Tomokazu Koshiba

The Arabidopsis cytochrome P450 CYP707A encodes ABA 8′‐hydroxylases: key enzymes in ABA catabolism

The hormonal action of abscisic acid (ABA) in plants is controlled by the precise balance between its biosynthesis and catabolism. In plants, ABA 8′‐hydroxylation is thought to play a predominant role in ABA catabolism. ABA 8′‐hydroxylase was shown to be a cytochrome P450 (P450); however, its corresponding gene had not been identified. Through phylogenetic and DNA microarray analyses during seed imbibition, the candidate genes for this enzyme were narrowed down from 272 Arabidopsis P450 genes. These candidate genes were functionally expressed in yeast to reveal that members of the CYP707A family, CYP707A1–CYP707A4, encode ABA 8′‐hydroxylases. Expression analyses revealed that CYP707A2 is responsible for the rapid decrease in ABA level during seed imbibition. During drought stress conditions, all CYP707A genes were upregulated, and upon rehydration a significant increase in mRNA level was observed. Consistent with the expression analyses, cyp707a2 mutants exhibited hyperdormancy in seeds and accumulated six‐fold greater ABA content than wild type. These results demonstrate that CYP707A family genes play a major regulatory role in controlling the level of ABA in plants.

Tissue-Specific Localization of an Abscisic Acid Biosynthetic Enzyme, AAO3, in Arabidopsis

Arabidopsis aldehyde oxidase 3 (AAO3) is an enzyme involved in abscisic acid (ABA) biosynthesis in response to drought stress. Since the enzyme catalyzes the last step of the pathway, ABA production sites may be determined by the presence of AAO3. Here, AAO3 localization was investigated using AAO3 promoter:AAO3-GFP transgenic plants and by an immunohistochemical technique. AAO3-GFP protein exhibited an activity to produce ABA from abscisic aldehyde, and the transgene restored the wilty phenotype of the aao3 mutant. GFP-fluorescence was detected in the root tips, vascular bundles of roots, hypocotyls and inflorescence stems, and along the leaf veins. Intense immunofluorescence signals were localized in phloem companion cells and xylem parenchyma cells. Faint but significant GFP- and immuno-fluorescence signals were observed in the leaf guard cells. In situ hybridization with antisense AAO3 mRNA showed AAO3 mRNA expression in the guard cells of dehydrated leaves. These results indicate that the ABA synthesized in vascular systems is transported to various target tissues and cells, and also that the guard cells themselves are able to synthesize ABA.

Interaction of Auxin and ERECTA in Elaborating Arabidopsis Inflorescence Architecture Revealed by the Activation Tagging of a New Member of the YUCCA Family Putative Flavin Monooxygenases

The aboveground body of higher plants has a modular structure of repeating units, or phytomers. As such, the position, size, and shape of the individual phytomer dictate the plant architecture. The Arabidopsis (Arabidopsis thaliana) ERECTA (ER) gene regulates the inflorescence architecture by affecting elongation of the internode and pedicels, as well as the shape of lateral organs. A large-scale activation-tagging genetic screen was conducted in Arabidopsis to identify novel genes and pathways that interact with the ER locus. A dominant mutant, super1-D, was isolated as a nearly complete suppressor of a partial loss-of-function allele er-103. We found that SUPER1 encodes YUCCA5, a novel member of the YUCCA family of flavin monooxygenases. The activation tagging of YUCCA5 conferred increased levels of free indole acetic acid, increased auxin response, and mild phenotypic characteristics of auxin overproducers, such as elongated hypocotyls, epinastic cotyledons, and narrow leaves. Both genetic and cellular analyses indicate that auxin and the ER pathway regulate cell division and cell expansion in a largely independent but overlapping manner during elaboration of inflorescence architecture.

A plant growth retardant, uniconazole, is a potent inhibitor of ABA catabolism in Arabidopsis

Plant growth retardants (PGRs) reduce the shoot growth of plants by inhibiting gibberellin biosynthesis. In this study, we performed detailed analyses of the inhibitory effects of PGRs on Arabidopsis abscisic acid (ABA) 8′-hydroxylase, a major ABA catabolic enzyme, recently identified as CYP707As. In an in vitro assay with CYP707A3 microsomes expressed in insect cells, uniconazole-P inhibited CYP707A3 activity more effectively than paclobutrazol or tetcyclacis, whereas the other PGRs tested did not inhibit it significantly. Uniconazole-P was found to be a strong competitive inhibitor (K i=8.0 nM) of ABA 8′-hydroxylase. Uniconazole-P-treated Arabidopsis plants showed enhanced drought tolerance. In uniconazole-P-treated plants, endogenous ABA levels increased 2-fold as compared with the control, and co-application of GA4 did not suppress the effects, indicating that the effects were not due to gibberellin deficiency. Thus uniconazole-P effectively inhibits ABA catabolism in Arabidopsis plants. We also discuss the structure-activity relationship of the azole-type compounds on ABA 8′-hydroxylase inhibitory activity.

Abscisic acid and stress treatment are essential for the acquisition of embryogenic competence by carrot somatic cells

Studies of carrot embryogenesis have suggested that abscisic acid (ABA) is involved in somatic embryogenesis. A relationship between endogenous ABA and the induction of somatic embryogenesis was demonstrated using stress-induced system of somatic embryos. The embryonic-specific genes C-ABI3 and embryogenic cell proteins (ECPs) were expressed during stress treatment prior to the formation of somatic embryos. The stress-induction system for embryogenesis was clearly distinguished by two phases: the acquisition of embryogenic competence and the formation of a somatic embryo. Somatic embryo formation was inhibited by the application of fluridone (especially at 10−4xa0M), a potent inhibitor of ABA biosynthesis, during stress treatment. The inhibitory effect of fluridone was nullified by the simultaneous application of fluridone and ABA. The level of endogenous ABA increased transiently during stress. However, somatic embryogenesis was not significantly induced by the application of only ABA to the endogenous level, in the absence of stress. These results suggest that the induction of somatic embryogenesis, in particular the acquisition of embryogenic competence, is caused not only by the presence of ABA but also by physiological responses that are directly controlled by stresses.

Effect of ABA upon anthocyanin synthesis in regenerated torenia shoots

To elucidate the mechanism of anthocyanin synthesis induction concomitant with chlorophyll degradation, we established a system in which anthocyanin synthesis and degradation of chlorophyll in regenerated torenia (Torenia fournieri) shoots was induced on medium containing 7% sucrose. Here, we studied the effect of several plant-growth regulators on anthocyanin synthesis and the degradation of chlorophyll in the torenia shoot regenerating system. Exogenous abscisic acid (ABA) could induce anthocyanin synthesis and chlorophyll senescence in regenerating torenia shoots on the medium containing a low concentration of sucrose (1.5%). We determined the changes in the amount of endogenous ABA in the regenerated shoots during the process of anthocyanin synthesis on the medium containing 7% sucrose. It was revealed that the 7% sucrose treatment elevated endogenous ABA levels before the induction of anthocyanin synthesis and chlorophyll degradation. However, while retransfer to the 1.5% sucrose medium resulted in a gradual decrease in the ABA level and a failure of induction of anthocyanin synthesis, normal shoot regeneration. These results suggest that changes in the amount of endogenous ABA may play an important role in the induction of anthocyanin synthesis and chlorophyll degradation in regenerated torenia shoots.

Aldehyde oxidase (AO) in the root nodules of Lupinus albus and Medicago truncatula: identification of AO in meristematic and infection zones

Phytohormones are involved in the organogenesis of legume root nodules. The source of the auxin indole-3-acetic acid (IAA) in nodules has not been clearly determined. We studied the enzyme aldehyde oxidase (AO; EC 1.2.3.1), that catalyzes the last step of IAA biosynthesis in plants, in the nodules of Lupinus albus and Medicago truncatula. Primordia and young lupin nodules and mature M. truncatula nodules showed AO activity bands after native polyacrylamide gel electrophoresis. Gel activity analyses using indole-3-aldehyde as substrate indicated that the nodules of white lupin and M. truncatula have the capability to synthesize IAA via the indole-3-pyruvic acid pathway. Immunolocalization and in situ hybridization experiments revealed that AO is preferentially expressed in the meristematic and the invasion zones in Medicago nodules and in the lateral meristematic zone of Lupinus nodules. High IAA immunolabeling was also detected in the meristematic and invasion zones. Low expression levels and no AO activity were detected in lupin Fix- nodules that displayed restricted growth and early senescence. We propose that local synthesis of IAA in the root nodule meristem and modulation of AO expression and activity are involved in regulation of nodule development.

Isolation of gametes and central cells from Oryza sativa L.

In vitro fertilization system of higher plants has been well established using maize gametes and central cells, which can produce embryos and endosperms. In the present study, procedures for isolating gametes and central cells from rice (Oryza sativa L. cv. Nipponbare), a model plant, are reported with the goal of establishing rice in vitro fertilization system. Egg cells and central cells were isolated by manual manipulation of enzyme-treated unpollinated ovules, and an alternative direct isolation method for egg cells that does not use enzymatic treatment was also established. Fluorescent visualization of the granular structures in the cytoplasm of isolated egg cells and the nucleoli in two polar nuclei of isolated central cells suggest that these cells are reliable gametes and central cells. For sperm cell isolation, the contents of rice pollen grains were released by osmotic pressure-induced bursting of the grains. In addition, electrofusion with isolated gametes was successfully conducted.

Red light causes a reduction in IAA levels at the apical tip by inhibiting de novo biosynthesis from tryptophan in maize coleoptiles

When maize coleoptiles were unilaterally exposed to red light (7.9xa0μmol m−2s−1 for 5xa0min), 3xa0h after treatment IAA levels in coleoptiles decreased in all regions, from top to basal, with levels about 60% of dark controls. Localized irradiation in the 5xa0mm top zone was sufficient to cause the same extent of IAA reduction in the tips to that in the tips of whole irradiated shoots. When coleoptiles were treated with N-1-naphthylphthalamic acid (NPA), an accumulation of IAA in the tip and a decrease of diffusible IAA from tips were simultaneously detected. IAA accumulation in red-light treated coleoptiles by NPA was much lower than that of dark controls. NPA treatment did not affect the content of conjugated IAA in either dark or light treated coleoptile tips. When 13C1115N2-tryptophan (Trp) was applied to the top of coleoptiles, substantial amounts of stable isotope were incorporated into free IAA in dark and red-light treated coleoptile tips. The ratio of incorporation was slightly lower in red-light treated coleoptile tips than that in dark controls. The label could not be detected in conjugated IAA. The rate of basipetal transport of IAA was about 10xa0mm h−1 and the velocity was not affected by red light. These results strongly suggest that red light does not affect the rates of conversion of free IAA to the conjugate form or of the basipetal transport, but just reduces the IAA level in the tips, probably inhibited by IAA biosynthesis from Trp in this region.

Possible involvement of abscisic acid in the induction of secondary somatic embryogenesis on seed-coat-derived carrot somatic embryos

When seed coats (pericarps) were picked from 14-day-old carrot (Daucus carota) seedlings and cultured on agar plates, embryogenic cell clusters were produced very rapidly at a high frequency on the open side edge. Embryo induction progressed without auxin treatment; indeed treatment caused the formation of non-embryogenic callus. The embryogenic tissues (primary embryos) developed normally until the torpedo stage; however, after this a number of secondary somatic embryos were produced in the hypocotyl and root regions. “Tertiary” embryos were formed on some of the secondary embryos, but many developed into normal plantlets. The primary embryos contained significantly higher levels of abscisic acid (ABA) than the hypocotyl-derived normal and seed-coat-derived secondary embryos. Fluridone inhibited the induction of secondary embryogenesis, while exogenously supplied ABA induced not only “tertiary” embryogenesis on the seed-coat-derived secondary embryos, but also secondary embryos on the hypocotyl-derived normal somatic embryos. These results indicate that ABA is one of the important endogenous factors for the induction of secondary embryogenesis on carrot somatic embryos. Higher levels of indole-3-acetic acid (IAA) in primary embryos also suggest the presence of some concerted effect of ABA and IAA on the induction of secondary embryogenesis in primary embryos.