Antisense-transformation reveals novel roles for class I beta-1,3-glucanase in tobacco seed after-ripening and photodormancy

Little is known about the molecular basis for seed dormancy, after-ripening, and radicle emergence through the covering layers during germination. In tobacco, endosperm rupture occurs after testa rupture and is the limiting step in seed germination. Class I beta-1,3-glucanase (betaGLU I), which is induced in the micropylar endosperm just prior to its penetration by the radicle, is believed to help weaken the endosperm wall. Evidence is presented here for a second site of betaGLU I action during after-ripening. Tobacco plants were transformed with antisense betaGLU I constructs with promoters thought to direct endosperm-specific expression. Unexpectedly, these transformants were unaffected in endosperm rupture and did not exhibit reduced betaGLU I expression during germination. Nevertheless, antisense betaGLU I transformation delayed the onset of testa rupture in light-imbibed, after-ripened seeds and inhibited the after-ripening-mediated release of photodormancy. It is proposed that betaGLU I expression in the dry seed contributes to the after-ripening-mediated release of seed dormancy.     

Seed after-ripening and over-expression of class I beta-1,3-glucanase confer maternal effects on tobacco testa rupture and dormancy release

'Coat-imposed' seed dormancy of many non-endospermic and endospermic species is released during after-ripening. After-ripening-mediated promotion of tobacco ( Nicotiana tabacum L.) seed germination is mainly due to a promotion of testa rupture and a similar promotion of subsequent endosperm rupture. Treatment of after-ripened or freshly harvested mature seeds with abscisic acid (ABA) delays endosperm rupture and inhibits the induction of class I beta-1,3-glucanase (betaGlu I) in the micropylar endosperm, but does not affect the kinetics of testa rupture. After-ripening-mediated release of photodormancy is correlated with a decreased gibberellin (GA) requirement for testa rupture during dark-imbibition. Reciprocal crosses between wild-type tobacco and sense-betaGlu I transformant lines showed that betaGlu I over-expression in the seed covering layers can replace the promoting effect of after-ripening on testa rupture in light, but only if the mother plant is a sense-betaGlu I line. This maternal effect supports the model of two sites for betaGlu I action: (i) betaGlu I contribution to the after-ripening-mediated release of dormancy in the dry seed state, which is manifested in the promotion and ABA-insensitivity of testa rupture during imbibition. (ii) ABA-sensitive expression of betaGlu I in the micropylar endosperm, which contributes to endosperm rupture. The importance of GA-signaling and testa characteristics appear to be a common feature during the after-ripening-mediated release of coat-imposed dormancy in endospermic and non-endospermic seeds.     

Brassinosteroids and gibberellins promote tobacco seed germination by distinct pathways

Seed germination of Nicotiana tabacum L. cv. Havana 425 is determined by the balance of forces between the growth potential of the embryo and the mechanical restraint of the micropylar endosperm. In contrast to the gibberellin GA4, the brassinosteroid (BR) brassinolide (BL) did not release photodormancy of dark-imbibed photodormant seeds. Brassinolide promoted seedling elongation and germination of non-photodormant seeds, but did not appreciably affect the induction of class I beta-1,3-glucanase (betaGLU I) in the micropylar endosperm. Brassinolide, but not GA4, accelerated endosperm rupture of tobacco seeds imbibed in the light. Brassinolide and GA4 promoted endosperm rupture of dark-imbibed non-photodormant seeds, but only GA4 enhanced betaGLU I induction. Promotion of endosperm rupture by BL was dose-dependent and 0.01 microM BL was most effective. Brassinolide and GA4 promoted abscisic acid (ABA)-inhibited dark-germination of non-photodormant seeds, but only GA4 replaced light in inducing betaGLU I. These results indicate that BRs and GAs promote tobacco seed germination by distinct signal transduction pathways and distinct mechanisms. Gibberellins and light seem to act in a common pathway to release photodormancy, whereas BRs do not release photodormancy. Induction of betaGLU I in the micropylar endosperm and promotion of release of 'coat-enhanced' dormancy seem to be associated with the GA-dependent pathway, but not with BR signalling. It is proposed that BRs promote seed germination by directly enhancing the growth potential of the emerging embryo in a GA- and betaGLU I-independent manner.     

Storage behavior of Chionanthus retusus seed and asynchronous development of the radicle and shoot apex during germination in relation to germination inhibitors, including abscisic acid and four phenolic glucosides

Studies on seed storage of Chionanthus retusus Lindl. & Paxt. revealed an orthodox behavior, one which showed both desiccation and freezing tolerance. An epicotyl after-ripening dormancy was expressed in C. retusus seeds by slow growth of the shoot apex relative to more rapid growth of the radicle when seeds were germinated at 30/20 degrees C. Although these seeds exhibit radicle protrusion, they must be after-ripened for another 8-10 weeks at 30/20 degrees C in order to obtain normal shoot growth. Removal of the endosperm, however, quickly stimulated cotyledon and shoot emergence without the additional after-ripening. Water-soluble glucoside phenolics, GL-3, Nuzhenide, ligustroside and oleoside dimethyl ester are present at relatively high levels in endosperm of freshly harvested seeds. These glucoside phenolics are excreted from the endosperm during subsequent after-ripening. Embryo and endosperm tissue from seed germinating at 30/20 degrees C (germination being defined by protrusion of the radicle) had a 10 times lower abscisic acid (ABA) content than similar tissues from freshly harvested mature seed. However, no shoot growth occurred even with the 10-fold reduction in ABA and a concomitant increase in endogenous gibberellins A1, A4 and A20. Thus, epicotyl dormancy during the first 8 weeks of after-ripening at 30/20 degrees C may be controlled by factors other than high ABA, i.e., the slow development of the shoot apex following radicle protrusion may be controlled more by high levels of glucoside phenolics than by diminished ABA and elevated GA levels.     

Class I [beta]-1,3-Glucanases in the Endosperm of Tobacco during Germination

Rupture of the seed coat and rupture of the endosperm are separate events in the germination of Nicotiana tabacum L. cv Havana 425 seeds. Treatment with 10-5 M abscisic acid (ABA) did not appreciably affect seed-coat rupture but greatly delayed subsequent endosperm rupture by more than 100 h and resulted in the formation of a novel structure consisting of the enlarging radicle with a sheath of greatly elongated endosperm tissue. Therefore, ABA appears to act primarily by delaying endosperm rupture and radicle emergence. Measurements of [beta]-1,3-glucanase activity, antigen content, and mRNA accumulation together with reporter gene experiments showed that induction of class I [beta]-1,3-glucanase genes begins just prior to the onset of endosperm rupture but after the completion of seed-coat rupture. This induction was localized exclusively in the micropylar region of the endosperm, where the radicle will penetrate. ABA treatment markedly inhibited the rate of [beta]-1,3-glucanase accumulation but did not delay the onset of induction. Independent of the ABA concentration used, onset of endosperm rupture was correlated with the same [beta]-1,3-glucanase content/seed. These results suggest that ABA-sensitive class I [beta]-1,3-glucanases promote radicle penetration of the endosperm, which is a key limiting step in tobacco seed germination.     

Water uptake and distribution in germinating tobacco seeds investigated in vivo by nuclear magnetic resonance imaging

The regulation of water uptake of germinating tobacco (Nicotiana tabacum) seeds was studied spatially and temporally by in vivo (1)H-nuclear magnetic resonance (NMR) microimaging and (1)H-magic angle spinning NMR spectroscopy. These nondestructive state-of-the-art methods showed that water distribution in the water uptake phases II and III is inhomogeneous. The micropylar seed end is the major entry point of water. The micropylar endosperm and the radicle show the highest hydration. Germination of tobacco follows a distinct pattern of events: rupture of the testa is followed by rupture of the endosperm. Abscisic acid (ABA) specifically inhibits endosperm rupture and phase III water uptake, but does not alter the spatial and temporal pattern of phase I and II water uptake. Testa rupture was associated with an increase in water uptake due to initial embryo elongation, which was not inhibited by ABA. Overexpression of beta-1,3-glucanase in the seed-covering layers of transgenic tobacco seeds did not alter the moisture sorption isotherms or the spatial pattern of water uptake during imbibition, but partially reverted the ABA inhibition of phase III water uptake and of endosperm rupture. In vivo (13)C-magic angle spinning NMR spectroscopy showed that seed oil mobilization is not inhibited by ABA. ABA therefore does not inhibit germination by preventing oil mobilization or by decreasing the water-holding capacity of the micropylar endosperm and the radicle. Our results support the proposal that different seed tissues and organs hydrate at different extents and that the micropylar endosperm region of tobacco acts as a water reservoir for the embryo.     

Proteomic analysis of seed dormancy in Arabidopsis

The mechanisms controlling seed dormancy in Arabidopsis (Arabidopsis thaliana) have been characterized by proteomics using the dormant (D) accession Cvi originating from the Cape Verde Islands. Comparative studies carried out with freshly harvested dormant and after-ripened non-dormant (ND) seeds revealed a specific differential accumulation of 32 proteins. The data suggested that proteins associated with metabolic functions potentially involved in germination can accumulate during after-ripening in the dry state leading to dormancy release. Exogenous application of abscisic acid (ABA) to ND seeds strongly impeded their germination, which physiologically mimicked the behavior of D imbibed seeds. This application resulted in an alteration of the accumulation pattern of 71 proteins. There was a strong down-accumulation of a major part (90%) of these proteins, which were involved mainly in energetic and protein metabolisms. This feature suggested that exogenous ABA triggers proteolytic mechanisms in imbibed seeds. An analysis of de novo protein synthesis by two-dimensional gel electrophoresis in the presence of [(35)S]-methionine disclosed that exogenous ABA does not impede protein biosynthesis during imbibition. Furthermore, imbibed D seeds proved competent for de novo protein synthesis, demonstrating that impediment of protein translation was not the cause of the observed block of seed germination. However, the two-dimensional protein profiles were markedly different from those obtained with the ND seeds imbibed in ABA. Altogether, the data showed that the mechanisms blocking germination of the ND seeds by ABA application are different from those preventing germination of the D seeds imbibed in basal medium.     

濒危植物巴东木莲种子休眠与萌发特性的研究

巴东木莲(Manglietia patungensis)为我国特有种, 属国家重点保护植物。为找出其生殖环节中的致危因素, 作者对巴东木莲种子休眠与后熟过程中的形态和萌发特性进行了研究。结果表明, 巴东木莲种胚发育不完全可能是种子休眠的主要原因, 在其后熟过程中胚不断分化、发育成熟; 种皮具有较好的透性, 与休眠的关系不大; 种子不同部位均存在萌发抑制物, 胚乳中高含量的萌发抑制物是影响胚萌发的重要因素。内源激素ABA和IAA在巴东木莲种子休眠与萌发过程中起着重要作用, ABA是引起休眠的关键因素, IAA有助于种子的萌发, IAA/ABA相对含量的变化对种子的休眠和萌发产生重要影响。巴东木莲种子的休眠是由种子本身的形态和生理特点引起的综合休眠, 在4℃低温保湿条件下才能完成其形态和生理后熟过程, 而自然条件下, 巴东木莲种子成熟时正值秋季少雨, 很容易失水而不能完成其后熟过程而失去生活力, 这可能是导致该物种自然更新困难的重要原因。     

The Time Required for Dormancy Release in Arabidopsis Is Determined by DELAY OF GERMINATION1 Protein Levels in Freshly Harvested Seeds

Seed dormancy controls the start of a plant's life cycle by preventing germination of a viable seed in an unfavorable season. Freshly harvested seeds usually show a high level of dormancy, which is gradually released during dry storage (after-ripening). Abscisic acid (ABA) has been identified as an essential factor for the induction of dormancy, whereas gibberellins (GAs) are required for germination. The molecular mechanisms controlling seed dormancy are not well understood. DELAY OF GERMINATION1 (DOG1) was recently identified as a major regulator of dormancy in Arabidopsis thaliana. Here, we show that the DOG1 protein accumulates during seed maturation and remains stable throughout seed storage and imbibition. The levels of DOG1 protein in freshly harvested seeds highly correlate with dormancy. The DOG1 protein becomes modified during after-ripening, and its levels in stored seeds do not correlate with germination potential. Although ABA levels in dog1 mutants are reduced and GA levels enhanced, we show that DOG1 does not regulate dormancy primarily via changes in hormone levels. We propose that DOG1 protein abundance in freshly harvested seeds acts as a timer for seed dormancy release, which functions largely independent from ABA.     

Abscisic acid,phaseic acid and gibberellin contents associated with dormancy and germination in barley

Analyses of abscisic acid (ABA), ent -kaurenoids and gibberellins (GAs) showed that there were major changes in the contents of these compounds associated with germination of after-ripened barley ( Hordeum vulgare cv. Schooner and cv. Proctor) grain but not in hydrated dormant grain. Embryos from dormant and after-ripened dry grain contained similar amounts of ABA, of ent -kaurenoids and of GAs, determined by gas chromatography-mass spectrometry-selected ion monitoring. In embryos of after-ripened grain, ABA content decreased rapidly after hydration and ABA appeared to be metabolized (inactivated) to phaseic acid (PA) rather than diffusing into the endosperm or the surrounding medium as previously thought. Similar changes in ABA occurred in hydrated dormant grain during germination in darkness. Accumulation of ent -kaurenoids and GAs, including GA_1, the first biologically active GA in the early 13-hydroxylation biosynthetic pathway, occurred to a much greater extent in after-ripened than in dormant grain and these changes occurred mainly after 18 h of hydration when ABA had already decreased and germination was occurring. The block in ent -kaurenoid and GA synthesis in dormant grain appeared to occur prior to ent -kaurene in the biosynthetic pathway. These results are consistent with the view that ABA is the primary effector of dormancy and that after-ripening involves the development of the ability to reduce the amount of ABA quickly following hydration. Accumulation of GAs does not appear to be causally related to loss of dormancy but it does appear to be related to germination.     

Endosperm-limited Brassicaceae seed germination: abscisic acid inhibits embryo-induced endosperm weakening of Lepidium sativum (cress) and endosperm rupture of cress and Arabidopsis thaliana

The endosperm is a barrier for radicle protrusion of many angiosperm seeds. Rupture of the testa (seed coat) and rupture of the endosperm are two sequential events during the germination of Lepidium sativum L. and Arabidopsis thaliana (L.) Heyhn. Abscisic acid (ABA) specifically inhibits the endosperm rupture of these two closely related Brassicaceae species. Lepidium seeds are large enough to allow the direct measurement of endosperm weakening by the puncture force method. We found that the endosperm weakens prior to endosperm rupture and that ABA delays the onset and decreases the rate of this weakening process in a dose-dependent manner. An early embryo signal is required and sufficient to induce endosperm weakening, which afterwards appears to be an organ-autonomous process. Gibberellins can replace this embryo signal; de novo gibberellin biosynthesis occurs in the endosperm and weakening is regulated by the gibberellin/ABA ratio. Our results suggest that the control of radicle protrusion during the germination of Brassicaceae seeds is mediated, at least in part, by endosperm weakening. We propose that Lepidium is an emerging Brassicaceae model system for endosperm weakening and that the complementary advantages of Lepidium and Arabidopsis can be used in parallel experiments to investigate the molecular mechanisms of endosperm weakening.     

After-ripening and phytochrome action in seeds of dormant lines of wild oat (Avena fatua)

The effects of a short exposure to red, far-red or alternate red/far-red light on the germination of seeds after-ripened for different periods of time were studied in dormant lines of wild oat ( Avena fatua L.). Three stages were distinguishable in the after-ripening period in the response of germination to light. Seeds stayed dormant and showed no response to light during stage I. Phytochrome-mediated germination was observed in seeds during stage II. The phytochrome action disappeared during stage III, i.e. seeds fully germinated following treatments of all light qualities. When the seeds were imbibed in polyethylene glycol solutions, dark germination was reduced and phytochrome again had an effect, which suggested the involvement of phytochrome in water uptake of the seed.     

Seed germination of GA-insensitive sleepy1 mutants does not require RGL2 protein disappearance in Arabidopsis

We explore the roles of gibberellin (GA) signaling genes SLEEPY1 (SLY1) and RGA-LIKE2 (RGL2) in regulation of seed germination in Arabidopsis thaliana, a plant in which the hormone GA is required for seed germination. Seed germination failure in the GA biosynthesis mutant ga1-3 is rescued by GA and by mutations in the DELLA gene RGL2, suggesting that RGL2 represses seed germination. RGL2 protein disappears before wild-type seed germination, consistent with the model that GA stimulates germination by causing the SCF(SLY1) E3 ubiquitin ligase complex to trigger ubiquitination and destruction of RGL2. Unlike ga1-3, the GA-insensitive sly1 mutants show variable seed dormancy. Seed lots with high seed dormancy after-ripened slowly, with stronger alleles requiring more time. We expected that if RGL2 negatively controls seed germination, sly1 mutant seeds that germinate well should accumulate lower RGL2 levels than those failing to germinate. Surprisingly, RGL2 accumulated at high levels even in after-ripened sly1 mutant seeds with 100% germination, suggesting that RGL2 disappearance is not a prerequisite for seed germination in the sly1 background. Without GA, several GA-induced genes show increased accumulation in sly1 seeds compared with ga1-3. It is possible that the RGL2 repressor of seed germination is inactivated by after-ripening of sly1 mutant seeds.     

Wheat seed proteins regulated by imbibition independent of dormancy status

Seed dormancy is an important trait in wheat (Trticum aestivum L.) and it can be released by germination-stimulating treatments such as after-ripening. Previously, we identified proteins specifically associated with after-ripening mediated developmental switches of wheat seeds from the state of dormancy to germination. Here, we report seed proteins that exhibited imbibition induced co-regulation in both dormant and after-ripened seeds of wheat, suggesting that the expression of these specific proteins/protein isoforms is not associated with the maintenance or release of seed dormancy in wheat.     

PHYSIOLOGY OF LIGHT-REQUIRING GERMINATION IN ERAGROSTIS SEEDS

The photorequirement for the germination of Eragrostis seedsdecreases with the progress of their after-ripening, and thegermination occurs whether in continuous light or in darknessat the final stage of after-ripening. The dehydration of seedsor the puncturing of seed coats also results in a decrease ofphotorequirement for germination. The rate of water absorptionof seeds increases with the germination capacity under continuousdark condition. However, there is no correlation between therespiration rate and the germination capacity; respiration isstimulated in the punctured seeds, but not in the after-ripenedseeds. The after-ripened or punctured seeds which no longer have aphotorequirement become light-sensitive again, when they areallowed to germinate in the air of low oxygen concentrations.The assumption is presented that the permeability to oxygenof the seed coat may be a factor controlling the seed germinationof this species. (Received August 21, 1964; )     

Dormancy and Germination of Abscisic Acid-Deficient Tomato Seeds : Studies with the sitiens Mutant

The role of abscisic acid (ABA) in the dormancy induction of tomato (Lycopersicon esculentum) seeds was studied by comparison of the germination behavior of the ABA-deficient sitiens mutant with that of the isogenic wild-type genotype. Freshly harvested mutant seeds, in contrast to wild-type seeds, always readily germinate and even exhibit viviparous germination in overripe fruits. Crosses between mutant and wild-type and self-pollination of heterozygous plants show that in particular the ABA fraction of embryo and endosperm is decisive for the induction of dormancy. After-ripened wild-type seeds fully germinate in water but are more sensitive toward osmotic inhibition than mutant seeds. Germination of both wild-type and mutant seeds is equally sensitive toward inhibition by exogenous ABA. ABA content of mature wild-type seeds is about 10-fold the level found in mutant seeds. Nevertheless, it is argued that the differences in dormancy between the seeds of both genotypes are not a result of actual ABA levels in the mature seeds or fruits but a result of differences in ABA levels during seed development. It is hypothesized that the high levels of ABA that occur during seed development in wild-type seeds induce an inhibition of cell elongation of the radicle that can still be observed after long periods of dry storage.