The pathway of auxin biosynthesis in plants

The plant hormone auxin, which is predominantly represented by indole-3-acetic acid (IAA), is involved in the regulation of plant growth and development. Although IAA was the first plant hormone identified, the biosynthetic pathway at the genetic level has remained unclear. Two major pathways for IAA biosynthesis have been proposed: the tryptophan (Trp)-independent and Trp-dependent pathways. In Trp-dependent IAA biosynthesis, four pathways have been postulated in plants: (i) the indole-3-acetamide (IAM) pathway; (ii) the indole-3-pyruvic acid (IPA) pathway; (iii) the tryptamine (TAM) pathway; and (iv) the indole-3-acetaldoxime (IAOX) pathway. Although different plant species may have unique strategies and modifications to optimize their metabolic pathways, plants would be expected to share evolutionarily conserved core mechanisms for auxin biosynthesis because IAA is a fundamental substance in the plant life cycle. In this review, the genes now known to be involved in auxin biosynthesis are summarized and the major IAA biosynthetic pathway distributed widely in the plant kingdom is discussed on the basis of biochemical and molecular biological findings and bioinformatics studies. Based on evolutionarily conserved core mechanisms, it is thought that the pathway via IAM or IPA is the major route(s) to IAA in plants.     

Activation of a flavin monooxygenase gene <Emphasis Type="Italic">YUCCA7</Emphasis> enhances drought resistance in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin regulates diverse molecular and physiological events at the cellular and organismal levels during plant growth and development in response to environmental stimuli. It acts either through distinct signaling pathways or in concert with other growth hormones. Its biological functions are adjusted by modulating biosynthesis, conjugate formation, and polar transport and distribution. Several tryptophan-dependent and -independent auxin biosynthetic pathways have been proposed. Recent studies have shown that a few flavin monooxygenase enzymes contribute to the tryptophan-dependent auxin biosynthesis. Here, we show that activation of a flavin monooxygenase gene YUCCA7 (YUC7), which belongs to the tryptophan-dependent auxin biosynthetic pathway, enhances drought resistance. An Arabidopsis activation-tagged mutant yuc7-1D exhibited phenotypic changes similar to those observed in auxin-overproducing mutants, such as tall, slender stems and curled, narrow leaves. Accordingly, endogenous levels of total auxin were elevated in the mutant. The YUC7 gene was induced by drought, primarily in the roots, in an abscisic acid (ABA)-dependent manner. The yuc7-1D mutant was resistant to drought, and drought-responsive genes, such as RESPONSIVE TO DESSICATION 29A (RD29A) and COLD-REGULATED 15A (COR15A), were up-regulated in the mutant. Interestingly, whereas stomatal aperture and production of osmoprotectants were not discernibly altered, lateral root growth was significantly promoted in the yuc7-1D mutant when grown under drought conditions. These observations support that elevation of auxin levels in the roots enhances drought resistance possibly by promoting root growth.     

Auxin biosynthesis in maize

For the biosynthesis of the phytohormone indole-3-acetic acid (IAA), a number of tryptophan-dependent and -independent pathways have been discussed. Maize is an appropriate model system to analyze IAA biosynthesis particularly because high quantities of IAA conjugates are stored in the endosperm. This allowed precursor feeding experiments in a kernel culture system followed by retrobiosynthetic NMR analysis, which strongly suggested that tryptophan-dependent IAA synthesis is the predominant route for auxin biosynthesis in the maize kernel. Two nitrilases ZmNIT1 and ZmNIT2 are expressed in seeds. ZmNIT2 efficiently hydrolyzes indole-3-acetonitrile (IAN) to IAA and thus could be involved in auxin biosynthesis. Redundant pathways, e.g., via indole-3-acetaldehyde could imply that multiple mutants will be necessary to obtain IAA-deficient plants and to conclusively identify relevant genes for IAA biosynthesis.     

Indole-3-glycerol phosphate, a branchpoint of indole-3-acetic acid biosynthesis from the tryptophan biosynthetic pathway in Arabidopsis thaliana

The phytohormone indole-3-acetic acid (IAA) plays a vital role in plant growth and development as a regulator of numerous biological processes. Its biosynthetic pathways have been studied for decades. Recent genetic and in vitro labeling evidence indicates that IAA in Arabidopsis thaliana and other plants is primarily synthesized from a precursor that is an intermediate in the tryptophan (Trp) biosynthetic pathway. To determine which intermediate(s) acts as the possible branchpoint for the Trp-independent IAA biosynthesis in plants, we took an in vivo approach by generating antisense indole-3-glycerol phosphate synthase (IGS) RNA transgenic plants and using available Arabidopsis Trp biosynthetic pathway mutants trp2-1 and trp3-1. Antisense transgenic plants display some auxin deficient-like phenotypes including small rosettes and reduced fertility. Protein gel blot analysis indicated that IGS expression was greatly reduced in the antisense lines. Quantitative analyses of IAA and Trp content in antisense IGS transgenic plants and Trp biosynthetic mutants revealed striking differences. Compared with wild-type plants, the Trp content in all the transgenic and mutant plants decreased significantly. However, total IAA levels were significantly decreased in antisense IGS transgenic plants, but remarkably increased in trp3-1 and trp2-1 plants. These results suggest that indole-3-glycerol phosphate (IGP) in the Arabidopsis Trp biosynthetic pathway serves as a branchpoint compound in the Trp-independent IAA de novo biosynthetic pathway.     

The rice FISH BONE gene encodes a tryptophan aminotransferase,which affects pleiotropic auxin‐related processes

Auxin is a fundamental plant hormone and its localization within organs plays pivotal roles in plant growth and development. Analysis of many Arabidopsis mutants that were defective in auxin biosynthesis revealed that the indole‐3‐pyruvic acid (IPA) pathway, catalyzed by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) families, is the major biosynthetic pathway of indole‐3‐acetic acid (IAA). In contrast, little information is known about the molecular mechanisms of auxin biosynthesis in rice. In this study, we identified a auxin‐related rice mutant, fish bone (fib). FIB encodes an orthologue of TAA genes and loss of FIB function resulted in pleiotropic abnormal phenotypes, such as small leaves with large lamina joint angles, abnormal vascular development, small panicles, abnormal organ identity and defects in root development, together with a reduction in internal IAA levels. Moreover, we found that auxin sensitivity and polar transport activity were altered in the fib mutant. From these results, we suggest that FIB plays a pivotal role in IAA biosynthesis in rice and that auxin biosynthesis, transport and sensitivity are closely interrelated.     

A mutation affecting the synthesis of 4-chloroindole-3-acetic acid

Traditionally, schemes depicting auxin biosynthesis in plants have been notoriously complex. They have involved up to four possible pathways by which the amino acid tryptophan might be converted to the main active auxin, indole-3-acetic acid (IAA), while another pathway was suggested to bypass tryptophan altogether. It was also postulated that different plants use different pathways, further adding to the complexity. In 2011, however, it was suggested that one of the four tryptophan-dependent pathways, via indole-3-pyruvic acid (IPyA), is the main pathway in Arabidopsis thaliana,¹ although concurrent operation of one or more other pathways has not been excluded. We recently showed that, for seeds of Pisum sativum (pea), it is possible to go one step further.² Our new evidence indicates that the IPyA pathway is the only tryptophan-dependent IAA synthesis pathway operating in pea seeds. We also demonstrated that the main auxin in developing pea seeds, 4-chloroindole-3-acetic acid (4-Cl-IAA), which accumulates to levels far exceeding those of IAA, is synthesized via a chlorinated version of the IPyA pathway.     

The Arabidopsis YUCCA1 flavin monooxygenase functions in the indole-3-pyruvic acid branch of auxin biosynthesis

The effects of auxins on plant growth and development have been known for more than 100 years, yet our understanding of how plants synthesize this essential plant hormone is still fragmentary at best. Gene loss- and gain-of-function studies have conclusively implicated three gene families, CYTOCHROME P450 79B2/B3 (CYP79B2/B3), YUCCA (YUC), and TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE-RELATED (TAA1/TAR), in the production of this hormone in the reference plant Arabidopsis thaliana. Each of these three gene families is believed to represent independent routes of auxin biosynthesis. Using a combination of pharmacological, genetic, and biochemical approaches, we examined the possible relationships between the auxin biosynthetic pathways defined by these three gene families. Our findings clearly indicate that TAA1/TARs and YUCs function in a common linear biosynthetic pathway that is genetically distinct from the CYP79B2/B3 route. In the redefined TAA1-YUC auxin biosynthetic pathway, TAA1/TARs are required for the production of indole-3-pyruvic acid (IPyA) from Trp, whereas YUCs are likely to function downstream. These results, together with the extensive genetic analysis of four pyruvate decarboxylases, the putative downstream components of the TAA1 pathway, strongly suggest that the enzymatic reactions involved in indole-3-acetic acid (IAA) production via IPyA are different than those previously postulated, and a new and testable model for how IAA is produced in plants is needed.     

Mediation of Herbicide Effects by Hormone Interactions

Chemical manipulation of the phytohormone system involves the use of herbicides for weed control in modern crop production. In the latter case, only compounds interacting with the auxin system have gained practical importance. Auxin herbicides mimic the overdose effects of indole-3-acetic acid (IAA), the principal natural auxin in higher plants. With their ability to control, particularly, dicotyledonous weeds in cereal crops, the synthetic auxins have been among the most successful herbicides used in agriculture. A newly discovered sequential hormone interaction plays a decisive role in their mode of action. The induction of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase in ethylene biosynthesis is the primary target process, following auxin herbicide signalling. Although the exact molecular target site has yet to be identified, it appears likely to be at the level of auxin receptor(s) for perception or signalling, leading ultimately to species- and organ-specific de novo enzyme synthesis. In sensitive dicots, ethylene causes epinastic growth and tissue swelling. Ethylene also triggers the biosynthesis of abscisic acid (ABA), mainly through the stimulated cleavage of xanthophylls to xanthoxal, catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED). ABA mediates stomatal closure which limits photosynthetic activity and biomass production, accompanied by an overproduction of reactive oxygen species. Growth inhibition, senescence and tissue decay are the consequences. Recent results suggest that ethylene-triggered ABA is not restricted to the action of auxin herbicides. It may function as a module in the signalling of a variety of stimuli leading to plant growth regulation. An additional phenomenon is caused by the auxin herbicide quinclorac which also controls grass weeds. Here, quinclorac induces the accumulation of phytotoxic levels of cyanide, a co-product of ethylene, which ultimately derives from herbicide-induced ACC synthase activity in the tissue. Phytotropins are a further group of hormone-related compounds which are used as herbicides. They inhibit polar auxin transport by interacting with a regulatory protein, the NPA-binding protein, of the auxin efflux carrier. This causes an abnormal accumulation of IAA and applied synthetic auxins in plant meristems. Growth inhibition, loss of tropic responses and, in combination with auxin herbicides, synergistic effects are the consequences.     

Current aspects of auxin biosynthesis in plants

Auxin is an important plant hormone essential for many aspects of plant growth and development. Indole-3-acetic acid (IAA) is the most studied auxin in plants, and its biosynthesis pathway has been investigated for over 70 years. Although the complete picture of auxin biosynthesis remains to be elucidated, remarkable progress has been made recently in understanding the mechanism of IAA biosynthesis. Genetic and biochemical studies demonstrate that IAA is mainly synthesized from l-tryptophan (Trp) via indole-3-pyruvate by two-step reactions in Arabidopsis. While IAA is also produced from Trp via indole-3-acetaldoxime in Arabidopsis, this pathway likely plays an auxiliary role in plants of the family Brassicaceae. Recent studies suggest that the Trp-independent pathway is not a major route for IAA biosynthesis, but they reveal an important role for a cytosolic indole synthase in this pathway. In this review, I summarize current views and future prospects of IAA biosynthesis research in plants.     

Cloning and Biochemical Characterization of <Emphasis Type="Italic">ToFZY</Emphasis>, a Tomato Gene Encoding a Flavin Monooxygenase Involved in a Tryptophan-dependent Auxin Biosynthesis Pathway

Indole-3-acetic acid (IAA), the main endogenous auxin, has been known for decades to be a key regulator for plant growth and development. Multiple routes have been proposed for IAA biosynthesis but physiologic roles or relevance of the different routes are still unclear. Recently, four members of the Arabidopsis thaliana YUC gene family have been implicated in an additional requirement of IAA involved in floral organ and vascular tissue formation. The loss-of-function yuc1yuc4 double mutants in Arabidopsis displayed phenotypes similar to the previously described loss-of-function floozy mutants in petunia (fzy). Moreover, it has been demonstrated that YUC1 encodes a flavin monooxygenase (FMO) that catalyzes a rate-limiting step of a tryptophan-dependent auxin biosynthesis pathway: the conversion of tryptamine to N-hydroxyl-tryptamine. Here we report on the genetic study of ToFZY, the putative tomato ortholog of YUC4 and FZY, including gene and cDNA sequence comparison and a preliminary expression analysis. In addition, we describe a novel conserved amino acid motif that may be considered a hallmark potentially useful for the identification of new YUC-like FMOs. We also demonstrate that ToFZY encodes a protein with the same enzymatic activity as YUC1. Finally, we provide evidence suggesting that the ToFZY gene belongs to a multigenic family whose members may exhibit a temporal and spatial specialization similar to that described in A. thaliana.     

Auxin synthesis in Agrobacterium tumefaciens and A. tumefaciens-transformed plant tissue

Problems of IAA biosynthesis, IAA precursors and IAA biosynthetic pathways in Agrobacterium tumefaciens-transformed plant tissue, especially in tobacco tissue culture are reviewed. The knowkedge about levels of IAA and the IAA biosynthetic pathways in Agrobacterium tumefaciens itself is also summarized.Dedical to the late Professor M. Kutáek, a pioneer in auxin biosynthesis research.     

Molecular Characterization of an Aux/IAA of Catharanthus roseus

In Catharanthus roseus cells, auxins are known to negatively regulate the biosynthesis of monoterpenoid indole alkaloids (MIA), a class of valuable secondary metabolites. Despite extensive studies of this regulation, no protein of the auxin signaling pathway has been isolated to date in this plant. We therefore decided to clone and characterize a C. roseus Aux/IAA protein that belongs to a family of gene expression repressors mediating auxin effects. Using PCR, a cDNA encoding the first C. roseus Aux/IAA was cloned and named CrIAA1. The deduced amino acid sequence has four highly conserved domains that are typical of the Aux/IAA protein family and has high homology to the Aux/IAA isoforms of Arabidopsis (>67%). The CrIAA1 gene expression, monitored by real-time PCR, was found to be dramatically induced by auxin treatment in C. roseus cells. Using GFP imagery and a bimolecular fluorescence complementation assay, we found that CrIAA1 can form oligomers in the nucleus. We also found that CrIAA1 is quickly degraded following auxin treatments, suggesting that auxin regulates CrIAA1 availability via a feedback mechanism. These results should help to elucidate the molecular nature of the processes responsible for the auxin-mediated regulation of MIA biosynthesis in C. roseus.     

Differential inhibition of indole-3-acetic acid and tryptophan biosynthesis by indole analogues. I. Tryptophan dependent IAA biosynthesis

Maize liquid endosperm extracts contain the enzymes necessary for all of the steps of the plant IAA biosynthetic pathway from tryptophan, and provide a means to assay the pathway in vitro. We have analyzed the reactions in the presence of a series of indole and indole-like analogues in order to evaluate the potential of these compounds to act as inhibitors of IAA biosynthesis. Such inhibitors will be useful to investigate the tryptophan to IAA pathway, to determine the precursors and intermediates involved, and to select for mutants in this process. A number of such compounds were tested using in vitro enzyme assays for both the tryptophan dependent IAA biosynthesis pathway and for tryptophan synthase activity. Some compounds showed strong inhibition of IAA biosynthesis while having only a slight effect on the reaction rate of tryptophan synthase . These results: (1) show that IAA biosynthesis can be selectively inhibited relative to tryptophan biosynthesis; (2) suggest potential ways to screen for IAA biosynthetic pathway mutations in plants; and (3) provide additional tools for studies of IAA biosynthesis in plants.     

Auxin signaling through SCFTIR1/AFBs mediates feedback regulation of IAA biosynthesis

We previously reported that exogenous application of auxin to Arabidopsis seedlings resulted in downregulation of indole-3-acetic acid (IAA) biosynthesis genes in a feedback manner. In this study, we investigated the involvement of the SCF^TIR1/AFB-mediated signaling pathway in feedback regulation of the indole-3-pyruvic acid-mediated auxin biosynthesis pathway in Arabidopsis. Application of PEO-IAA, an inhibitor of the IAA signal transduction pathway, to wild-type seedlings resulted in increased endogenous IAA levels in roots. Endogenous IAA levels in the auxin-signaling mutants axr2-1, axr3-3, and tir1-1afb1-1afb2-1afb3-1 also increased. Furthermore, YUCCA (YUC) gene expression was repressed in response to auxin treatment, and expression of YUC7 and YUC8 increased in response to PEO-IAA treatment. YUC genes were also induced in auxin-signaling mutants but repressed in TIR1-overexpression lines. These observations suggest that the endogenous IAA levels are regulated by auxin biosynthesis in a feedback manner, and the Aux/IAA and SCF^TIR1/AFB-mediated auxin-signaling pathway regulates the expression of YUC genes.     

Yucasin is a potent inhibitor of YUCCA,a key enzyme in auxin biosynthesis

Indole‐3–acetic acid (IAA), an auxin plant hormone, is biosynthesized from tryptophan. The indole‐3–pyruvic acid (IPyA) pathway, involving the tryptophan aminotransferase TAA1 and YUCCA (YUC) enzymes, was recently found to be a major IAA biosynthetic pathway in Arabidopsis. TAA1 catalyzes the conversion of tryptophan to IPyA, and YUC produces IAA from IPyA. Using a chemical biology approach with maize coleoptiles, we identified 5–(4–chlorophenyl)‐4H‐1,2,4–triazole‐3–thiol (yucasin) as a potent inhibitor of IAA biosynthesis in YUC‐expressing coleoptile tips. Enzymatic analysis of recombinant AtYUC1‐His suggested that yucasin strongly inhibited YUC1‐His activity against the substrate IPyA in a competitive manner. Phenotypic analysis of Arabidopsis YUC1 over‐expression lines (35S::YUC1) demonstrated that yucasin acts in IAA biosynthesis catalyzed by YUC. In addition, 35S::YUC1 seedlings showed resistance to yucasin in terms of root growth. A loss‐of‐function mutant of TAA1, sav3–2, was hypersensitive to yucasin in terms of root growth and hypocotyl elongation of etiolated seedlings. Yucasin combined with the TAA1 inhibitor l –kynurenine acted additively in Arabidopsis seedlings, producing a phenotype similar to yucasin‐treated sav3–2 seedlings, indicating the importance of IAA biosynthesis via the IPyA pathway in root growth and leaf vascular development. The present study showed that yucasin is a potent inhibitor of YUC enzymes that offers an effective tool for analyzing the contribution of IAA biosynthesis via the IPyA pathway to plant development and physiological processes.     

The Endosperm-Specific Expression of <i>YUCCA</i> Genes Enhances Rice Grain Filling

Grain filling is a crucial process that affects yield in rice (Oryza sativa L.). Auxin biosynthesis and signaling are closely related to rice yield; therefore, it is important to understand the effects of auxin biosynthesis on rice grain filling to improve crop yield. In this study, we used physiological and molecular strategies to identify the roles of auxin in rice grain filling. Exogenous application of auxin (IAA) or auxin analogues (2, 4-D) to young spikelets and flag leaves improved the seed-setting rate and yield per spike. Furthermore, real-time quantitative PCR assays confirmed that nine members of the OsYUCCA family of auxin biosynthetic genes were upregulated during grain filling, implication that auxin biosynthesis plays a major role in grain development. The specific expression of either Arabidopsis AtYUCCA1 or OsYUCCA2 in the endosperm or leaves resulted in increased expression of OsIAA genes and auxin content of seeds, as well as increased grain filling and seed-setting rate. This result establishes that the auxin content in grains and leaves is important for grain development. Our findings further highlight the potential applications for improving rice yield by elevating targeted gene expression in specific tissues.