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Disruption of either the auxin transporter PIN-FORMED 1 (PIN1) or the protein kinase PINOID (PID) leads to the development of pin-like inflorescences. Previous studies have shown that phosphoregulation of PIN1 by AGC kinases including PID directs auxin flux to drive organ initiation. Here, we report unexpected findings on the genetic interactions between these two genes. We deleted the first 2/3 of the PIN1 coding sequence using CRISPR/Cas9, and the resulting pin1 mutant (pin1-27) was a strong allele. Surprisingly, heterozygous pin1-27 suppressed two independent pid null mutants, whereas homozygous pin1-27 enhanced the phenotypes of the pid mutants during embryogenesis. Furthermore, we show that deletion of either the hydrophilic loop or the second half of PIN1 also abolished PIN1 function, yet those heterozygous pin1 mutants were also capable of rescuing pid nulls. Moreover, we inserted green fluorescent protein (GFP) into the hydrophilic loop of PIN1 through CRISPR-mediated homology-directed repair (HDR). The GFP signal and pattern in the PIN1-GFPHDR line are similar to those in the previously reported PIN1-GFP transgenic lines. Interestingly, the PIN1-GFPHDR line also rescued various pid null mutant alleles in a semidominant fashion. We conclude that decreasing the number of functional PIN1 copies is sufficient to suppress the pid mutant phenotype, suggesting that PIN1 is likely part of a larger protein complex required for organogenesis.

The phytohormone auxin, indole-3-acetic acid (IAA), plays a prominent role in plant development. Auxin homeostasis is coordinately regulated by auxin synthesis, transport, and inactivation; however, the physiological contribution of auxin inactivation to auxin homeostasis has not been determined. The GH3 IAA-amino acid conjugating enzymes play a central role in auxin inactivation. Chemical inhibition of GH3 proteins in planta is challenging because the inhibition of these enzymes leads to IAA overaccumulation that rapidly induces GH3 expression. Here, we report the characterization of a potent GH3 inhibitor, kakeimide, that selectively targets IAA-conjugating GH3 proteins. Chemical knockdown of the auxin inactivation pathway demonstrates that auxin turnover is very rapid (about 10 min) and indicates that both auxin biosynthesis and inactivation dynamically regulate auxin homeostasis.

Auxin is a key plant growth regulator for diverse developmental processes in plants. Indole-3-acetic acid (IAA) is a primary plant auxin that regulates the formation of various organs. Plants also produce phenylacetic acid (PAA), another natural auxin, which occurs more abundantly than IAA in various plant species. Although it has been demonstrated that the two auxins have distinct transport characteristics, the metabolic pathways and physiological roles of PAA in plants remain unsolved. In this study, we investigated the role of Arabidopsis UDP-glucosyltransferase UGT84B1 in IAA and PAA metabolism. We demonstrated that UGT84B1, which converts IAA to IAA-glucoside (IAA-Glc), can also catalyze the conversion of PAA to PAA-glucoside (PAA-Glc), with a higher catalytic activity in vitro. Furthermore, we showed a significant increase in both the IAA and PAA levels in the ugt84b1 null mutants. However, no obvious developmental phenotypes were observed in the ugt84b1 mutants under laboratory growth conditions. Moreover, the overexpression of UGT84B1 resulted in auxin-deficient root phenotypes and changes in the IAA and PAA levels. Our results indicate that UGT84B1 plays an important role in IAA and PAA homeostasis in Arabidopsis.

The phytohormone auxin regulates a wide range of developmental processes in plants. Indole-3-acetic acid (IAA) is the main auxin that moves in a polar manner and forms concentration gradients, whereas phenylacetic acid (PAA), another natural auxin, does not exhibit polar movement. Although these auxins occur widely in plants, the differences between IAA and PAA metabolism remain largely unknown. In this study, we investigated the role of Arabidopsis IAA CARBOXYL METHYLTRANSFERASE 1 (IAMT1) in IAA and PAA metabolism. IAMT1 proteins expressed in Escherichia coli could convert both IAA and PAA to their respective methyl esters. Overexpression of IAMT1 caused severe auxin-deficient phenotypes and reduced the levels of IAA, but not PAA, in the root tips of Arabidopsis, suggesting that IAMT1 exclusively metabolizes IAA in vivo. We generated iamt1 null mutants via CRISPR/Cas9-mediated genome editing and found that the single knockout mutants had normal auxin levels and did not exhibit visibly altered phenotypes. These results suggest that other proteins, namely the IAMT1 homologs in the SABATH family of carboxyl methyltransferases, may also regulate IAA levels in Arabidopsis.

Indole-3-acetamide (IAM) is the first confirmed auxin biosynthetic intermediate in some plant pathogenic bacteria. Exogenously applied IAM or production of IAM by overexpressing the bacterial iaaM gene in Arabidopsis causes auxin overproduction phenotypes. However, it is still inconclusive whether plants use IAM as a key precursor for auxin biosynthesis. Herein, we reported the isolation IAMHYDROLASE1 (IAMH1) gene in Arabidopsis from a forward genetic screen for IAM-insensitive mutants that display normal auxin sensitivities. IAMH1 has a close homolog named IAMH2 that is located right next to IAMH1 on chromosome IV in Arabidopsis. We generated iamh1 iamh2 double mutants using our CRISPR/Cas9 gene editing technology. We showed that disruption of the IAMH genes rendered Arabidopsis plants resistant to IAM treatments and also suppressed the iaaM overexpression phenotypes, suggesting that IAMH1 and IAMH2 are the main enzymes responsible for converting IAM into indole-3-acetic acid (IAA) in Arabidopsis. The iamh double mutants did not display obvious developmental defects, indicating that IAM does not play a major role in auxin biosynthesis under normal growth conditions. Our findings provide a solid foundation for clarifying the roles of IAM in auxin biosynthesis and plant development.

The plant pathogen Agrobacterium tumefaciens infects plants and introduces the transferred-DNA (T-DNA) region of the Ti-plasmid into nuclear DNA of host plants to induce the formation of tumors (crown galls). The T-DNA region carries iaaM and iaaH genes for synthesis of the plant hormone auxin, indole-3-acetic acid (IAA). It has been demonstrated that the iaaM gene encodes a tryptophan 2-monooxygenase which catalyzes the conversion of tryptophan to indole-3-acetamide (IAM), and the iaaH gene encodes an amidase for subsequent conversion of IAM to IAA. In this article, we demonstrate that A. tumefaciens enhances the production of both IAA and phenylacetic acid (PAA), another auxin which does not show polar transport characteristics, in the formation of crown galls. Using liquid chromatography-tandem mass spectroscopy, we found that the endogenous levels of phenylacetamide (PAM) and PAA metabolites, as well as IAM and IAA metabolites, are remarkably increased in crown galls formed on the stem of tomato plants, implying that two distinct auxins are simultaneously synthesized via the IaaM-IaaH pathway. Moreover, we found that the induction of the iaaM gene dramatically elevated the levels of PAM, PAA and its metabolites, along with IAM, IAA and its metabolites, in Arabidopsis and barley. From these results, we conclude that A. tumefaciens enhances biosynthesis of two distinct auxins in the formation of crown galls.

A well-developed root system in rice and other crops can ensure plants to efficiently absorb nutrients and water. Auxin is a key regulator for various aspect of root development, but the detailed molecular mechanisms by which auxin controls crown root development in rice are not understood. We show that overexpression of a YUC gene, which encodes the rate-limiting enzyme in auxin biosynthesis, causes massive proliferation of crown roots. On the other hand, we find that disruption of TAA1, which functions upstream of YUC genes, greatly reduces crown root development. We find that YUC overexpression-induced crown root proliferation requires the presence of the transcription factor WOX11. Moreover, the crown rootless phenotype of taa1 mutants was partially rescued by overexpression of WOX11. Furthermore, we show that WOX11 expression is induced in OsYUC1 overexpression lines, but is repressed in the taa1 mutants. Our results indicate that auxin synthesized by the TAA/YUC pathway is necessary and sufficient for crown root development in rice. Auxin activates WOX11 transcription, which subsequently drives crown root initiation and development, establishing the YUC-Auxin-WOX11 module for crown root development in rice.

Phytohormone auxin is a master regulator in plant growth and development. Regulation of cellular auxin level plays a central role in plant development. Auxin polar transport system modulates an auxin gradient that determines plant developmental process in response to environmental conditions and developmental programs. Photolabile caged auxins allow optical control of artificial auxin gradients at cellular resolution. Especially, two-photon uncaging system achieves high spatiotemporal control of photolysis reaction at two-photon cross-section. However, the development of caged versions of auxin has been limited by the instability of the caged auxins to higher plant metabolic activities. Here, we describe the synthesis and application of highly stable caged auxins, 4-methoxy-7-nitroindolinyl (MNI)-caged auxins. Natural auxin, indole 3-acetic acid, and two synthetic auxins, 1-NAA and 2,4-D were caged by MNI caging group. MNI-caged auxins showed a high stability in planta and a rapid release the original auxin when photolyzed. We demonstrated that optical control of auxin-responsive gene expression and auxin-related physiological responses by using MNI-caged auxins. We anticipate that MNI-caged auxins will be an effective tool for high-resolution control of endogenous auxin level.

Auxin binding protein 1 (ABP1) has been studied for decades. It has been suggested that ABP1 functions as an auxin receptor and has an essential role in many developmental processes. Here we present our unexpected findings that ABP1 is neither required for auxin signaling nor necessary for plant development under normal growth conditions. We used our ribozyme-based CRISPR technology to generate an Arabidopsis abp1 mutant that contains a 5-bp deletion in the first exon of ABP1, which resulted in a frameshift and introduction of early stop codons. We also identified a T-DNA insertion abp1 allele that harbors a T-DNA insertion located 27 bp downstream of the ATG start codon in the first exon. We show that the two new abp1 mutants are null alleles. Surprisingly, our new abp1 mutant plants do not display any obvious developmental defects. In fact, the mutant plants are indistinguishable from wild-type plants at every developmental stage analyzed. Furthermore, the abp1 plants are not resistant to exogenous auxin. At the molecular level, we find that the induction of known auxin-regulated genes is similar in both wild-type and abp1 plants in response to auxin treatments. We conclude that ABP1 is not a key component in auxin signaling or Arabidopsis development.

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.

Inactivation of the phytohormone auxin plays important roles in plant development, and several enzymes have been implicated in auxin inactivation. In this study, we show that the predominant natural auxin, indole-3-acetic acid (IAA), is mainly inactivated via the GH3-ILR1-DAO pathway. IAA is first converted to IAA-amino acid conjugates by GH3 IAA-amidosynthetases. The IAA-amino acid conjugates IAA-aspartate (IAA-Asp) and IAA-glutamate (IAA-Glu) are storage forms of IAA and can be converted back to IAA by ILR1/ILL amidohydrolases. We further show that DAO1 dioxygenase irreversibly oxidizes IAA-Asp and IAA-Glu into 2-oxindole-3-acetic acid-aspartate (oxIAA-Asp) and oxIAA-Glu, which are subsequently hydrolyzed by ILR1 to release inactive oxIAA. This work established a complete pathway for the oxidative inactivation of auxin and defines the roles played by auxin homeostasis in plant development. Auxin inactivation plays important roles in plant development. Here the authors show that the main route of IAA inactivation in Arabidopsis is via conjugation by GH3 IAA-amidosynthetases followed by DAO1 dioxygenase-mediated oxidation of the conjugated forms and hydrolysis by ILR1 to release inactive oxIAA. [ABSTRACT FROM AUTHOR]

Auxin is a key regulator of plant growth and development. Local auxin biosynthesis and intercellular transport generates regional gradients in the root that are instructive for processes such as specification of developmental zones that maintain root growth and tropic responses. Here we present a toolbox to study auxin-mediated root development that features: (i) the ability to control auxin synthesis with high spatio-temporal resolution and (ii) single-cell nucleus tracking and morphokinetic analysis infrastructure. Integration of these two features enables cutting-edge analysis of root development at single-cell resolution based on morphokinetic parameters under normal growth conditions and during cell-type-specific induction of auxin biosynthesis. We show directional auxin flow in the root and refine the contributions of key players in this process. In addition, we determine the quantitative kinetics of Arabidopsis root meristem skewing, which depends on local auxin gradients but does not require PIN2 and AUX1 auxin transporter activities. Beyond the mechanistic insights into root development, the tools developed here will enable biologists to study kinetics and morphology of various critical processes at the single cell-level in whole organisms. Auxin gradients regulate plant root growth and development. Here the authors manipulate auxin synthesis in specific root cell types and use single-cell nucleus tracking and morphokinetics to map directional auxin flow in the root and quantify the kinetics of meristem skewing. [ABSTRACT FROM AUTHOR]

Root meristem activity is the most critical process influencing root development. Although several factors that regulate meristem activity have been identified in rice, studies on the enhancement of meristem activity in roots are limited. We identified a T-DNA activation tagging line of a zinc-finger homeobox gene, OsZHD2 , which has longer seminal and lateral roots due to increased meristem activity. The phenotypes were confirmed in transgenic plants overexpressing OsZHD2. In addition, the overexpressing plants showed enhanced grain yield under low nutrient and paddy field conditions. OsZHD2 was preferentially expressed in the shoot apical meristem and root tips. Transcriptome analyses and quantitative real-time PCR experiments on roots from the activation tagging line and the wild type showed that genes for ethylene biosynthesis were up-regulated in the activation line. Ethylene levels were higher in the activation lines compared with the wild type. ChIP assay results suggested that OsZHD2 induces ethylene biosynthesis by controlling ACS5 directly. Treatment with ACC (1-aminocyclopropane-1-carboxylic acid), an ethylene precursor, induced the expression of the DR5 reporter at the root tip and stele, whereas treatment with an ethylene biosynthesis inhibitor, AVG (aminoethoxyvinylglycine), decreased that expression in both the wild type and the OsZHD2 overexpression line. These observations suggest that OsZHD2 enhances root meristem activity by influencing ethylene biosynthesis and, in turn, auxin. [ABSTRACT FROM AUTHOR]

Summary: YUC flavin monooxygenases catalyze the rate‐limiting step of auxin biosynthesis. Here we report the vacuolar targeting and degradation of GFP‐YUC1. GFP‐YUC1 fusion expressed in Arabidopsis protoplasts or transgenic plants was primarily localized in vacuoles. Surprisingly, we established that GFP‐YUC1, a soluble protein, was sorted to vacuoles through the ESCRT pathway, which has long been recognized for sorting and targeting integral membrane proteins. We further show that GFP‐YUC1 was ubiquitinated and in this form GFP‐YUC1 was targeted for degradation, a process that was also stimulated by elevated auxin levels. Our findings revealed a molecular mechanism of GFP‐YUC1 degradation and demonstrate that the ESCRT pathway can recognize both soluble and integral membrane proteins as cargoes. [ABSTRACT FROM AUTHOR]

Gametophytic development in Arabidopsis depends on nutrients and cell wall materials from sporophytic cells. However, it is not clear whether hormones and signaling molecules from sporophytic tissues are also required for gametophytic development. Herein, we show that auxin produced by the flavin monooxygenases YUC2 and YUC6 in the sporophytic microsporocytes is essential for early stages of pollen development. The first asymmetric mitotic division (PMI) of haploid microspores is the earliest event in male gametophyte development. Microspore development in yuc2yuc6 double mutants arrests before PMI and consequently yuc2yuc6 fail to produce viable pollens. Our genetic analyses reveal that YUC2 and YUC6 act as sporophytic genes for pollen formation. We further show that ectopic production of auxin in tapetum, which provides nutrients for pollen development, fails to rescue the sterile phenotypes of yuc2yuc6. In contrast, production of auxin in either microsporocytes or microspores rescued the defects of pollen development in yuc2yuc6 double mutants. Our results demonstrate that local auxin biosynthesis in sporophytic microsporocytic cells and microspore controls male gametophyte development during the generation transition from sporophyte to male gametophyte. [ABSTRACT FROM AUTHOR]

Auxin responses have been arbitrarily divided into two categories: genomic and non-genomic effects. Genomic effects are largely mediated by SCF TIR1/AFB -Aux/IAA auxin receptor complexes whereas it has been postulated that AUXIN BINDING PROTEIN 1 (ABP1) controls the non-genomic effects. However, the roles of ABP1 in auxin signaling and plant development were recently called into question. In this paper, we present recent progress in understanding the SCF TIR1/AFB -Aux/IAA pathway. In more detail, we discuss the current understanding of ABP1 research and provide an updated view of ABP1-related genetic materials. Further, we propose a model in which auxin efflux carriers may play a role in auxin perception and we briefly describe recent insight on processes downstream of auxin perception. [ABSTRACT FROM AUTHOR]

The shoot apical meristem ( SAM) produces all of the plant's aerial organs. The SAM is established either during embryogenesis or experimentally in in vitro tissue culture. Although several factors including the Class I KNOTTED1- LIKE HOMEOBOX ( KNOXI) proteins, auxin, and cytokinin are known to play essential roles in SAM development, the underlying mechanisms of SAM formation and maintenance are still largely not understood. Herein we demonstrate that Os ARID3, a member of the rice ( Oryza sativa) AT-rich Interaction Domain ( ARID) family, is required for SAM development. Disruption of Os ARID3 leads to a defective SAM, early seedling lethality, and impaired capacity of in vitro shoot regeneration. We show that the expression levels of several KNOXI genes and the biosynthetic genes for auxin and cytokinin are significantly altered in the Osarid3 mutant calli. Moreover, we determine that auxin concentrations are increased, whereas cytokinin levels are decreased, in Osarid3 calli. Furthermore, chromatin immunoprecipitation results demonstrate that Os ARID3 binds directly to the KNOXI gene OSH71, the auxin biosynthetic genes Os YUC1 and Os YUC6, and the cytokinin biosynthetic genes Os IPT2 and Os IPT7. We also show through electrophoretic mobility shift assays that Os ARID3 specifically binds to the AT-rich DNA sequences of the identified target genes. We conclude that Os ARID3 is an AT-rich specific DNA-binding protein and that it plays a major role in SAM development in rice. [ABSTRACT FROM AUTHOR]

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 ( Me JA). The similar response of the yuc8 and yuc9 mutants to Me JA 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. [ABSTRACT FROM AUTHOR]

The directional transport of the phytohormone auxin depends on the phosphorylation status and polar localization of PIN-FORMED (PIN) auxin efflux proteins. While PINIOD (PID) kinase is directly involved in the phosphorylation of PIN proteins, the phosphatase holoenzyme complexes that dephosphorylate PIN proteins remain elusive. Here, we demonstrate that mutations simultaneously disrupting the function of Arabidopsis thaliana   FyPP1 (for Phytochrome-associated serine/threonine protein phosphatase1) and FyPP3 , two homologous genes encoding the catalytic subunits of protein phosphatase6 (PP6), cause elevated accumulation of phosphorylated PIN proteins, correlating with a basal-to-apical shift in subcellular PIN localization. The changes in PIN polarity result in increased root basipetal auxin transport and severe defects, including shorter roots, fewer lateral roots, defective columella cells, root meristem collapse, abnormal cotyledons (small, cup-shaped, or fused cotyledons), and altered leaf venation. Our molecular, biochemical, and genetic data support the notion that FyPP1/3, SAL (for SAPS DOMAIN-LIKE), and PP2AA proteins (RCN1 [for ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1] or PP2AA1, PP2AA2, and PP2AA3) physically interact to form a novel PP6-type heterotrimeric holoenzyme complex. We also show that FyPP1/3, SAL, and PP2AA interact with a subset of PIN proteins and that for SAL the strength of the interaction depends on the PIN phosphorylation status. Thus, an Arabidopsis PP6-type phosphatase holoenzyme acts antagonistically with PID to direct auxin transport polarity and plant development by directly regulating PIN phosphorylation. [ABSTRACT FROM AUTHOR]

Indole-3-acetic acid (IAA), the main naturally occurring auxin, is essential for almost every aspect of plant growth and development. However, only recently have studies finally established the first complete auxin biosynthesis pathway that converts tryptophan (Trp) to IAA in plants. Trp is first converted to indole-3-pyruvate (IPA) by the TAA family of amino transferases and subsequently IAA is produced from IPA by the YUC family of flavin monooxygenases. The two-step conversion of Trp to IAA is the main auxin biosynthesis pathway that plays an essential role in many developmental processes. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM AUTHOR]

The phytohormone auxin plays critical roles in the regulation of plant growth and development. Indole-3-acetic acid (IAA) has been recognized as the major auxin for more than 70 y. Although several pathways have been proposed, how auxin is synthesized in plants is still unclear. Previous genetic and enzymatic studies demonstrated that both TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) flavin monooxygenase-like proteins are required for biosynthesis of IAA during plant development, but these enzymes were placed in two independent pathways. In this article, we demonstrate that the TAA family produces indole-3-pyruvic acid (IPA) and the YUC family functions in the conversion of IPA to IAA in Arabidopsis (Arabidopsis thaliana) by a quantification method of IPA using liquid chromatography-electrospray ionization-tandem MS. We further show that YUC protein expressed in Escherichia coli directly converts IPA to IAA. Indole-3-acetaldehyde is probably not a precursor of IAA in the IPA pathway. Our results indicate that YUC proteins catalyze a rate-limiting step of the IPA pathway, which is the main IAA biosynthesis pathway in Arabidopsis. [ABSTRACT FROM AUTHOR]

Flavin monooxygenases (FMOs) play critical roles in plant growth and development by synthesizing auxin and other signaling molecules. However, the structure and function relationship within plant FMOs is not understood. Here we defined the important residues and domains of the Arabidopsis YUC1 FMO, a key enzyme in auxin biosynthesis. We previously showed that simultaneous inactivation of YUC1 and its homologue YUC4 caused severe defects in vascular and floral development. We mutagenized the yuc4 mutant and screened for mutants with phenotypes similar to those of yuc1 yuc4 double mutants. Among the isolated mutants, five of them contained mutations in the YUC1 gene. Interestingly, the mutations identified in the new yuc1 alleles were concentrated in the two GXGXXG motifs that are highly conserved among the plant FMOs. One such motif presumably binds to flavin adenine dinucleotide (FAD) cofactor and the other binds to nicotinamide adenine dinucleotide phosphate (NADPH). We also identified the Ser139 to Phe conversion in yuc1, a mutation that is located between the two nucleotide‐binding sites. By analyzing a series of yuc1 mutants, we identified key residues and motifs essential for the functions of YUC1 FMO. [ABSTRACT FROM AUTHOR]

• Auxin is essential for many aspects of plant growth and development, including the determination of lateral organ shapes. • Here, the characterization of a dominant Arabidopsis thaliana mutant spl-D (SPOROCYTELESS dominant), and the roles of SPL in auxin homeostasis and plant development, are reported. • The spl-D mutant displayed a severe up-curling leaf phenotype caused by increased expression of SPOROCYTELESS/NOZZLE (SPL/NZZ), a putative transcription factor gene that was previously linked to sporocyte formation. The spl-D plants also displayed pleiotropic developmental defects including fewer lateral roots, simpler venation patterns, and reduced shoot apical dominance. The leaf and floral phenotypes of spl-D and SPL over-expression lines were reminiscent of yucca (yuc) triple and quadruple mutants, suggesting that SPL may regulate auxin homeostasis. Consistent with this hypothesis, it was found that over-expression of SPL led to down-regulation of the auxin reporter DR5-GUS, and that many auxin-responsive genes were down-regulated in spl-D leaves. Interestingly, the expression of YUC2 and YUC6, two key genes in auxin biosynthesis, was significantly repressed in spl-D plants. • Taken together with the genetic and phenotypic analysis of spl-D/yuc6-D double mutant, these data suggest that SPL may regulate auxin homeostasis by repressing the transcription of YUC2 and YUC6 and participate in lateral organ morphogenesis. [ABSTRACT FROM AUTHOR]

Auxin is a plant hormone that regulates many aspects of plant growth and development. We used a chemical genetics approach to identify SIR1, a regulator of many auxin-inducible genes. The sir1 mutant was resistant to sirtinol, a small molecule that activates many auxin-inducible genes and promotes auxin-related developmental phenotypes. SIR1 is predicted to encode a protein composed of a ubiquitin-activating enzyme E1-like domain and a Rhodanese-like domain homologous to that of prolyl isomerase. We suggest a molecular context for how the auxin signal is propagated to exert its biological effects. [ABSTRACT FROM AUTHOR]

We identify an Arabidopsis pyridoxal-phosphate-dependent aminotransferase, VAS1, whose loss-of-function simultaneously increases amounts of the phytohormone auxin and the ethylene precursor 1-aminocyclopropane-1-carboxylate. VAS1 uses the ethylene biosynthetic intermediate methionine as an amino donor and the auxin biosynthetic intermediate indole-3-pyruvic acid as an amino acceptor to produce L-tryptophan and 2-oxo-4-methylthiobutyric acid. Our data indicate that VAS1 serves key roles in coordinating the amounts of these two vital hormones. [ABSTRACT FROM AUTHOR]

Gretchen Hagen 3 (GH3) amido synthetases conjugate amino acids to a carboxyl group of small molecules including hormones auxin, jasmonate, and salicylic acid. The Arabidopsis genome harbors 19 GH3 genes, whose exact roles in plant development have been difficult to define because of genetic redundancy among the GH3 genes. Here we use CRISPR/Cas9 gene editing technology to delete the Arabidopsis group II GH3 genes, which are able to conjugate indole-3-acetic acid (IAA) to amino acids. We show that plants lacking the eight group II GH3 genes (gh3 octuple mutants) accumulate free IAA and fail to produce IAA-Asp and IAA-Glu conjugates. Consequently, gh3 octuple mutants have extremely short roots, long and dense root hairs, and long hypocotyls. Our characterization of gh3 septuple mutants, which provide sensitized backgrounds, reveals that GH3.17 and GH3.9 play prominent roles in root elongation and seed production, respectively. We show that GH3 functions correlate with their expression patterns, suggesting that local deactivation of auxin also contributes to maintaining auxin homeostasis. Moreover, this work provides a method for elucidating functions of individual members of a gene family, whose members have overlapping functions. • The eight Arabidopsis GH3 amido synthetase genes responsible for conjugating auxin to amino acids were completely deleted by CRISPR/Cas9 gene editing. • Arabidopsis plants without the auxin conjugating enzymes over-accumulate auxin and fail to make IAA-Glu and IAA-Asp conjugates, demonstrating the essential roles of GH3 amido synthetases in auxin homeostasis. • Analysis of plants with just one auxin-conjugating GH3 gene defined unique roles of GH3.17 and GH3.9 in plant development. • The method for defining functions of individual genes in a gene family can be adopted for studying other gene families. [ABSTRACT FROM AUTHOR]