An unusual spliced variant of DELLA protein, a negative regulator of gibberellin signaling, in lettuce.

DELLA proteins are negative regulators of the signaling of gibberellin (GA), a phytohormone regulating plant growth. DELLA degradation is triggered by its interaction with GID1, a soluble GA receptor, in the presence of bioactive GA. We isolated cDNA from a spliced variant of LsDELLA1 mRNA in lettuce, and named it LsDELLA1sv. It was deduced that LsDELLA1sv encodes truncated LsDELLA1, which has DELLA and VHYNP motifs at the N terminus but lacks part of the C-terminal GRAS domain. The recombinant LsDELLA1sv protein interacted with both Arabidopsis GID1 and lettuce GID1s in the presence of GA. A yeast two-hybrid assay suggested that LsDELLA1sv interacted with LsDELLA1. The ratio of LsDELLA1sv to LsDELLA1 transcripts was higher in flower samples at the late reproductive stage and seed samples (dry seeds and imbibed seeds) than in the other organ samples examined. This study suggests that LsDELLA1sv is a possible modulator of GA signaling in lettuce.

Characterization of a loss-of-function mutant of gibberellin biosynthetic gene LsGA3ox1 in lettuce.

A previous study generated lettuce (Lactuca sativa) mutant lines tagged by retrotransposon Tnt1 from tobacco (Nicotiana tabacum) and identified a homozygous mutant, Tnt6a, that exhibited severe dwarf phenotype. Here we show that Tnt1 is inserted into the intron of gibberellin biosynthetic gene LsGA3ox1 in Tnt6a mutants. Expression analysis suggests that LsGA3ox1 is nearly knocked out in the Tnt6a mutants.

Germination of photoblastic lettuce seeds is regulated via the control of endogenous physiologically active gibberellin content, rather than of gibberellin responsiveness.

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.

Drought induction of Arabidopsis 9-cis-epoxycarotenoid dioxygenase occurs in vascular parenchyma cells.

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.

Phytochrome- and gibberellin-mediated regulation of abscisic acid metabolism during germination of photoblastic lettuce seeds.

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.

Deactivation of gibberellin by 2-oxidation during germination of photoblastic lettuce seeds.

Gibberellin (GA) plays an important role in the induction of germination of photoblastic lettuce (Lactuca sativa L. cv. Grand Rapids) seeds. We have previously shown that gene expression of a GA 3-oxidase (Ls3h1) increased after a red light treatment, resulting in an increase in the endogenous content of GA1, bioactive GA. Since the metabolism of GAs is also important for determining the endogenous levels of bioactive GAs, cDNAs encoding GA 2-oxidases (LsGA2ox1 and LsGA2ox2, for L. sativa GA 2-oxidase), which catalyze the deactivation of GAs, were isolated from lettuce seeds to investigate the regulation of these genes by light. An expression analysis shows that the mRNA levels of both enzymes was not markedly altered under different light conditions during germination. However, the amount of LsGA2ox2 transcripts was decreased to approximately half the level by red light. This reduction might play a role in the increase in GA1 level by red light in the lettuce seeds.