Yusuke Kakei

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

Key messageThe IPyA pathway, the major auxin biosynthesis pathway, is transcriptionally regulated through a negative feedback mechanism in response to active auxin levels.AbstractThe 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.

Transcriptome analysis of hormone-induced gene expression in Brachypodium distachyon.

Brachypodium distachyon is a new model plant closely related to wheat and other cereals. In this study, we performed a comprehensive analysis of hormone-regulated genes in Brachypodium distachyon using RNA sequencing technology. Brachypodium distachyon seedlings were treated with eight phytohormones (auxin, cytokinine, brassinosteroid, gibberelline, abscisic acid, ethylene, jasmonate and salicylic acid) and two inhibitors, Brz220 (brassinosteroid biosynthesis inhibitor) and prohexadione (gibberelline biosynthesis inhibitor). The expressions of 1807 genes were regulated in a phytohormone-dependent manner. We compared the data with the phytohormone responses that have reported in rice. Transcriptional responses to hormones are conserved between Bracypodium and rice. Transcriptional regulation by brassinosteroid, gibberellin and ethylene was relatively weaker than those by other hormones. This is consistent with the data obtained from comprehensive analysis of hormone responses reported in Arabidopsis. Brachypodium and Arabidopsis also shared some common transcriptional responses to phytohormones. Alternatively, unique transcriptional responses to phytohormones were observed in Brachypodium. For example, the expressions of ACC synthase genes were up-regulated by auxin treatment in rice and Arabidopsis, but no orthologous ACC synthase gene was up-regulated in Brachypodium. Our results provide information useful to understand the diversity and similarity of hormone-regulated transcriptional responses between eudicots and monocots.

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.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.

BPG3 is a novel chloroplast protein that involves the greening of leaves and related to brassinosteroid signaling

Brassinosteroids are plant steroid hormones that regulate plant organs and chloroplast development. The detailed molecular mechanism for plant development by BR signaling is yet to be revealed, and many points regarding the relationship between BR signaling and chloroplast development remain unknown. We identify here the dominant mutant Brz-insensitive-pale green3-1D (bpg3-1D) from the Arabidopsis FOX lines that show reduced sensitivity to the chlorophyll accumulation promoted by the BR biosynthesis inhibitor, Brassinazole (Brz), in the light. BPG3 encodes a novel chloroplast protein that is evolutionally conserved in bacteria, algae, and higher plants. The expression of BPG3 was induced by light and Brz. The inhibition of electron transport in photosystem II of the chloroplasts was detected in bpg3-1D. These results suggest that BPG3 played an important role in regulating photosynthesis in the chloroplast under BR signaling. Graphical Abstract BR-inhibitor Brz activated chloroplast functions. bpg3 (Brz-insensitive-pale green3) mutant is disrupted in the signaling from BR-deficient condition to chloroplast activation.

Extreme suppression of lateral floret development by a single amino acid change in the VRS1 transcription factor

Extreme suppression of lateral floret development in deficiens barley is the result of a single amino acid substitution in the homeodomain-leucine zipper class I transcription factor VRS1. Increasing grain yield is an endless challenge for cereal crop breeding. In barley (Hordeum vulgare), grain number is controlled mainly by Six-rowed spike 1 (Vrs1), which encodes a homeodomain leucine zipper class I transcription factor. However, little is known about the genetic basis of grain size. Here, we show that extreme suppression of lateral florets contributes to enlarged grains in deficiens barley. Through a combination of fine-mapping and resequencing of deficiens mutants, we have identified that a single amino acid substitution at a putative phosphorylation site in VRS1 is responsible for the deficiens phenotype. deficiens mutant alleles confer an increase in grain size, a reduction in plant height, and a significant increase in thousand grain weight in contemporary cultivated germplasm. Haplotype analysis revealed that barley carrying the deficiens allele (Vrs1.t1) originated from two-rowed types carrying the Vrs1.b2 allele, predominantly found in germplasm from northern Africa. In situ hybridization of histone H4, a marker for cell cycle or proliferation, showed weaker expression in the lateral spikelets compared with central spikelets in deficiens. Transcriptome analysis revealed that a number of histone superfamily genes were up-regulated in the deficiens mutant, suggesting that enhanced cell proliferation in the central spikelet may contribute to larger grains. Our data suggest that grain yield can be improved by suppressing the development of specific organs that are not positively involved in sink/source relationships.

Genome-wide analysis of specific alterations in transcript structure and accumulation caused by nutrient deficiencies in Arabidopsis thaliana

The alteration of transcript structure contributes to transcriptome plasticity. In this study, we analyzed the genome-wide response of exon combination patterns to deficiencies in 12 different nutrients in Arabidopsis thaliana roots. RNA sequencing analysis and bioinformatics using a simulation survey revealed more than 600 genes showing varying exon combinations. The overlap between genes showing differential expression (DE) and genes showing differential exon combination (DC) was notably low. Additionally, gene ontology analysis showed that gene functions were not shared between the DE and DC genes, suggesting that the genes showing DC had different roles than those showing DE. Most of the DC genes were nutrient specific. For example, two homologs of the MYB transcription factor genes MYB48 and MYB59 showed differential alternative splicing only in response to low levels of potassium. Alternative splicing of those MYB genes modulated DNA-binding motifs, and MYB59 is reportedly involved in the inhibition of root elongation. Therefore, the increased abundance of MYB isoforms with an intact DNA-binding motif under low potassium may be involved in the active inhibition of root elongation. Overall, we provide global and comprehensive data for DC genes affected by nutritional deficiencies, which contribute to elucidating an unknown mechanism involved in adaptation to nutrient deficiency.

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.