthesis, as demonstrated by the increased auxin contents and auxin-dependent phenotypes displayed by gain-of-function mutants as well as the significantly decreased indole-3-acetic acid (IAA) levels in yuc8 and yuc8/9 knockout lines. Gene expression data obtained by qPCR analysis and microscopic examination of promoter-reporter lines reveal an oxylipin-mediated regulation of YUC9 expression that is dependent on the COI1 signal transduction pathway. In support of these findings, the roots of the analyzed yuc knockout mutants displayed a reduced response to methyl jasmonate (MeJA). The similar response of the yuc8 and yuc9 mutants to MeJA in cotyledons and hypocotyls suggests functional overlap of YUC8 and YUC9 in aerial tissues, while their function in roots shows some specificity, probably in part related to different spatio-temporal expression patterns of the two genes. These results provide evidence for an intimate functional relationship between oxylipin signaling and auxin homeostasis.

Auxin overproduction in shoots cannot rescue auxin deficiencies in Arabidopsis roots.

Auxin plays an essential role in root development. It has been a long-held dogma that auxin required for root development is mainly transported from shoots into roots by polarly localized auxin transporters. However, it is known that auxin is also synthesized in roots. Here we demonstrate that a group of YUCCA (YUC) genes, which encode the rate-limiting enzymes for auxin biosynthesis, plays an essential role in Arabidopsis root development. Five YUC genes (YUC3, YUC5, YUC7, YUC8 and YUC9) display distinct expression patterns during root development. Simultaneous inactivation of the five YUC genes (yucQ mutants) leads to the development of very short and agravitropic primary roots. The yucQ phenotypes are rescued by either adding 5 nM of the natural auxin, IAA, in the growth media or by expressing a YUC gene in the roots of yucQ. Interestingly, overexpression of a YUC gene in shoots in yucQ causes the characteristic auxin overproduction phenotypes in shoots; however, the root defects of yucQ are not rescued. Our data demonstrate that localized auxin biosynthesis in roots is required for normal root development and that auxin transported from shoots is not sufficient for supporting root elongation and root gravitropic responses.

Pattern of auxin and cytokinin responses for shoot meristem induction results from the regulation of cytokinin biosynthesis by AUXIN RESPONSE FACTOR3.

De novo organ regeneration is an excellent biological system for the study of fundamental questions regarding stem cell initiation, cell fate determination, and hormone signaling. Despite the general belief that auxin and cytokinin responses interact to regulate de novo organ regeneration, the molecular mechanisms underlying such a cross talk are little understood. Here, we show that spatiotemporal biosynthesis and polar transport resulted in local auxin distribution in Arabidopsis (Arabidopsis thaliana), which in turn determined the cytokinin response during de novo shoot regeneration. Genetic and pharmacological interference of auxin distribution disrupted the cytokinin response and ATP/ADP ISOPENTENYLTRANSFERASE5 (AtIPT5) expression, affecting stem cell initiation and meristem formation. Transcriptomic data suggested that AUXIN RESPONSE FACTOR3 (ARF3) mediated the auxin response during de novo organ regeneration. Indeed, mutations in ARF3 caused ectopic cytokinin biosynthesis via the misexpression of AtIPT5, and this disrupted organ regeneration. We further showed that ARF3 directly bound to the promoter of AtIPT5 and negatively regulated AtIPT5 expression. The results from this study thus revealed an auxin-cytokinin cross talk mechanism involving distinct intermediate signaling components required for de novo stem cell initiation and shed new light on the mechanisms of organogenesis in planta.

Conversion of tryptophan to indole-3-acetic acid by TRYPTOPHAN AMINOTRANSFERASES OF ARABIDOPSIS and YUCCAs in Arabidopsis.

Auxin is an essential hormone, but its biosynthetic routes in plants have not been fully defined. In this paper, we show that the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) family of amino transferases converts tryptophan to indole-3-pyruvate (IPA) and that the YUCCA (YUC) family of flavin monooxygenases participates in converting IPA to indole-3-acetic acid, the main auxin in plants. Both the YUCs and the TAAs have been shown to play essential roles in auxin biosynthesis, but it has been suggested that they participate in two independent pathways. Here, we show that all of the taa mutant phenotypes, including defects in shade avoidance, root resistance to ethylene and N-1-naphthylphthalamic acid (NPA), are phenocopied by inactivating YUC genes. On the other hand, we show that the taa mutants in several known auxin mutant backgrounds, including pid and npy1, mimic all of the well-characterized developmental defects caused by combining yuc mutants with the auxin mutants. Furthermore, we show that overexpression of YUC1 partially suppresses the shade avoidance defects of taa1 and the sterile phenotypes of the weak but not the strong taa mutants. In addition, we discovered that the auxin overproduction phenotypes of YUC overexpression lines are dependent on active TAA genes. Our genetic data show that YUC and TAA work in the same pathway and that YUC is downstream of TAA. The yuc mutants accumulate IPA, and the taa mutants are partially IPA-deficient, indicating that TAAs are responsible for converting tryptophan to IPA, whereas YUCs play an important role in converting IPA to indole-3-acetic acid.

REVEILLE1, a Myb-like transcription factor, integrates the circadian clock and auxin pathways.

The circadian clock modulates expression of a large fraction of the Arabidopsis genome and affects many aspects of plant growth and development. We have discovered one way in which the circadian system regulates hormone signaling, identifying a node that links the clock and auxin networks. Auxin plays key roles in development and responses to environmental cues, in part through regulation of plant growth. We have characterized REVEILLE1 (RVE1), a Myb-like, clock-regulated transcription factor that is homologous to the central clock genes CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). Despite this homology, inactivation of RVE1 does not affect circadian rhythmicity but instead causes a growth phenotype, indicating this factor is a clock output affecting plant development. CCA1 regulates growth via the bHLH transcription factors PHYTOCHROME INTERACTING FACTOR4 (PIF4) and PIF5, but RVE1 acts independently of these genes. RVE1 instead controls auxin levels, promoting free auxin production during the day but having no effect during the night. RVE1 positively regulates the expression of the auxin biosynthetic gene YUCCA8 (YUC8), providing a mechanism for its growth-promoting effects. RVE1 is therefore a node that connects two important signaling networks that coordinate plant growth with rhythmic changes in the environment.

NPY genes and AGC kinases define two key steps in auxin-mediated organogenesis in Arabidopsis.

Auxin is an essential regulator of plant organogenesis. Most key genes in auxin biosynthesis, transport, and signaling belong to gene families, making it difficult to conduct genetic analysis of auxin action in plant development. Herein we report the functional analysis of several members of 2 gene families (NPY/ENP/MAB4 genes and AGC kinases) in auxin-mediated organogenesis and their relationships with the YUC family of flavin monooxygenases that are essential for auxin biosynthesis. We show that 5 NPY genes (NPY1 to NPY5) and 4 AGC kinases (PID, PID2, WAG1, and WAG2) have distinct, yet overlapping, expression patterns. Disruption of NPY1 does not cause obvious defects in organogenesis, but npy1 npy3 npy5 triple mutants failed to make flower primordia, a phenotype that is also observed when AGC kinase PID is compromised. Inactivation of YUC1 and YUC4 in npy1 background also phenocopies npy1 npy3 npy5 and pid. Simultaneous disruption of PID and its 3 closest homologs (PID2, WAG1, and WAG2) completely abolishes the formation of cotyledons, which phenocopies npy1 pid double mutants and yuc1 yuc4 pid triple mutants. Our results demonstrate that NPY genes and AGC kinases define 2 key steps in a pathway that controls YUC-mediated organogenesis in Arabidopsis.

Effect of combined arsenic and lead exposure on their uptake and translocation in Indian mustard.

Phytoremediation makes use of hyperaccumulating plants to remove potentially toxic elements (PTEs) from soil selectively. Most researches examining hyperaccumulators focused on how they act on a single PTE contaminant. However, there is more than one kind of PTEs in most contaminated soils. Phytoremediation approaches could be less effective in environments containing multiple PTEs contaminants. Here we examine arsenic (As) and lead (Pb) accumulation in Indian Mustard (Brassica juncea) from solutions with one or both pollutants. Indian mustard accumulates As or Pb when exposed in the single liquid exposure of As or Pb, and the highest concentrations of As and Pb in Indian Mustard reach 1,786 mg/kg and 47,200 mg/kg, respectively. But the absorption efficiencies of As and Pb decrease (by >90% for As, and ∼10-30% for Pb) when both As and Pb are present. The translocation of As and Pb from the root to leaf is also impeded by 36%-88% for As and 55-85% for Pb when treated with both PTEs. In As and Pb co-treatment, significant negative correlations between As (V) and P and between Pb and other elements (including K, Mg and Ca) were found in Indian mustard. X-ray absorption near edge (XANES) spectroscopy and subcellular extraction experiments indicate that much of the accumulated Pb bound within lead phosphate particles, and often located within the cell wall. Pb could decrease the percentage of water-soluble As and increase protein combined As in subcellular levels within Indian mustard. Based on these data, we suggest that the competition between Pb and monovalent and divalent nutrients (e.g., Ca(II), Mg(II) and K(I)), and the formation of lead phosphates within cell walls play critical roles in decreasing As and Pb co-uptake efficiencies for Indian mustard.

NPY1, a BTB-NPH3-like protein, plays a critical role in auxin-regulated organogenesis in Arabidopsis.

Auxin is an essential regulator for plant development. To elucidate the mechanisms by which auxin regulates plant development, we isolated an Arabidopsis mutant naked pins in yuc mutants 1 (npy1) that develops pin-like inflorescences and fails to initiate any flowers in yuc1 yuc4, a background that is defective in auxin biosynthesis. The phenotypes of npy1 yuc1 yuc4 triple mutants closely resemble those of Arabidopsis mutants pin-formed1 (pin1), pinoid (pid), and monopteros (mp), which are defective in either auxin transport or auxin signaling. NPY1 belongs to a large family of proteins and is homologous to NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3), a BTB/POZ protein that regulates phototropic responses along with the protein kinase PHOT1 (Phototropin 1). We demonstrate that NPY1 works with the protein kinase PID, which is homologous to PHOT1, to regulate auxin-mediated plant development. The npy1 pid double mutants fail to form any cotyledons, a phenotype that is also observed in yuc1 yuc4 pid triple mutants. Interestingly, both auxin-regulated organogenesis and phototropic responses require an auxin response factor (ARF). Disruption of ARF7/NPH4 leads to nonphototropic hypocotyls and arf5/mp forms pin-like inflorescences. Whereas the PHOT1/NPH3 pathway is regulated by light, our data suggest that the PID/NPY1 pathway may be regulated by auxin synthesized by the YUC flavin monooxygenases. Our findings put YUCs, PID, and NPY1 into a genetic framework for further dissecting the mechanisms of auxin action in plant development.

Arabidopsis AGC protein kinases IREH1 and IRE3 control root skewing.

AGC protein kinases play important roles in plant growth and development. Several AGC kinases in Arabidopsis have been functionally characterized. However, the "AGC Other" subfamily, including IRE, IREH1, IRE3 and IRE4, has not been well understood. Here, we reported that ireh1 mutants displayed a root skewing phenotype, which can be enhanced by ire3 mutation. IREH1 and IRE3 were expressed in roots, consistent with their function in controlling root skewing. The fluorescence intensities of the microtubule marker KNpro:EGFP-MBD were decreased in ireh1, ire3 and ireh1 ire3 mutants compared to wild type. The microtubule arrangements in ireh1 and ireh1 ire3 mutants were also altered. IREH1 physically interacted with IRE3 in vitro and in planta. Thus, our findings demonstrate that IREH1 and IRE3 protein kinases play important roles in controlling root skewing, and maintaining microtubule network in Arabidopsis.

Possible Interactions between the Biosynthetic Pathways of Indole Glucosinolate and Auxin.

Glucosinolates (GLS) are a group of plant secondary metabolites mainly found in Cruciferous plants, share a core structure consisting of a ß-thioglucose moiety and a sulfonated oxime, but differ by a variable side chain derived from one of the several amino acids. These compounds are hydrolyzed upon cell damage by thioglucosidase (myrosinase), and the resulting degradation products are toxic to many pathogens and herbivores. Human beings use these compounds as flavor compounds, anti-carcinogens, and bio-pesticides. GLS metabolism is complexly linked to auxin homeostasis. Indole GLS contributes to auxin biosynthesis via metabolic intermediates indole-3-acetaldoxime (IAOx) and indole-3-acetonitrile (IAN). IAOx is proposed to be a metabolic branch point for biosynthesis of indole GLS, IAA, and camalexin. Interruption of metabolic channeling of IAOx into indole GLS leads to high-auxin production in GLS mutants. IAN is also produced as a hydrolyzed product of indole GLS and metabolized to IAA by nitrilases. In this review, we will discuss current knowledge on involvement of GLS in auxin homeostasis.

NPY genes play an essential role in root gravitropic responses in Arabidopsis.

Plants can sense the direction of gravity and orient their growth to ensure that roots are anchored in soil and that shoots grow upward. Gravitropism has been studied extensively using Arabidopsis genetics, but the exact mechanisms for gravitropism are not fully understood. Here, we demonstrate that five NPY genes play a key role in Arabidopsis root gravitropism. NPY genes were previously identified as regulators of auxin-mediated organogenesis in a genetic pathway with the AGC kinases PID, PID2, WAG1, and WAG2. We show that all five NPY genes are highly expressed in primary root tips. The single npy mutants do not display obvious gravitropism defects, but the npy1 npy2 npy3 npy4 npy5 quintuple mutants show dramatic gravitropic phenotypes. Systematic analysis of all the npy double, triple, and quadruple combinations demonstrates that the five NPY genes all contribute to gravitropism. Our work indicates that gravitropism, phototropism, and organogenesis use analogous mechanisms in which at least one AGC kinase, one NPH3/NPY gene, and one ARF are required.

An allelic mutant series of ATM3 reveals its key role in the biogenesis of cytosolic iron-sulfur proteins in Arabidopsis.

The ATP-binding cassette transporters of mitochondria (ATMs) are highly conserved proteins, but their function in plants is poorly defined. Arabidopsis (Arabidopsis thaliana) has three ATM genes, namely ATM1, ATM2, and ATM3. Using a collection of insertional mutants, we show that only ATM3 has an important function for plant growth. Additional atm3 alleles were identified among sirtinol-resistant lines, correlating with decreased activities of aldehyde oxidases, cytosolic enzymes that convert sirtinol into an auxin analog, and depend on iron-sulfur (Fe-S) and molybdenum cofactor (Moco) as prosthetic groups. In the sirtinol-resistant atm3-3 allele, the highly conserved arginine-612 is replaced by a lysine residue, the negative effect of which could be mimicked in the yeast Atm1p ortholog. Arabidopsis atm3 mutants displayed defects in root growth, chlorophyll content, and seedling establishment. Analyses of selected metal enzymes showed that the activity of cytosolic aconitase (Fe-S) was strongly decreased across the range of atm3 alleles, whereas mitochondrial and plastid Fe-S enzymes were unaffected. Nitrate reductase activity (Moco, heme) was decreased by 50% in the strong atm3 alleles, but catalase activity (heme) was similar to that of the wild type. Strikingly, in contrast to mutants in the yeast and mammalian orthologs, Arabidopsis atm3 mutants did not display a dramatic iron homeostasis defect and did not accumulate iron in mitochondria. Our data suggest that Arabidopsis ATM3 may transport (1) at least two distinct compounds or (2) a single compound required for both Fe-S and Moco assembly machineries in the cytosol, but not iron.

Auxin synthesized by the YUCCA flavin monooxygenases is essential for embryogenesis and leaf formation in Arabidopsis.

Auxin plays a key role in embryogenesis and seedling development, but the auxin sources for the two processes are not defined. Here, we demonstrate that auxin synthesized by the YUCCA (YUC) flavin monooxygenases is essential for the establishment of the basal body region during embryogenesis and the formation of embryonic and postembryonic organs. Both YUC1 and YUC4 are expressed in discrete groups of cells throughout embryogenesis, and their expression patterns overlap with those of YUC10 and YUC11 during embryogenesis. The quadruple mutants of yuc1 yuc4 yuc10 yuc11 fail to develop a hypocotyl and a root meristem, a phenotype similar to those of mp and tir1 afb1 afb2 afb3 auxin signaling mutants. We further show that YUC genes play an essential role in the formation of rosette leaves by analyzing combinations of yuc mutants and the polar auxin transport mutants pin1 and aux1. Disruption of YUC1, YUC4, or PIN1 alone does not abolish leaf formation, but the triple mutant yuc1 yuc4 pin1 fails to form leaves and flowers. Furthermore, disruption of auxin influx carrier AUX1 in the quadruple mutant yuc1 yuc2 yuc4 yuc6, but not in wild-type background, phenocopies yuc1 yuc4 pin1, demonstrating that auxin influx is required for plant leaf and flower development. Our data demonstrate that auxin synthesized by the YUC flavin monooxygenases is an essential auxin source for Arabidopsis thaliana embryogenesis and postembryonic organ formation.