Prof

Tomokazu Koshiba

e-mail

koshiba-tomokazu@c.metro-u.ac.jp

Asc Prof

Takashi Okamoto

e-mail

okamoto-takashi@c.metro-u.ac.jp

Ast Prof

Toshiko Furukawa

e-mail

furukawa-toshiko(at)tmu.ac.jp

Regulation of auxin (IAA) biosynthesis and its site(s)

The plant hormone auxin was discovered in the late 1920s following studies on phototropism and the growth of grass coleoptiles, and indole-3-acetic acid (IAA) has been identified as a major native auxin in many plants. IAA is now recognized as a master signaling substance involved in almost all aspects of plant growth and development, and also in plant environmental responses. During the last decade, the study of molecular mechanisms of IAA action has greatly advanced, but regulation of IAA biosynthesis and homeostasis have not yet been accurately determined in plants.

In monocots, such as oats, rice and maize, coleoptile tips have long been used as a good material for investigating IAA production as well as polar and lateral transport. IAA produced in the coleoptile tips was proved to move to lower regions and act as a regulator for growth or gravi- and photo-tropic curvature. So far, it is indicated that in the coleoptile tip region there are at least two different signaling pathways involved in the perception of environmental stimuli; one regulates the direction of IAA transport and another attenuates the amount of IAA itself.

Our laboratory has found that the region within 2 mm of the coleoptile tip is definitely the site of IAA biosynthesis from tryptophan, and the IAA production is not affected by conjugated IAA. The IAA synthesized in the tips does not stay there but is immediately transported to the lower regions.

We are now investigating (1) the mechanism of IAA movement in response to gravi- and light-stimuli, (2) the precise site(s) (tissue or cell) of IAA biosynthesis in the maize coleoptile, and (3) intra-cellular IAA distribution and movement. Our final goal is to identify the IAA biosynthetic gene that actually functions in plants.

Regulation of abscisic acid (ABA) biosynthesis and its transport in response to water stress

The plant hormone abscisic acid (ABA) plays a central role in many aspects of plant growth, including seed development, dormancy and germination as well as plant stress responses, such as drought, salinity and low temperature. Under drought stress, an increase in endogenous ABA level is generally observed in various plant species. Most plant species adapt to water shortage by accumulating endogenous ABA to control the expression of numerous stress-responsive genes and cause stomata to close to restrict transpiration. The framework of the ABA biosynthetic pathway in plants has been well established in the last decade. In higher plants, ABA is synthesized from a C40 carotenoid, zeaxanthin. In the plastid, a multi-step process of conversion from zeaxanthin to the C15 intermediate, xanthoxin, is catalyzed by zeaxanthin epoxidase (ZEP), an unidentified violaxanthin isomerase and 9'-cis-epoxycarotenoid dioxygenase (NCED). Successively, xanthoxin is converted to ABA via abscisic aldehyde in the cytosol, catalyzed by a short-chain dehydrogenase/reductase (SDR) and aldehyde oxidase (AO). A variety of studies have indicated that NCED is a key regulatory enzyme in the pathway controlling ABA production for almost all aspects of ABA related plant responses.

However, it still remains to be resolved which tissues/cells are actually producing ABA upon environmental responses. We are focusing on clarifying the spatial and temporal expression profiles of AtNCED3, AtABA2 and AAO3 at the protein and cellular levels in Arabidopsis. Our recent results are indicating that ABA is predominantly synthesized in vascular tissues and stomatal cells. We are also interested in the molecular mechanism of water stress sensing that triggers the rapid ABA biosynthesis in plants.

Investigations for embryogenesis of angiosperms using in vitro fertilization system, a tool to dissect cell specification from a higher plant zygote

Generally, the life of sexual organisms starts from a single cell, the fertilized egg cell or zygote. By cell division and growth this single cell finally gives rise to the mature organism, which contains different cell types, tissues and organs. The zygote, the progenitor cell, is the origin for the formation of cells of different developmental fates and the main body axes. In flowering plants (angiosperms) little is known about the underlying mechanisms of these processes and, in spite of its fundamental importance, regulation of early embryonic development is only poorly understood. New data suggest that the asymmetric division of the zygote separates determinants of apical and basal cell fates and that programs of transcription are initiated in the domains of single cells of the early embryo.

Investigations into the molecular mechanisms of early embryogenesis in higher plants have mostly been conducted through mutant analyses using Arabidopsis, since the plant lends itself to embryogenesis studies in addition to being widely used as a genetic model organism. The fixed pattern of embryo formation in Arabidopsis makes it possible to trace the origin of seedling structures back to the region in the early embryo. Another reason why mutant analyses have been employed for embryogenesis research is the difficulty associated with directly addressing the female gamete, zygote and early embryo in the embryo sac, which is deeply embedded in the ovular tissue. In the late 1980s, technical advances in isolating viable gametes led to successful in vitro combination of male and female gametes, and an in vitro fertilization system was developed using maize gametes. Maize zygotes produced in vitro by electrical fusion of an egg cell with a sperm cell also develop into an asymmetrical two-celled embryo, proembryo and transition-phase embryo via zygotic embryogenesis in a similar manner to that in planta. A major benefit of the in vitro gamete fusion and subsequent culture of zygotes is that the first unequal division of zygotes can be observed directly, and the zygote and two-celled embryo can be used as materials for further analyses. Using the in vitro fertilization system, we are currently focusing on elucidation of the molecular mechanisms involved in the formation of polarity of egg/zygote cells, and in the first unequal cell division of zygotes.

Regulation of rice PR10 (RSOsPR10) expression and its function in relation to environmental stresses

Plants are constantly exposed to a variety of biotic and abiotic stresses. To survive these challenges, plants have developed elaborate mechanisms to perceive external signals and to manifest adaptive responses with proper physiological changes. At the molecular level, the perception of environmental stimuli and the subsequent activation of defense responses require a complex interplay of signaling cascades. One area in which the details of the defense responses are becoming more evident is in the plant's production of pathogen-related (PR) proteins. PR proteins have been defined as plant proteins that are induced during pathogen infection or wounding.

Among 14 different classes of PR protein families, PR10 proteins are primarily small, acidic intracellular proteins of about 16 kDa. Many biotic stresses have been shown to activate PR10 protein expression, suggesting their importance during plant defense responses. However, the exact biological functions of the PR10 proteins are still unclear. A rice PR10 protein was first characterized as a probenazole-inducible protein (PBZ1). The biotic and abiotic stress-inducible nature of the PR10 genes has been investigated including pathogen infection, salt tolerance, UV irradiation, and ozone stress. Some plant hormones and defense-related signaling molecules have been reported to regulate OsPR10 gene expression, such as jasmonic acid, salicylic acid, abscisic acid (ABA), and kinetin. A different type of rice PR10 gene, JIOsPR10, was also identified, which was shown to be up-regulated by jasmonic acid and salicylic acid, and by pathogen infection. However, there has been no direct evidence to show their physiological activity and function in plant-defense mechanisms.

During the course of proteome analysis in rice roots and isolation of stress response proteins, we found a protein, RSOsPR10, that was induced by salt and drought stresses. We cloned the full-length cDNA and revealed that the gene encoded a novel PR10 protein. Now we are investigating the regulation mechanism of the gene expression of RSOsPR10 in relation to abiotic and biotic stresses and the defense-related signaling molecules for RSOsPR10 expression.
We have produced RSOsPR10 introducing transgenic rice to investigate the physiological function of this gene in defense responses in rice. Overexpression lines showed high tolerance against drought and hight salt stress.
Furthermore, we ingestigated the localization of the protein in rice root after NaCl treatment. The protein existed around vascular system.

Related Links

Recent Publications

1. Main papers, reviews and books. Please check "Related Links" for the whole of the list.
3. - Auxin,ABA -
4. Melhorn, V., Matsumi, K., Koiwai, H., Ikegami, K., Okamoto, M., Nambara, E., Bittner, F., Koshiba, T. (2007) Transient expression of AtNCED3 and AAO3 genes in guard cells causes the stomatal closure in Vicia faba L. J. Plant Res. 121: 125-131.
5. Okamoto, M., Kuwahara, A., Seo, M., Kushiro, T., Asami, T., Hirai, N., Kamiya, Y., Koshiba, T., Nambara, E. (2006) CYP707A1 and CYP707A2, which encode ABA 8�f-hydroxylases, are indispensable for a proper control of seed dormancy and germination in Arabidopsis. Plant Physiol. 141: 97-107.
6. Nishimura, T., Mori, Y., Furukawa, T., Kadota, A., Koshiba, T. (2006) Red light causes a reduction in IAA levels at the apical tip by inhibiting de novo biosynthesis from tryptophan in maize coleoptiles. Planta 224: 1427-1435.
7. Seo, M., Aoki, H., Koiwai, H., Kamiya, Y., Nambara, E., Koshiba, T. (2004) Comparative studies on the Arabidopsis aldehyde oxidase (AAO) gene family revealed a major role of AAO3 in ABA biosynthesis in seeds. Plant Cell Physiol., 45:1694-1703.
8. Kushiro, T., Okamoto, M., Nakabayashi, K., Yamagishi, K., Kitamura, S., Asami, T., Hirai, N., Koshiba, T., Kamiya, Y., Nambara, E. (2004) The Arabidopsis cytochrome P450 CYP707A encodes ABA 8�f-hydroxylases: key enzymes in ABA catabolism. EMBO J., 23:1647-1656.
9. Koiwai, H, Nakaminami, K., Seo, M, Mitsuhashi, W, Toyomasu, T, Koshiba, T. (2004) Tissue-specific localization of an abscisic acid biosynthetic enzyme, AAO3, in Arabidopsis. Plant Physiol. 134: 1697-1707.
10. Cheng, W.-H., Endo, A., Zhou, L., Penney, J., Chen, H.-C., Arroyo, A., Leon, P., Nambara, E., Asami, T., Seo, M., Koshiba, T., Sheen, J. (2002) A unique short-chain dehydrogenase/reductase in Arabidopsis glucose signaling and abscisic acid biosynthesis and functions. Plant Cell, 14: 2723-2743.
11. Seo, M., Koshiba, T. (2002) Complex regulation of ABA biosynthesis in plants. Trends in Plant Science, 7: 41-48.
12. Seo, M., Peeters, A.J.M., Koiwai, H., Oritani, T., Marion-Poll, A., Zeevaart, J.A.D., Koornneef, M., Kamiya, Y., Koshiba, T. (2000) The Arabidopsis aldehyde oxidase 3 (AAO3) gene product catalyzes the final step in abscisic acid biosynthesis in leaves. Proc. Natl. Acad. Sci. USA., 97: 12908-12913.
13. Koshiba, T., Saito, E., Ono, N., Yamamoto, N., Sato, M. (1996) Purification and properties of flavin- and molybdenum-containing aldehyde oxidase from coleoptiles of maize. Plant Physiol., 110: 781-789.
14. Koshiba, T., Kamiya, Y., Iino, M. (1995) Biosynthesis of indole-3-acetic acid from L-tryptophan in coleoptile tips of maize (Zea mays L.). Plant Cell Physiol., 36: 1503-1510.
15. Koshiba, T., Ballas, N., Wong, L.-M., Theologis, A. (1995) Transcriptional regulation of PS-IAA4/5 and PS-IAA6 eary gene expression by indoleacetic acid and protein synthesis inhibitors in pea (Pisum sativum). J. Mol. Biol., 253: 396-413.
16. - embryogenesis of angiosperms using in vitro fertilization system -
17. Uchiumi, T., Uemura, I., Okamoto, T. (2007) Establishment of an in vitro fertilization system in rice (Oryza sativa L.). Planta 226:581-589.
18. Uchiumi, T., Komatsu, S., Koshiba, T., Okamoto, T. (2006) Isolation of gametes and central cells from Oryza sativa L. Sex. Plant Reprod. 19: 37-45.
19. Okamoto, T., Scholten, S., Lo�Nrz H., Kranz, E. (2005) Identification of genes that are up- or down-regulated in the apical or basal cell of maize two-celled embryos and monitoring their expressions during zygote development by a cell manipulation- and PCR-based approach. Plant Cell Physiol., 46:332-338.
20. Okamoto, T., Higuchi, K. Shinkawa, T., Isobe, T., Lorz, H., Koshiba, T., Kranz, E. (2004) Identification of major proteins in maize egg cells. Plant Cell Physiol., 45: 1406-1412.
21. - PR10 -
22. Hashimoto, M., Kiseleva, L., Sawa, S., Furukawa, T., Komatsu, S., Koshiba, T. (2004) A novel rice PR10 protein, RSOsPR10, specifically induced in roots by biotic and abiotic stresses, possibly via the jasmonic acid signaling pathway. Plant Cell Physiol., 45: 550-559.