The regulation of abscisic acid (ABA) biosynthesis is essential for plant responses to drought stress. In this study, we examined the tissue-specific localization of ABA biosynthetic enzymes in turgid and dehydrated Arabidopsis (Arabidopsis thaliana) plants using specific antibodies against 9-cis-epoxycarotenoid dioxygenase 3 (AtNCED3), AtABA2, and Arabidopsis aldehyde oxidase 3 (AAO3). Immunohistochemical analysis revealed that in turgid plants, AtABA2 and AAO3 proteins were localized in vascular parenchyma cells most abundantly at the boundary between xylem and phloem bundles, but the AtNCED3 protein was undetectable in these tissues. In water-stressed plants, AtNCED3 was detected exclusively in the vascular parenchyma cells together with AtABA2 and AAO3. In situ hybridization using the antisense probe for AtNCED3 showed that the drought-induced expression of AtNCED3 was also restricted to the vascular tissues. Expression analysis of laser-microdissected cells revealed that, among nine drought-inducible genes examined, the early induction of most genes was spatially restricted to vascular cells at 1 h and then some spread to mesophyll cells at 3 h. The spatial constraint of AtNCED3 expression in vascular tissues provides a novel insight into plant systemic response to drought stresses.

Fusicoccins are a class of diterpene glucosides produced by the plant-pathogenic fungus Phomopsis amygdali. As modulators of 14-3-3 proteins, fusicoccins function as potent activators of plasma membrane H ؉ -ATPase in plants and also exhibit unique biological activity in animal cells. Despite their well studied biological activities, no genes encoding fusicoccin biosynthetic enzymes have been identified. Cyclic diterpenes are commonly synthesized via cyclization of a C20 precursor, geranylgeranyl diphosphate (GGDP), which is produced through condensation of the universal C5 isoprene units dimethylallyl diphosphate and isopentenyl diphosphate by prenyltransferases. We found that (؉)-fusicocca-2,10 (14)-diene, a tricyclic hydrocarbon precursor for fusicoccins, is biosynthesized from the C5 isoprene units by an unusual multifunctional enzyme, P. amygdali fusicoccadiene synthase (PaFS), which shows both prenyltransferase and terpene cyclase activities. The functional analysis of truncated mutants and site-directed mutagenesis demonstrated that PaFS consists of two domains: a terpene cyclase domain at the N terminus and a prenyltransferase domain at the C terminus. These findings suggest that fusicoccadiene can be produced efficiently in the fungus by using the C5 precursors, irrespective of GGDP availability. In fact, heterologous expression of PaFS alone resulted in the accumulation of fusicocca-2,10 (14)-diene in Escherichia coli cells, whereas no product was detected in E. coli cells expressing Gibberella fujikuroi ent-kaurene synthase, another fungal diterpene cyclase that also uses GGDP as a substrate but does not contain a prenyltransferase domain. Genome walking suggested that fusicoccin biosynthetic enzymes are encoded as a gene cluster near the PaFS gene.biosynthesis ͉ cyclase ͉ prenyltransferase F usicoccins (FC) A (1, 2) and J (3, 4) ( Fig. 1) are the major metabolites produced by the plant-pathogenic fungus Phomopsis (Fusicoccum) amygdali (Del.) (5). Fusicoccin A and the structurally related cotylenins (CN) (6-8), e.g., cotylenin A (Fig.

Germination of lettuce (Lactuca sativa L.) seed is regulated by phytochrome. The requirement for red light is circumvented by the application of gibberellin (GA). We have previously shown that the endogenous content of GA 1 , the main bioactive GA in lettuce seeds, increases after red-light treatment. To clarify which step of GA 1 synthesis is regulated by phytochrome, cDNAs encoding GA 20-oxidases (Ls20ox1 and Ls20ox2, for L. sativa GA 20-oxidase) and 3␤-hydroxylases (Ls3h1 and Ls3h2 for L. sativa GA 3␤-hydroxylase) were isolated from lettuce seeds by reverse-transcription polymerase chain reaction. Functional analysis of recombinant proteins expressed in Escherichia coli confirmed that the Ls20ox and Ls3h encode GA 20-oxidases and 3␤-hydroxylases, respectively. Northern-blot analysis showed that Ls3h1 expression was dramatically induced by red-light treatment within 2 h, and that this effect was canceled by a subsequent far-red-light treatment. Ls3h2 mRNA was not detected in seeds that had been allowed to imbibe under any light conditions. Expression of the two Ls20ox genes was induced by initial imbibition alone in the dark. The level of Ls20ox2 mRNA decreased after the red-light treatment, whereas that of Ls20ox1 was unaffected by light. These results suggest that red light promotes GA 1 synthesis in lettuce seeds by inducing Ls3h1 expression via phytochrome action.

SummaryRice (Oryza sativa L.) produces ent-copalyl diphosphate (ent-CDP) and syn-CDP as precursors for several classes of phytoalexins and the phytohormones, gibberellins (GAs). It has recently been shown that a loss-offunction mutation of OsCPS1, a gene encoding a putative ent-CDP synthase, results in a severely GA-deficient dwarf phenotype in rice. To clarify the biological functions of the ent-and syn-CDP synthases involved in the biosynthesis of phytoalexins and/or GAs, we isolated two cDNAs, OsCyc1 and OsCyc2, encoding putative diterpene cyclases from ultraviolet (UV)-irradiated rice leaves (cv. Nipponbare). The production of phytoalexins in rice leaves is known to be highly induced by UV treatment. Using a bacterial expression system, we demonstrated that OsCyc1 encodes syn-CDP synthase and that OsCyc2 and OsCPS1 encode ent-CDP synthase. The level of expression of the OsCyc1 and OsCyc2 transcripts in rice leaves increased drastically in response to UV treatment, whereas expression of the OsCPS1 transcript was not induced by UV light. These results suggest that OsCyc1, OsCyc2 and OsCPS1 are responsible for the biosynthesis of momilactones A and B and oryzalexin S, oryzalexins A-F and phytocassanes A-E, and GAs, respectively. Our results strongly suggest the presence of two ent-CDP synthase isoforms in rice, one that participates in the biosynthesis of GAs and a second that is involved in the biosynthesis of phytoalexins.

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.

SUMMARYPlants frequently possess operon-like gene clusters for specialized metabolism. Cultivated rice, Oryza sativa, produces antimicrobial diterpene phytoalexins represented by phytocassanes and momilactones, and the majority of their biosynthetic genes are clustered on chromosomes 2 and 4, respectively. These labdanerelated diterpene phytoalexins are biosynthesized from geranylgeranyl diphosphate via ent-copalyl diphosphate or syn-copalyl diphosphate. The two gene clusters consist of genes encoding diterpene synthases and chemical-modification enzymes including P450s. In contrast, genes for the biosynthesis of gibberellins, which are labdane-related phytohormones, are scattered throughout the rice genome similar to other plant genomes. The mechanism of operon-like gene cluster formation remains undefined despite previous studies in other plant species. Here we show an evolutionary insight into the rice gene clusters by a comparison with wild Oryza species. Comparative genomics and biochemical studies using wild rice species from the AA genome lineage, including Oryza barthii, Oryza glumaepatula, Oryza meridionalis and the progenitor of Asian cultivated rice Oryza rufipogon indicate that gene clustering for biosynthesis of momilactones and phytocassanes had already been accomplished before the domestication of rice. Similar studies using the species Oryza punctata from the BB genome lineage, the distant FF genome lineage species Oryza brachyantha and an outgroup species Leersia perrieri suggest that the phytocassane biosynthetic gene cluster was present in the common ancestor of the Oryza species despite the different locations, directions and numbers of their member genes. However, the momilactone biosynthetic gene cluster evolved within Oryza before the divergence of the BB genome via assembly of ancestral genes.

Rice (Oryza sativa L.) produces diterpene phytoalexins, such as momilactones, oryzalexins, and phytocassanes. Using rice genome information and in vitro assay with recombinant enzymes, we identified genes (OsKS4 and OsKS10) encoding the type-A diterpene cyclases 9beta-pimara-7,15-diene synthase and ent-sandaracopimaradiene synthase which are involved in the biosynthesis of momilactones A, B and oryzalexins A-F respectively. Transcript levels of these two genes increased remarkably after ultraviolet (UV) treatment, which is consistent with elevated production of phytoalexins by UV. These two genes might prove powerful tools for understanding plant defense mechanisms in rice.

SummaryWe have isolated and characterized a cDNA encoding a novel diterpene cyclase, OsDTC1, from suspensioncultured rice cells treated with a chitin elicitor. OsDTC1 functions as ent-cassa-12,15-diene synthase, which is considered to play a key role in the biosynthesis of (À)-phytocassanes recently isolated as rice diterpenoid phytoalexins. The expression of OsDTC1 mRNA was also con®rmed in ultraviolet (UV)-irradiated rice leaves. In addition, we identi®ed ent-cassa-12,15-diene, a putative diterpene hydrocarbon precursor of (À)-phytocassanes, as an endogenous compound in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves. The OsDTC1 cDNA isolated here will be a useful tool to investigate the regulatory mechanisms of the biosyntheis of (À)-phytocassanes in rice.