Indole-3-pyruvic acid regulates TAA1 activity, which plays a key role in coordinating the two steps of auxin biosynthesis.

Auxin biosynthesis involves two types of enzymes: the Trp aminotransferases (TAA/TARs) and the flavin monooxygenases (YUCCAs). This two-step pathway is highly conserved throughout the plant kingdom and is essential for almost all of the major developmental processes. Despite their importance, it is unclear how these enzymes are regulated and how their activities are coordinated. Here, we show that TAA1/TARs are regulated by their product indole-3-pyruvic acid (IPyA) (or its mimic KOK2099) via negative feedback regulation in Arabidopsis thaliana. This regulatory system also functions in rice and tomato. This negative feedback regulation appears to be achieved by both the reversibility of Trp aminotransferase activity and the competitive inhibition of TAA1 activity by IPyA. The Km value of IPyA is 0.7 µM, and that of Trp is 43.6 µM; this allows IPyA to be maintained at low levels and prevents unfavorable nonenzymatic indole-3-acetic acid (IAA) formation from IPyA in vivo. Thus, IPyA levels are maintained by the push (by TAA1/TARs) and pull (by YUCCAs) of the two biosynthetic enzymes, in which TAA1 plays a key role in preventing the over- or under-accumulation of IPyA. TAA1 prefer Ala among various amino acid substrates in the reverse reaction of auxin biosynthesis, allowing TAA1 to show specificity for converting Trp and pyruvate to IPyA and Ala, and the reverse reaction.

Effect of an auxin biosynthesis inhibitor, p-phenoxyphenyl boronic acid, on auxin biosynthesis and development in rice.

p-Phenoxyphenyl boronic acid (PPBo) is a specific inhibitor of auxin biosynthesis in Arabidopsis. We examined the inhibitory activity of PPBo in rice. The activity of OsYUCCA, a key enzyme for auxin biosynthesis, was inhibited by PPBo in vitro. The endogenous indole-3-acetic acid (IAA) level and the expression levels of auxin-response genes were significantly reduced in PPBo-treated rice seedlings, which showed typical auxin-deficiency phenotypes. Seminal root growth was promoted by 1 µM PPBo, which was reversed by co-treatment of IAA and PPBo. By contrast, the inhibition of root growth by 10 µM PPBo was not recovered by IAA. The root meristem morphology and cell division were restored by IAA at 60 µM, but that concentration may be too high to support root growth. In conclusion, PPBo is an inhibitor of auxin biosynthesis that targets YUCCA in rice.

Insertion of a transposon-like sequence in the 5'-flanking region of the YUCCA gene causes the stony hard phenotype.

Melting-flesh peaches produce large amounts of ethylene, resulting in rapid fruit softening at the late-ripening stage. In contrast, stony hard peaches do not soften and produce little ethylene. The indole-3-acetic acid (IAA) level in stony hard peaches is low at the late-ripening stage, resulting in low ethylene production and inhibition of fruit softening. To elucidate the mechanism of low IAA concentration in stony hard peaches, endogenous levels of IAA and IAA intermediates or metabolites were analysed by ultra-performance liquid chromatography-tandem mass spectrometry. Although the IAA level was low, the indole-3-pyruvic acid (IPyA) level was high in stony hard peaches at the ripening stage. These results indicate that YUCCA activity is reduced in ripening stony hard peaches. The expression of one of the YUCCA isogenes in peach, PpYUC11, was suppressed in ripening stony hard peaches. Furthermore, an insertion of a transposon-like sequence was found upstream of the PpYUC11 gene in the 5'-flanking region. Analyses of the segregation ratio of the stony hard phenotype and genotype in F1 progenies indicated that the transposon-inserted allele of PpYUC11, hd-t, correlated with the stony hard phenotype. On the basis of the above findings, we propose that the IPyA pathway (YUCCA pathway) is the main auxin biosynthetic pathway in ripening peaches of 'Akatsuki' and 'Manami' cultivars. Because IAA is not supplied from storage forms, IAAde novo synthesis via the IPyA pathway (YUCCA pathway) in mesocarp tissues is responsible for auxin generation to support fruit softening, and its disruption can lead to the stony hard phenotype.

Aminooxy-naphthylpropionic acid and its derivatives are inhibitors of auxin biosynthesis targeting l-tryptophan aminotransferase: structure-activity relationships.

We previously reported l-α-aminooxy-phenylpropionic acid (AOPP) to be an inhibitor of auxin biosynthesis, but its precise molecular target was not identified. In this study we found that AOPP targets TRYPTOPHAN AMINOTRANSFERASE of ARABIDOPSIS 1 (TAA1). We then synthesized 14 novel compounds derived from AOPP to study the structure-activity relationships of TAA1 inhibitors in vitro. The aminooxy and carboxy groups of the compounds were essential for inhibition of TAA1 in vitro. Docking simulation analysis revealed that the inhibitory activity of the compounds was correlated with their binding energy with TAA1. These active compounds reduced the endogenous indole-3-acetic acid (IAA) content upon application to Arabidopsis seedlings. Among the compounds, we selected 2-(aminooxy)-3-(naphthalen-2-yl)propanoic acid (KOK1169/AONP) and analyzed its activities in vitro and in vivo. Arabidopsis seedlings treated with KOK1169 showed typical auxin-deficient phenotypes, which were reversed by exogenous IAA. In vitro and in vivo experiments indicated that KOK1169 is more specific for TAA1 than other enzymes, such as phenylalanine ammonia-lyase. We further tested 41 novel compounds with aminooxy and carboxy groups to which we added protection groups to increase their calculated hydrophobicity. Most of these compounds decreased the endogenous auxin level to a greater degree than the original compounds, and resulted in a maximum reduction of about 90% in the endogenous IAA level in Arabidopsis seedlings. We conclude that the newly developed compounds constitute a class of inhibitors of TAA1. We designated them 'pyruvamine'.

Biochemical and Chemical Biology Study of Rice OsTAR1 Revealed that Tryptophan Aminotransferase is Involved in Auxin Biosynthesis: Identification of a Potent OsTAR1 Inhibitor, Pyruvamine2031.

IAA, a major form of auxin, is biosynthesized from l-tryptophan via the indole-3-pyruvic acid (IPyA) pathway in Arabidopsis. Tryptophan aminotransferases (TAA1/TARs) catalyze the first step from l-tryptophan to IPyA. In rice, the importance of TAA/TARs or YUC homologs in auxin biosynthesis has been suggested, but the enzymatic activities and involvement of the intermediate IPyA in auxin biosynthesis remain elusive. In this study, we obtained biochemical evidence that the rice tryptophan aminotransferase OsTAR1 converts l-tryptophan to IPyA, and has a Km of 82.02 µM and a Vmax of 10.92 µM min-1 m-1, comparable with those in Arabidopsis. Next, we screened for an effective inhibitor of OsTAR1 from our previously reported inhibitor library for TAA1/TARs, designated pyruvamine (PVM). Differing from previous observations in Arabidopsis, hydroxy-type PVMs, e.g. PVM2031 (previous name KOK2031), had stronger inhibitory effects in rice than the methoxy-type. PVM2031 inhibited recombinant OsTAR1 in vitro. The Ki of PVM2031 was 276 nM. PVM2031 treatment of rice seedlings resulted in morphological changes in vivo, such as reduced lateral root density. Exogenous IAA rescued this growth inhibition, suggesting that the inhibitory effect is auxin specific. Furthermore, rice roots showed reduced IAA levels concomitant with reduced levels of IPyA in the presence of the inhibitors, suggesting that the IPyA pathway is an auxin biosynthesis pathway in rice. Since PVM2031 showed stronger inhibitory effects on rice auxin biosynthesis than known tryptophan aminotransferase inhibitors, we propose that the hydroxy-type PVM2031 is an effective tool for biochemical analysis of the function of auxin biosynthesis in rice roots.

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 SCFTIR1/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 SCFTIR1/AFB-mediated auxin-signaling pathway regulates the expression of YUC genes.

Small-molecule auxin inhibitors that target YUCCA are powerful tools for studying auxin function.

Auxin is essential for plant growth and development, this makes it difficult to study the biological function of auxin using auxin-deficient mutants. Chemical genetics have the potential to overcome this difficulty by temporally reducing the auxin function using inhibitors. Recently, the indole-3-pyruvate (IPyA) pathway was suggested to be a major biosynthesis pathway in Arabidopsis thaliana L. for indole-3-acetic acid (IAA), the most common member of the auxin family. In this pathway, YUCCA, a flavin-containing monooxygenase (YUC), catalyzes the last step of conversion from IPyA to IAA. In this study, we screened effective inhibitors, 4-biphenylboronic acid (BBo) and 4-phenoxyphenylboronic acid (PPBo), which target YUC. These compounds inhibited the activity of recombinant YUC in vitro, reduced endogenous IAA content, and inhibited primary root elongation and lateral root formation in wild-type Arabidopsis seedlings. Co-treatment with IAA reduced the inhibitory effects. Kinetic studies of BBo and PPBo showed that they are competitive inhibitors of the substrate IPyA. Inhibition constants (Ki ) of BBo and PPBo were 67 and 56 nm, respectively. In addition, PPBo did not interfere with the auxin response of auxin-marker genes when it was co-treated with IAA, suggesting that PPBo is not an inhibitor of auxin sensing or signaling. We propose that these compounds are a class of auxin biosynthesis inhibitors that target YUC. These small molecules are powerful tools for the chemical genetic analysis of auxin function.

Transcriptional feedback regulation of YUCCA genes in response to auxin levels in Arabidopsis.

KEY MESSAGE: The IPyA pathway, the major auxin biosynthesis pathway, is transcriptionally regulated through a negative feedback mechanism in response to active auxin levels. The phytohormone auxin plays an important role in plant growth and development, and levels of active free auxin are determined by biosynthesis, conjugation, and polar transport. Unlike conjugation and polar transport, little is known regarding the regulatory mechanism of auxin biosynthesis. We discovered that expression of genes encoding indole-3-pyruvic acid (IPyA) pathway enzymes is regulated by elevated or reduced active auxin levels. Expression levels of TAR2, YUC1, YUC2, YUC4, and YUC6 were downregulated in response to synthetic auxins [1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D)] exogenously applied to Arabidopsis thaliana L. seedlings. Concomitantly, reduced levels of endogenous indole-3-acetic acid (IAA) were observed. Alternatively, expression of these YUCCA genes was upregulated by the auxin biosynthetic inhibitor kynurenine in Arabidopsis seedlings, accompanied by reduced IAA levels. These results indicate that expression of YUCCA genes is regulated by active auxin levels. Similar results were also observed in auxin-overproduction and auxin-deficient mutants. Exogenous application of IPyA to Arabidopsis seedlings preincubated with kynurenine increased endogenous IAA levels, while preincubation with 2,4-D reduced endogenous IAA levels compared to seedlings exposed only to IPyA. These results suggest that in vivo conversion of IPyA to IAA was enhanced under reduced auxin levels, while IPyA to IAA conversion was depressed in the presence of excess auxin. Based on these results, we propose that the IPyA pathway is transcriptionally regulated through a negative feedback mechanism in response to active auxin levels.

Analysis of a putative auxin biosynthesis inhibitor, indole-3-oxoethylphosphonic acid, in Arabidopsis.

Previously we identified indole-3-acetic acid (IAA) biosynthesis inhibitors that act on the conversion of l-tryptophan to indole-3-pyruvic acid in the IAA biosynthesis of Arabidopsis. In the present study, we synthesized a new compound, indole-3-oxoethylphosphonic acid (IOEP), and found that IOEP had an inhibitory effect on IAA biosynthesis in Arabidopsis. The results suggest that IOEP is a novel inhibitor of auxin biosynthesis in Arabidopsis.

Investigation of Auxin Biosynthesis and Action Using Auxin Biosynthesis Inhibitors.

Auxin plays important roles in almost all aspects of plant growth and development. Chemical genetics is an effective approach to understand auxin action, especially in nonmodel plant species, in which auxin-related mutants are not yet available. Among auxin-related chemical tools, we present approaches to utilize auxin biosynthesis inhibitors. The inhibitors are effective not only to understand auxin biosynthesis but also to understand auxin action. The effectiveness of the inhibitors can be assessed based on in vitro or in vivo assays. The in vitro assay employs enzyme inhibition assays. The in vivo assay employs UPLC-MS/MS-based analysis of endogenous IAA and its intermediates or metabolites.

Auxin biosynthesis inhibitors, identified by a genomics-based approach, provide insights into auxin biosynthesis.

Despite its importance in plant growth and development, the auxin biosynthetic pathway has remained elusive. In this study, we analyzed hormone series transcriptome data from AtGenExpress in Arabidopsis and found that aminoethoxyvinylglycine (AVG) had the strongest anti-auxin activity. We also identified other effective compounds such as L-amino-oxyphenylpropionic acid (AOPP) through additional screening. These inhibitors shared characteristics in that they inhibited pyridoxal enzymes and/or aminotransferases. They reduced endogenous IAA levels in both monocots and dicots. L-AOPP inhibited root development of Arabidopsis in main root elongation, gravitropism, root skewing and root hair formation. This inhibition was generally recovered after exogenous IAA treatment, and the recovery was almost completely to the level of non-inhibited seedlings. The compounds inhibited conversion from tryptophan to indole-3-pyruvic acid in enzyme extracts from Arabidopsis and wheat. Our data collectively suggest that the inhibitors directly blocked auxin biosynthesis, and that the major target site was tryptophan aminotransferase. This enzyme probably makes up one of the major biosynthesis pathways conserved among higher plants. Each inhibitor, however, demonstrated a different action spectrum in shoot and root of rice and tomato, indicating diversity in biosynthesis pathways between organs and species. Our results provide novel insights into auxin biosynthesis and action, and uncover structural characteristics of auxin biosynthesis inhibitors.

Arabidopsis seedlings over-accumulated indole-3-acetic acid in response to aminooxyacetic acid.

Previously we identified aminooxy compounds as auxin biosynthesis inhibitors. One of the compounds, aminooxyacetic acid (AOA) inhibited indole-3-acetic acid (IAA) biosynthesis in rice and tomato. Here, we found that AOA induced auxin over-accumulation in Arabidopsis. The results suggest that auxin-related metabolic pathways are divergent among these plant species.

Suppression of elongation and growth of tomato seedlings by auxin biosynthesis inhibitors and modeling of the growth and environmental response.

To develop a growth inhibitor, the effects of auxin inhibitors were investigated. Application of 30 µM L-α-aminooxy-ß-phenylpropionic acid (AOPP) or (S)-methyl 2-((1,3-dioxoisoindolin-2-yl)oxy)-3-phenylpropanoate (KOK1101), decreased the endogenous IAA levels in tomato seedlings at 8 days after sowing. Then, 10-1200 µM AOPP or KOK1101 were sprayed on the leaves and stem of 2-3 leaf stage tomato plants grown under a range of environmental conditions. We predicted plant growth and environmental response using a model based on the observed suppression of leaf enlargement. Spraying AOPP or KOK1101 decreased stem length and leaf area. Concentration-dependent inhibitions and dose response curves were observed. Although the effects of the inhibitors on dry weight varied according to the environmental conditions, the net assimilation rate was not influenced by the inhibitors. Accordingly, the observed decrease in dry weight caused by the inhibitors may result from decreased leaf area. Validation of the model based on observed data independent of the dataset showed good correlations between the observed and predicted values of dry weight and leaf area index.

Aspen pectate lyase PtxtPL1-27 mobilizes matrix polysaccharides from woody tissues and improves saccharification yield.

BACKGROUND: Wood cell walls are rich in cellulose, hemicellulose and lignin. Hence, they are important sources of renewable biomass for producing energy and green chemicals. However, extracting desired constituents from wood efficiently poses significant challenges because these polymers are highly cross-linked in cell walls and are not easily accessible to enzymes and chemicals. RESULTS: We show that aspen pectate lyase PL1-27, which degrades homogalacturonan and is expressed at the onset of secondary wall formation, can increase the solubility of wood matrix polysaccharides. Overexpression of this enzyme in aspen increased solubility of not only pectins but also xylans and other hemicelluloses, indicating that homogalacturonan limits the solubility of major wood cell wall components. Enzymatic saccharification of wood obtained from PL1-27-overexpressing trees gave higher yields of pentoses and hexoses than similar treatment of wood from wild-type trees, even after acid pretreatment. CONCLUSIONS: Thus, the modification of pectins may constitute an important biotechnological target for improved wood processing despite their low abundance in woody biomass.

The UGT73C5 of Arabidopsis thaliana glucosylates brassinosteroids.

Steroid hormones are essential for development, and the precise control of their homeostasis is a prerequisite for normal growth. UDP-glycosyltransferases (UGTs) are considered to play an important regulatory role in the activity of steroids in mammals and insects. This study provides an indication that a UGT accepting plant steroids as substrates functions in brassinosteroid (BR) homeostasis. The UGT73C5 of Arabidopsis thaliana catalyses 23-O-glucosylation of the BRs brassinolide (BL) and castasterone. Transgenic plants overexpressing UGT73C5 displayed BR-deficient phenotypes and contained reduced amounts of BRs. The phenotype, which was already apparent in seedlings, could be rescued by application of BR. In feeding experiments with BL, wild-type seedlings converted BL to the 23-O-glucoside; in the transgenic lines silenced in UGT73C5 expression, no 23-O-glucoside was detected, implying that this UGT is the only enzyme that catalyzes BL-23-O-glucosylation in seedlings. Plant lines in which UGT73C5 expression was altered also displayed hypocotyl phenotypes previously described for seedlings in which BR inactivation by hydroxylation was changed. These data support the hypothesis that 23-O-glucosylation of BL is a function of UGT73C5 in planta, and that glucosylation regulates BR activity.