The expression of tgas118, encoding a defensin in Lycopersicon esculentum, is regulated by gibberellin.

A flower specific cDNA, tgas118, has been isolated after differential screening of a gib-1 anther cDNA library of Lycopersicon esculentum. The corresponding mRNA was present in all tissues analysed. Northern blot analysis revealed that in wild-type tomato the gene was predominantly expressed throughout flower development, while in the gibberellin (GA)-deficient mutant of tomato (gib-1) the abundance declined. Treatment of the mutant with GA resulted in an accumulation of the tgas118 mRNA within hours in leaf and bud tissues. In the leaf, GA1, GA3 and GA9 were effective in enhancing the expression while GA4 was not. In addition to GA, wounding and dehydration also increased the accumulation of tgas118 mRNA in leaf tissue. In situ hybridization showed that application of 50 ng GA3 bud(-1) induced a similar spatial expression of the tgas118 mRNA in gib-1 buds 24 h post treatment to that found in wild-type flower buds. The deduced TGAS118 protein displays up to 77% similarity with defensins and as its expression is up-regulated by stimuli such as wounding it is proposed that it may play a role in protection against pathogens.     

Expression of two gibberellin-regulated cDNAs during early flower development in tomato (Solanum lycopersicon). Effect of grafting and paclobutrazol

To study the role of translocation of gibberellin (GA) intermediates or bioactive GAs from other plant parts to buds during early flower development in tomato ( Solanum lycopersicon ), the effect of grafting and paclobutrazol (PAC) treatment on the expression of tgas100 and tgas118 , two GA-regulated mRNAs, was analysed. Both mRNAs accumulated in a dose-dependent fashion. Application of 0.5 ng GA₃ per bud to developmentally arrested flower buds of a GA-deficient mutant of tomato ( gib-1 ) induced tgas100 expression, while the tgas118 abundance increased. For obtaining normal flower development through anthesis in the mutant, a single GA₃ treatment was required of at least 5 ng GA₃ per bud. In wild-type flower buds, PAC decreased the abundance of tgas100 and tgas118 mRNAs either when PAC was sprayed on whole plants or directly applied to buds. When only the wild-type buds were treated with PAC, the expression profiles characteristic for untreated buds were not restored by translocation of endogenous GAs. Grafting of gib-1 scions onto wild-type donor plants did not result in normal flower development or expression profiles like in wild-type buds. We conclude that the role of GA transport in early flower development of tomato is negligible and that the GAs required for development have to be synthesized in the flower bud itself.     

A gibberellin-regulated xyloglucan endotransglycosylase gene is expressed in the endosperm cap during tomato seed germination.

Xyloglucan endotransglycosylases (XETs) modify xyloglucans, major components of primary cell walls in dicots. A cDNA encoding an XET (LeXET4) was isolated from a germinating tomato (Lycopersicon esculentum Mill.) seed cDNA library. DNA gel blot analysis showed that LeXET4 is a single-copy gene in the tomato genome. LeXET4 mRNA was strongly expressed in germinating seeds, was much less abundant in stems, and was not detected in roots, leaves or flower tissues. During germination, LeXET4 mRNA was detected in seeds within 12 h of imbibition with maximum mRNA abundance at 24 h. Tissue prints showed that LeXET4 mRNA was localized exclusively to the endosperm cap region. Expression of LeXET4 was dependent on exogenous gibberellin (GA) in GA-deficient (gib-1 mutant) tomato seeds, while abscisic acid, a seed germination inhibitor, had no effect on LeXET4 mRNA expression in wild-type seeds. LeXET4 mRNA disappeared after radicle emergence, even though degradation of the lateral endosperm cell walls continued. The temporal, spatial and hormonal regulation pattern of LeXET4 gene expression suggests that XET has a role in endosperm cap weakening, a key process regulating tomato seed germination.     

Vacuolar H(+)-ATPase is expressed in response to gibberellin during tomato seed germination

Completion of germination (radicle emergence) by gibberellin (GA)-deficient (gib-1) mutant tomato (Lycopersicon esculentum Mill.) seeds is dependent upon exogenous GA, because weakening of the endosperm tissue enclosing the radicle tip requires GA. To investigate genes that may be involved in endosperm weakening or embryo growth, differential cDNA display was used to identify mRNAs differentially expressed in gib-1 seeds imbibed in the presence or absence of GA(4+7). Among these was a GA-responsive mRNA encoding the 16-kD hydrophobic subunit c of the V(0) membrane sector of vacuolar H(+)-translocating ATPases (V-ATPase), which we termed LVA-P1. LVA-P1 mRNA expression in gib-1 seeds was dependent on GA and was particularly abundant in the micropylar region prior to radicle emergence. Both GA dependence and tissue localization of LVA-P1 mRNA expression were confirmed directly in individual gib-1 seeds using tissue printing. LVA-P1 mRNA was also expressed in wild-type seeds during development and germination, independent of exogenous GA. Specific antisera detected protein subunits A and B of the cytoplasmic V(1) sector of the V-ATPase holoenzyme complex in gib-1 seeds only in the presence of GA, and expression was localized to the micropylar region. The results suggest that V-ATPase plays a role in GA-regulated germination of tomato seeds.     

Expression of an expansin is associated with endosperm weakening during tomato seed germination

Expansins are extracellular proteins that facilitate cell wall extension, possibly by disrupting hydrogen bonding between hemicellulosic wall components and cellulose microfibrils. In addition, some expansins are expressed in non-growing tissues such as ripening fruits, where they may contribute to cell wall disassembly associated with tissue softening. We have identified at least three expansin genes that are expressed in tomato (Lycopersicon esculentum Mill.) seeds during germination. Among these, LeEXP4 mRNA is specifically localized to the micropylar endosperm cap region, suggesting that the protein might contribute to tissue weakening that is required for radicle emergence. In gibberellin (GA)-deficient (gib-1) mutant seeds, which germinate only in the presence of exogenous GA, GA induces the expression of LeEXP4 within 12 hours of imbibition. When gib-1 seeds were imbibed in GA solution combined with 100 microM abscisic acid, the expression of LeEXP4 was not reduced, although radicle emergence was inhibited. In wild-type seeds, LeEXP4 mRNA accumulation was blocked by far-red light and decreased by low water potential but was not affected by abscisic acid. The presence of LeEXP4 mRNA during seed germination parallels endosperm cap weakening determined by puncture force analysis. We hypothesize that LeEXP4 is involved in the regulation of seed germination by contributing to cell wall disassembly associated with endosperm cap weakening.     

Class I chitinase and beta-1,3-glucanase are differentially regulated by wounding,methyl jasmonate,ethylene, and gibberellin in tomato seeds and leaves

Class I chitinase (Chi9) and beta-1,3-glucanase (GluB) genes are expressed in the micropylar endosperm cap of tomato (Lycopersicon esculentum) seeds just before radicle emergence through this tissue to complete germination. In gibberellin (GA)-deficient mutant (gib-1) seeds, expression of Chi9 and GluB mRNA and protein is dependent upon GA. However, as expression occurs relatively late in the germination process, we investigated whether the genes are induced indirectly in response to tissue wounding associated with endosperm cap weakening and radicle protrusion. Wounding and methyl jasmonate (MeJA) induced Chi9 expression, whereas ethylene, abscisic acid, sodium salicylate, fusicoccin, or beta-aminobutyric acid were without effect. Chi9 expression occurred only in the micropylar tissues when seeds were exposed to MeJA or were wounded at the chalazal end of the seed. Expression of Chi9, but not GluB, mRNA was reduced in germinating seeds of the jasmonate-deficient defenseless1 tomato mutant and could be restored by MeJA treatment. Chi9 expression during germination may be associated with "wounding" from cell wall hydrolysis and weakening in the endosperm cap leading to radicle protrusion, and jasmonate is involved in the signaling pathway for this response. Among these treatments and chemicals (other than GA), only MeJA and wounding induced a low level of GluB expression in gib-1 seeds. However, MeJA, wounding, and particularly ethylene induced both genes in leaves, whereas GA induced only Chi9 in leaves. Although normally expressed simultaneously during tomato seed germination, Chi9 and GluB genes are regulated distinctly and tissue specifically by hormones and wounding.     

Vacuolar H+-ATPase Is Expressed in Response to Gibberellin during Tomato Seed Germination

Completion of germination (radicle emergence) by gibberellin (GA)-deficient (gib-1) mutant tomato (Lycopersicon esculentum Mill.) seeds is dependent upon exogenous GA, because weakening of the endosperm tissue enclosing the radicle tip requires GA. To investigate genes that may be involved in endosperm weakening or embryo growth, differential cDNA display was used to identify mRNAs differentially expressed in gib-1 seeds imbibed in the presence or absence of GA₄₊₇. Among these was a GA-responsive mRNA encoding the 16-kD hydrophobic subunit c of the V₀ membrane sector of vacuolar H⁺-translocating ATPases (V-ATPase), which we termed LVA-P1. LVA-P1 mRNA expression in gib-1 seeds was dependent on GA and was particularly abundant in the micropylar region prior to radicle emergence. Both GA dependence and tissue localization of LVA-P1 mRNA expression were confirmed directly in individual gib-1 seeds using tissue printing. LVA-P1 mRNA was also expressed in wild-type seeds during development and germination, independent of exogenous GA. Specific antisera detected protein subunits A and B of the cytoplasmic V₁ sector of the V-ATPase holoenzyme complex in gib-1 seeds only in the presence of GA, and expression was localized to the micropylar region. The results suggest that V-ATPase plays a role in GA-regulated germination of tomato seeds.     

Characterization of the Arrest in Anther Development Associated with Gibberellin Deficiency of the gib-1 Mutant of Tomato

The role of gibberellins in flower bud development was investigated by studying the gib-1 mutant of tomato, Lycopersicon esculentum. This gibberellin-deficient mutant initiates flower buds, but floral development is not completed unless the mutant is treated with gibberellin. Treatment with other plant growth regulators does not induce normal flower development. Development of gib-1 flower buds, as measured by progress toward anthesis, ceases at a bud length of 2.5 millimeters; however, increase in size of the bud continues. Buds between 2.5 and 3.7 millimeters are developmentally arrested but still are capable of developing normally after treatment with gibberellic acid. Anthers of these developmentally arrested buds contain pollen mother cells that are in the G1 phase of premeiotic interphase. Following treatment of developmentally arrested buds with gibberellic acid, premeiotic DNA synthesis and callose accumulation in pollen mother cells are evident by 48 hours posttreatment, and within 66 hours, prophase I of meiosis- and meiosis-related changes in tapetum development are observable.     

Two Tomato Expansin Genes Show Divergent Expression and Localization in Embryos during Seed Development and Germination

Expansins are plant proteins that can induce extension of isolated cell walls and are proposed to mediate cell expansion. Three expansin genes were expressed in germinating tomato (Lycopersicon esculentum Mill.) seeds, one of which (LeEXP4) was expressed specifically in the endosperm cap tissue enclosing the radicle tip. The other two genes (LeEXP8 and LeEXP10) were expressed in the embryo and are further characterized here. LeEXP8 mRNA was not detected in developing or mature seeds but accumulated specifically in the radicle cortex during and after germination. In contrast, LeEXP10 mRNA was abundant at an early stage of seed development corresponding to the period of rapid embryo expansion; it then decreased during seed maturation and increased again during germination. When gibberellin-deficient (gib-1) mutant seeds were imbibed in water, LeEXP8 mRNA was not detected, but a low level of LeEXP10 mRNA was present. Expression of both genes increased when gib-1 seeds were imbibed in gibberellin. Abscisic acid did not prevent the initial expression of LeEXP8 and LeEXP10, but mRNA abundance of both genes subsequently decreased during extended incubation. The initial increase in LeEXP8, but not LeEXP10, mRNA accumulation was blocked by low water potential, but LeEXP10 mRNA amounts fell after longer incubation. When seeds were transferred from abscisic acid or low water potential solutions to water, abundance of both LeEXP8 and LeEXP10 mRNAs increased in association with germination. The tissue localization and expression patterns of both LeEXP8 and LeEXP10 suggest developmentally specific roles during embryo and seedling growth.     

AgNO3诱导的苦瓜纯雌系完全花分化与幼叶和花蕾中内源激素的关系

本文用酶联免疫检测（ELIsA）技术研究三叶-心期喷施AgNO3诱导苦瓜纯雌系完全花分化过程中花蕾与幼叶中内源激素含量变化。结果显示：喷施AgNO,后幼叶中IAA、GA,、ZR和ABA含量与喷水的相比,都是先下降后增加。AgNO3处理的花蕾中这4种激素含量在72h内没有一致变化规律,但变化幅度大于幼叶的,表明生殖器官的内源激素对苦瓜性别分化影响比营养器官的大。AgNO3处理后24-48h内,花蕾中这4种激素的含量明显低于喷水的,而其余时间则高于喷水的。此外,AgNO3处理的花蕾中ABA／IAA、ZR／I从和GA3／IAA比值也在2448h发生剧烈的变化,48h之后这些激素比值与喷水的相差不大。这些结果说明了AgNO3处理后24-48h是苦瓜纯雌系性别分化的关键时期,IAA可能是诱导纯雌系苦瓜雄性分化的关键激素。     

Characterisation of the procera mutant of tomato and the interaction of gibberellins with end-of-day far-red light treatments

The tomato ( Solanum lycopersicum L.) slender mutant procera ( pro ) was analysed for its relationship with gibberellin (GA) by combining it with GA deficiency due to the gib-1 mutation. The sensitivity to GA biosynthesis inhibitors and the GA content were measured in the pro gib-1 double mutant. In the gib-1 mutant background, the pro mutation strongly reduced the GA requirement for seed germination and stem growth and almost fully restored the morphological leaf defects of the gib-1 mutant. An end-of-day far-red light treatment, when applied to the various genotypes, indicated that GAs are required for a response to this treatment, but that it act independently of the Pro gene product.     

Molecular cloning and analysis of a pea cDNA that is expressed in darkness and very rapidly induced by gibberellic acid

Using cDNA representational difference analysis (cDNA RDA), we isolated a cDNA named GDA-1 from a cDNA library constructed with mRNA from short-day (SD) grown G2 pea apical tissue. The amino acid sequence deduced from GDA-1 shares partial identity with the B2 protein which is expressed during embryogenesis of carrot cells. Northern analysis showed that GDA-1 mRNA is abundant in SD-grown G2 pea apical buds. In long-day (LD) conditions, there was almost no detectable GDA-1 mRNA. When LD-grown G2 peas were kept in continuous darkness for 24 h, the GDA-1 mRNA content reached a level equivalent to about 50% of that in the SD samples. On the other hand, when SD-grown peas were transferred into the light for 24 h, the amount of hybridizable GDA-1 mRNA dropped to the same as that of LD-grown plants. GDA-1 expression was found to be independent of flower initiation time. GA₃ application in vitro resulted in rapid accumulation of GDA-1 mRNA in LD-grown G2 pea apical buds, which is compatible with its delaying effect on apical senescence. Time-course experiments revealed that GDA-1 is induced within 15 min of GA₃ application. Exogenous GA₃ did not influence the expression of GDA-1 in SD-grown G2 peas. Since both photoperiod and GA induce the expression of GDA-1, we speculate that they may activate similar signal transduction pathways in G2 peas. Our work also shows that photoperiod may change the efficiency of gibberellin perception by plants. Received: 27 March 1998 / Accepted: 2 June 1998     

Class I beta-1,3-glucanase and chitinase are expressed in the micropylar endosperm of tomato seeds prior to radicle emergence.

beta-1,3-Glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) mRNAs, proteins, and enzyme activities were expressed specifically in the micropylar tissues of imbibed tomato (Lycopersicon esculentum Mill.) seeds prior to radicle emergence. RNA hybridization and immunoblotting demonstrated that both enzymes were class I basic isoforms. beta-1,3-Glucanase was expressed exclusively in the endosperm cap tissue, whereas chitinase localized to both endosperm cap and radicle tip tissues. beta-1,3-Glucanase and chitinase appeared in the micropylar tissues of gibberellin-deficient gib-1 tomato seeds only when supplied with gibberellin. Accumulation of beta-1,3-glucanase mRNA, protein and enzyme activity was reduced by 100 microM abscisic acid, which delayed or prevented radicle emergence but not endosperm cap weakening. In contrast, expression of chitinase mRNA, protein, and enzyme activity was not affected by abscisic acid. Neither of these enzymes significantly hydrolyzed isolated tomato endosperm cap cell walls. Although both beta-1,3-glucanase and chitinase were expressed in tomato endosperm cap tissue prior to radicle emergence, we found no evidence that they were directly involved in cell wall modification or tissue weakening. Possible functions of these hydrolases during tomato seed germination are discussed.