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.

Germination of lettuce (Lactuca sativa) 'Grand Rapids' seeds is regulated by phytochrome. The action of phytochrome includes alterations in the levels of gibberellin (GA) and abscisic acid (ABA). To determine the molecular mechanism of phytochrome regulation of ABA metabolism, we isolated four lettuce cDNAs encoding 9-cis-epoxycarotenoid dioxygenase (biosynthesis; LsNCED1-LsNCED4) and four cDNAs for ABA 8#-hydroxylase (catabolism; LsABA8ox1-LsABA8ox4). Measurements of ABA and its catabolites showed that a decrease in ABA level coincided with a slight increase in the level of the ABA catabolite phaseic acid after red light treatment. Quantitative reverse transcription-polymerase chain reaction analysis indicated that ABA levels are controlled by phytochrome through down-regulation of LsNCED2 and LsNCED4 expression and up-regulation of LsABA8ox4 expression in lettuce seeds. Furthermore, the expression levels of LsNCED4 decreased after GA 1 treatment, whereas the levels of expression of the other two genes were unaffected. The LsNCED4 expression was also down-regulated by red light in lettuce seeds in which GA biosynthesis was suppressed by AMO-1618, a specific GA biosynthesis inhibitor. These results indicate that phytochrome regulation of ABA metabolism is mediated by both GA-dependent and -independent mechanisms. Spatial analysis showed that after red light treatment, the ABA decrease on the hypocotyl side was greater than that on the cotyledon side of lettuce seeds. Moreover, phytochrome-regulated expression of ABA and GA biosynthesis genes was observed on the hypocotyl side, rather than the cotyledon side, suggesting that this regulation occurs near the photoperceptive site.Plants undergo correct morphogenesis by responding to light and adapt to various forms of light. In addition to imbibition, some seeds require light to germinate and are called photoblastic seeds. Photoblastic seeds germinate after irradiation with red light (R; wavelength approximately 600-700 nm), and this effect of R is cancelled by successive irradiation with far-red light (FR; wavelength approximately 700-750 nm). The effects of R and FR are reversible. This type of germination, called photogermination, was discovered by experiments using lettuce (Lactuca sativa) 'Grand Rapids' seeds (Borthwick et al., 1952). The energy from wavelengths of R is mainly required for photosynthesis. This strategy allows survival of small seeds in which storage substances are low, such as the seeds of lettuce, tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), and Arabidopsis (Arabidopsis thaliana). The typical reversible photoreaction of lettuce seeds allowed Butler et al. (1959) to detect the pigment that acts as the R/FR receptor in extracts from etiolated cotyledons of Brassica rapa and etiolated shoots of Zea mays using differential spectrophotometry, and this photoreceptor was named phytochrome.Many studies to elucidate the mechanisms of photoblastic lettuce seed germination have been carried out for over a half century, including biochemica...

Phytochrome regulates lettuce (Lactuca sativa L. cv. Grand Rapids) seed germination via the control of the endogenous level of bioactive gibberellin (GA). In addition to the previously identified LsGA20ox1, LsGA20ox2, LsGA3ox1, LsGA3ox2, LsGA2ox1, and LsGA2ox2, five cDNAs were isolated from lettuce seeds: LsCPS, LsKS, LsKO1, LsKO2, and LsKAO. Using an Escherichia coli expression system and functional assays, it is shown that LsCPS and LsKS encode ent-copalyl diphosphate synthase and ent-kaurene synthase, respectively. Using a Pichia pastoris system, it was found that LsKO1 and LsKO2 encode ent-kaurene oxidases and LsKAO encodes ent-kaurenoic acid oxidase. A comprehensive expression analysis of GA metabolism genes using the quantitative reverse transcription polymerase chain reaction suggested that transcripts of LsGA3ox1 and LsGA3ox2, both of which encode GA 3-oxidase for GA activation, were primarily expressed in the hypocotyl end of lettuce seeds, were expressed at much lower levels than the other genes tested, and were potently up-regulated by phytochrome. Furthermore, LsDELLA1 and LsDELLA2 cDNAs that encode DELLA proteins, which act as negative regulators in the GA signalling pathway, were isolated from lettuce seeds. The transcript levels of these two genes were little affected by light. Lettuce seeds in which de novo GA biosynthesis was suppressed responded almost identically to exogenously applied GA, irrespective of the light conditions, suggesting that GA responsiveness is not significantly affected by light in lettuce seeds. It is proposed that lettuce seed germination is regulated mainly via the control of the endogenous content of bioactive GA, rather than the control of GA responsiveness.