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.     

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.     

Seed after-ripening is a discrete developmental pathway associated with specific gene networks in Arabidopsis

After-ripening (AR) is a time and environment regulated process occurring in the dry seed, which determines the germination potential of seeds. Both metabolism and perception of the phytohormone abscisic acid (ABA) are important in the initiation and maintenance of dormancy. However, molecular mechanisms that regulate the capacity for dormancy or germination through AR are unknown. To understand the relationship between ABA and AR, we analysed genome expression in Arabidopsis thaliana mutants defective in seed ABA synthesis (aba1-1) or perception (abi1-1). Even though imbibed mutant seeds showed no dormancy, they exhibited changes in global gene expression resulting from dry AR that were comparable with changes occurring in wild-type (WT) seeds. Core gene sets were identified that were positively or negatively regulated by dry seed storage. Each set included a gene encoding repression or activation of ABA function (LPP2 and ABA1, respectively), thereby suggesting a mechanism through which dry AR may modulate subsequent germination potential in WT seeds. Application of exogenous ABA to after-ripened WT seeds did not reimpose characteristics of freshly harvested seeds on imbibed seed gene expression patterns. It was shown that secondary dormancy states reinstate AR status-specific gene expression patterns. A model is presented that separates the action of ABA in seed dormancy from AR and dry storage regulated gene expression. These results have major implications for the study of genetic mechanisms altered in seeds as a result of crop domestication into agriculture, and for seed behaviour during dormancy cycling in natural ecosystems.     

Seed dormancy in Acer: Is there a common mechanism for all Acer species and what part is played in it by abscisic acid?

Seeds of Acer pseudoplatanus L. are usually considered to show only testa-imposed dormancy, but a transient embryo dormancy has also been identified at the time of fruit dispersal. Even embryos that did not show full dormancy at this stage possessed low germinative vigour. Removal of embryo dormancy and the development of increased germination potential did not require the low temperatures necessary for the removal of testa-imposed dormancy in this species. Germination rates of embryos from freshly harvested or briefly stored fruits were accelerated by removal of cotyledonary tissue, the most rapid responses occurring in isolated embryonic axes following complete removal of both cotyledons. Longer storage reduced this effect because of the increases in germinative vigour of whole embryos. Abscisic acid (ABA) reinforced embryo dormancy in embryos from freshly harvested or briefly stored fruits and also reduced germination rates in similarly derived isolated embryonic axes. This response to ABA also became progressively less marked as the storage period was extended. Loss of embryo dormancy was correlated with a reduction in endogenous abscisic acid levels in both whole embryos and cotyledons, suggesting that endogenous ABA contributes to the regulation of embryo dormancy in these seeds. There are no indications, however, that endogenous ABA is directly implicated in the low temperature processes associated with the removal of testa-imposed dormancy. The relevance of embryo dormancy in the intact seed of A. pseudoplatanus is discussed.     

Dormancy, ABA content and sensitivity of a barley mutant to ABA application during seed development and after ripening.

Assessment of dormancy inception, maintenance and release was studied for artificially dried immature seeds harvested throughout seed development in the barley cv. Triumph and its mutant line TL43. Each was grown in Spain and Scotland under low and high dormancy inducing conditions, respectively. Both TL43 and Triumph followed a similar pattern of release from dormancy across the seasons, although seeds of TL43 were able to germinate at an earlier seed development stage. Abscisic acid (ABA) content was also studied in immature grains throughout the seed development period. Total amount of ABA in seeds of Triumph and TL43 was higher in plants grown in Scotland than in Spain. However, no clear genotypic differences in ABA pattern in the course of grain development could be detected whilst significant genotypic differences were observed for germination percentage (GP). Endogenous ABA content alone throughout grain development did not explain genetic differences in GP within environments. Environmental and genetic differences in dormancy were also observed on mature seeds throughout the after-ripening period. The initial germination (GP(0)) played a key role in the sensitivity to ABA of post-harvest mature seeds. For the same after-ripening stage, TL43 was more insensitive to exogenous ABA than Triumph. However, ABA responses in seeds of the two genotypes with similar GP(0) at different after-ripening stages were comparable. Therefore, differences in exogenous ABA sensitivity of post-harvest mature grain of these two genotypes seemed to be determined by, or coincident with, the initial germination percentage.     

DOG1 expression is predicted by the seed-maturation environment and contributes to geographical variation in germination in Arabidopsis thaliana

Seasonal germination timing of Arabidopsis thaliana strongly influences overall life history expression and is the target of intense natural selection. This seasonal germination timing depends strongly on the interaction between genetics and seasonal environments both before and after seed dispersal. DELAY OF GERMINATION 1 (DOG1) is the first gene that has been identified to be associated with natural variation in primary dormancy in A. thaliana. Here, we report interaccession variation in DOG1 expression and document that DOG1 expression is associated with seed‐maturation temperature effects on germination; DOG1 expression increased when seeds were matured at low temperature, and this increased expression was associated with increased dormancy of those seeds. Variation in DOG1 expression suggests a geographical structure such that southern accessions, which are more dormant, tend to initiate DOG1 expression earlier during seed maturation and achieved higher expression levels at the end of silique development than did northern accessions. Although elimination of the synthesis of phytohormone abscisic acid (ABA) results in the elimination of maternal temperature effects on dormancy, DOG1 expression predicted dormancy better than expression of genes involved in ABA metabolism.     

Seed Dormancy in Acer: The Role of Testa-imposed and Embryo Dormancy in Acer velutinum

The embryo dormancy shown in freshly harvested samples of Acervelutinum seeds is weakly established and very short-lived.Loss of this embryo dormancy occurred during post-harvest fruitstorage at either 5 or 17 C. In contrast, the dormancy of intactfruits and seeds was overcome only during storage at the lowertemperature. Removal of the cotyledons from embryos of freshlyharvested fruits allowed more rapid germination of the embryonicaxes, indicating that the cotyledons exert an inhibitory effect,although the axes still retained a measure of innate dormancy.The inhibitory effect of the cotyledons became less marked withincreasing duration of fruit storage, this loss of inhibitoryeffect occurring at both storage temperatures. Applied ABA stronglysuppressed germinative capacity in intact embryos and isolatedembryonic axes from freshly harvested fruits, but when ABA wasapplied to embryos of fruits that had been stored for variousperiods at 5 or 17 C, the inhibitory effect was first weakenedand then lost with increased storage. Although dormancy in the seeds of A. velutinum may be describedas intermediate between testa-imposed dormancy and true dormancy,it is perhaps more properly included in the former category. Acer velutinum Boiss. var. vanvolxemii, abscisic acid, embryo dormancy, germination, seed storage, testa-imposed dormancy, tissue sensitivity     

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.     

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。     

Regulation of dormancy in barley by blue light and after-ripening: effects on abscisic acid and gibberellin metabolism

White light strongly promotes dormancy in freshly harvested cereal grains, whereas dark and after-ripening have the opposite effect. We have analyzed the interaction of light and after-ripening on abscisic acid (ABA) and gibberellin (GA) metabolism genes and dormancy in barley (Hordeum vulgare 'Betzes'). Analysis of gene expression in imbibed barley grains shows that different ABA metabolism genes are targeted by white light and after-ripening. Of the genes examined, white light promotes the expression of an ABA biosynthetic gene, HvNCED1, in embryos. Consistent with this result, enzyme-linked immunosorbent assays show that dormant grains imbibed under white light have higher embryo ABA content than grains imbibed in the dark. After-ripening has no effect on expression of ABA biosynthesis genes, but promotes expression of an ABA catabolism gene (HvABA8'OH1), a GA biosynthetic gene (HvGA3ox2), and a GA catabolic gene (HvGA2ox3) following imbibition. Blue light mimics the effects of white light on germination, ABA levels, and expression of GA and ABA metabolism genes. Red and far-red light have no effect on germination, ABA levels, or HvNCED1. RNA interference experiments in transgenic barley plants support a role of HvABA8'OH1 in dormancy release. Reduced HvABA8'OH1 expression in transgenic HvABA8'OH1 RNAi grains results in higher levels of ABA and increased dormancy compared to nontransgenic grains.     

Role of reactive oxygen species in the regulation of Arabidopsis seed dormancy

Freshly harvested seeds of Arabidopsis thaliana, Columbia (Col) accession were dormant when imbibed at 25°C in the dark. Their dormancy was alleviated by continuous light during imbibition or by 5 weeks of storage at 20°C (after-ripening). We investigated the possible role of reactive oxygen species (ROS) in the regulation of Col seed dormancy. After 24 h of imbibition at 25°C, non-dormant seeds produced more ROS than dormant seeds, and their catalase activity was lower. In situ ROS localization revealed that germination was associated with an accumulation of superoxide and hydrogen peroxide in the radicle. ROS production was temporally and spatially regulated: ROS were first localized within the cytoplasm upon imbibition of non-dormant seeds, then in the nucleus and finally in the cell wall, which suggests that ROS play different roles during germination. Imbibition of dormant and non-dormant seeds in the presence of ROS scavengers or donors, which inhibited or stimulated germination, respectively, confirmed the role of ROS in germination. Freshly harvested seeds of the mutants defective in catalase (cat2-1) and vitamin E (vte1-1) did not display dormancy; however, seeds of the NADPH oxidase mutants (rbohD) were deeply dormant. Expression of a set of genes related to dormancy upon imbibition in the cat2-1 and vet1-1 seeds revealed that their non-dormant phenotype was probably not related to ABA or gibberellin metabolism, but suggested that ROS could trigger germination through gibberellin signaling activation.     

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.     

Analysis of natural allelic variation at seed dormancy loci of Arabidopsis thaliana

Arabidopsis accessions differ largely in their seed dormancy behavior. To understand the genetic basis of this intraspecific variation we analyzed two accessions: the laboratory strain Landsberg erecta (Ler) with low dormancy and the strong-dormancy accession Cape Verde Islands (Cvi). We used a quantitative trait loci (QTL) mapping approach to identify loci affecting the after-ripening requirement measured as the number of days of seed dry storage required to reach 50% germination. Thus, seven QTL were identified and named delay of germination (DOG) 1-7. To confirm and characterize these loci, we developed 12 near-isogenic lines carrying single and double Cvi introgression fragments in a Ler genetic background. The analysis of these lines for germination in water confirmed four QTL (DOG1, DOG2, DOG3, and DOG6) as showing large additive effects in Ler background. In addition, it was found that DOG1 and DOG3 genetically interact, the strong dormancy determined by DOG1-Cvi alleles depending on DOG3-Ler alleles. These genotypes were further characterized for seed dormancy/germination behavior in five other test conditions, including seed coat removal, gibberellins, and an abscisic acid biosynthesis inhibitor. The role of the Ler/Cvi allelic variation in affecting dormancy is discussed in the context of current knowledge of Arabidopsis germination.