Gibberellin requirement for Arabidopsis seed germination is determined both by testa characteristics and embryonic abscisic acid

The mechanisms imposing a gibberellin (GA) requirement to promote the germination of dormant and non-dormant Arabidopsis seeds were analyzed using the GA-deficient mutant ga1, several seed coat pigmentation and structure mutants, and the abscisic acid (ABA)-deficient mutant aba1. Testa mutants, which exhibit reduced seed dormancy, were not resistant to GA biosynthesis inhibitors such as tetcyclacis and paclobutrazol, contrarily to what was found before for other non-dormant mutants in Arabidopsis. However, testa mutants were more sensitive to exogenous GAs than the wild-types in the presence of the inhibitors or when transferred to a GA-deficient background. The germination capacity of the ga1-1 mutant could be integrally restored, without the help of exogenous GAs, by removing the envelopes or by transferring the mutation to a tt background (tt4 and ttg1). The double mutants still required light and chilling for dormancy breaking, which may indicate that both agents can have an effect independently of GA biosynthesis. The ABA biosynthesis inhibitor norflurazon was partially efficient in releasing the dormancy of wild-type and mutant seeds. These results suggest that GAs are required to overcome the germination constraints imposed both by the seed coat and ABA-related embryo dormancy.     

Influence of the testa on seed dormancy, germination, and longevity in Arabidopsis

The testa of higher plant seeds protects the embryo against adverse environmental conditions. Its role is assumed mainly by controlling germination through dormancy imposition and by limiting the detrimental activity of physical and biological agents during seed storage. To analyze the function of the testa in the model plant Arabidopsis, we compared mutants affected in testa pigmentation and/or structure for dormancy, germination, and storability. The seeds of most mutants exhibited reduced dormancy. Moreover, unlike wild-type testas, mutant testas were permeable to tetrazolium salts. These altered dormancy and tetrazolium uptake properties were related to defects in the pigmentation of the endothelium and its neighboring crushed parenchymatic layers, as determined by vanillin staining and microscopic observations. Structural aberrations such as missing layers or a modified epidermal layer in specific mutants also affected dormancy levels and permeability to tetrazolium. Both structural and pigmentation mutants deteriorated faster than the wild types during natural aging at room temperature, with structural mutants being the most strongly affected.     

A Seed Shape Mutant of Arabidopsis That Is Affected in Integument Development

A seed shape mutant of Arabidopsis was isolated from an ethyl methanesulfonate-treated population. Genetic analysis revealed that the heart-shaped phenotype was maternally inherited, showing that this is a testa mutant. This indicated the importance of the testa for the determination of the seed shape. This recessive aberrant testa shape (ats) gene was located at position 59.0 on chromosome 5. A comparison was made between ovules and developing and mature seeds of the wild type and of the mutant using light and scanning electron microscopy. We showed that the mutant seed shape is determined during the first few days after fertilization, when the embryo occupies only a very small part of the seed. The integuments of ats ovules consisted of only three rather than five cell layers. In double mutants, the effect of ats was additive to other testa mutations, such as transparent testa, glabra (ttg), glabrous2 (gl2), and apetala2 (ap2). The ats mutation resulted in a reduced dormancy, which was maternally inherited. This effect of a testa mutation on germination was also seen in ttg seeds, in which the outer layer of the testa was disturbed. This indicated the importance of the testa as a factor in determining dormancy in Arabidopsis.     

The Induction of Seed Germination in Arabidopsis thaliana Is Regulated Principally by Phytochrome B and Secondarily by Phytochrome A

We examined whether spectrally active phytochrome A (PhyA) and phytochrome B (PhyB) play specific roles in the induction of seed germination in Arabidopsis thaliana (L.) Heynh., using PhyA- and PhyB-null mutants, fre1-1 (A. Nagatani, J.W. Reed, J. Chory [1993] Plant Physiol 102: 269-277) and hy3-Bo64 (J. Reed, P.Nagpal, D.S. Poole, M. Furuya, J. Chory [1993] Plant Cell 5: 147-157). When dormant seeds of each genotype imbibed in the dark on aqueous agar plates, the hy3 (phyB) mutant did not germinate, whereas the fre1 (phyA) mutant germinated at a rate of 50 to 60%, and the wild type (WT) germinated at a rate of 60 to 70%. By contrast, seeds of all genotypes germinated to nearly 100% when plated in continuous irradiation with white or red light. When plated in continuous far-red light, however, frequencies of seed germination of the WT and the fre1 and hy3 mutants averaged 14, nearly 0, and 47%, respectively, suggesting that PhyB in the red-absorbing form prevents PhyA-dependent germination under continuous far-red light. When irradiated briefly with red or far-red light after imbibition for 1 h, a typical photoreversible effect on seed germination was observed in the fre1 mutant and the WT but not in the hy3 mutant. In contrast, when allowed to imbibe in the dark for 24 to 48 h and exposed to red light, the seed germination frequencies of the hy3 mutant were more than 40%. Immunoblot analyses of the mutant seeds showed that PhyB apoprotein accumulated in dormant seeds of the WT and the fre1 mutant as much as in the seeds that had imbibed. In contrast, PhyA apoprotein, although detected in etiolated seedlings grown in the dark for 5 d, was not detectable in the dormant seeds of the WT and the hy3 mutant. The above physiological and immunochemical evidence indicates that PhyB in the far-red-absorbing form was stored in the Arabidopsis seeds and resulted in germination in the dark. Hence, PhyA does not play any role in dark germination but induces germination under continuous irradiation with far-red light. Finally, we examined seeds from a signal transduction mutant, det1, and a det1/hy3 double mutant. The det1 seeds exhibited photoreversible responses of germination on aqueous agar plates, and the det1/hy3 double mutant seeds did not. Hence, DET1 is likely to act in a distinct pathway from PhyB in the photoregulation of seed germination.     

Isolation and characterization of high temperature-resistant germination mutants of Arabidopsis thaliana

Temperature is a primary environmental cue for seed germination of many weeds and vegetables. To investigate the mechanism of germination regulation by temperature, we selected five high temperature (thermoinhibition)-resistant germination mutants (TRW lines) from 20,000 T-DNA insertion lines of Arabidopsis. Segregation analyses indicated that each of the five lines had single locus recessive mutations. The seeds of TRW134-15 and TRW187 showed reduced sensitivity to ABA and also to the gibberrellin biosynthesis inhibitor, paclobutrazol. Genetic and nucleotide sequencing analyses indicated that TRW187 is a new allele of abi3 (abi3-14). TRW71-1 exhibited a maternal effect for both thermoinhibition-resistant and transparent testa phenotypes, and genetic analysis revealed that the mutation was allelic to tt7 (tt7-4 sib). Interestingly, the seeds of reduced dormancy mutants rdo1, rdo2, rdo3 and rdo4 were also thermoinhibition tolerant, and all the TRW seeds showed reduced dormancy. Like rdo3, TRW13-1 had shorter siliques and slightly shorter stems than the wild type. The mutation of TRW13-1 was mapped to the bottom arm of chromosome 1 where rdo3 has also been mapped, but the two mutants are not allelic. We designated TRW13-1 as thermoinhibition-resistant germination 1 (trg1). We also mapped the ABA-insensitive mutation of TRW134-15 to the bottom arm of chromosome 5 and named it trg2. These results show that both embryo/endosperm and maternal factors contribute to germination inhibition at supraoptimal temperatures in Arabidopsis. In addition, we confirm the role of ABA in thermoinhibition of seed germination and a link between seed physiological dormancy and response to high temperature.     

Role of the testa in preventing cellular rupture during imbibition of legume seeds

Studies with the seeds of soybean, navy bean, pea, and peanut were made to determine the extent of leakage of intracellular enzymes during imbition. Embryos with intact testae from all four species were found to leak detectable activities of either intracellular enzymes of the cytosol (glucose-6-phosphate dehydrogenase) or enzymes found in both the cytosol and organelles (malate dehydrogenase, glutamate dehydrogenase, glutamate oxaloacetate transaminase, and NADP-isocitrate dehydrogenase) after 6 hours imbition at 25 C. Pea and peanut embryos with testae leaked considerably lower levels of activity for these enzymes than did those of soybean and bean. Leakage of mitochondrial marker enzymes (fumarase, cytochrome c oxidase, and adenylate kinase) was not detected from embryos with testae, suggesting that a differential diffusion of intracellular components out of cells occurred. Soybean and bean embryos without testae leaked high, and proportionally (per cent dry seed basis) similar, levels of all cytosol, cytosol-organelle, and mitochondrial marker enzymes and protein during imbibition, indicating that cell membranes were not differential to leakage and that they had ruptured. Pea and peanut embryos without testae leaked detectable activities of all cytosol and cytosol-organelle enzymes, although fumarase was the only detectable mitochondrial marker enzyme leaked, suggesting that some degree of differential leakage may have occurred in these species. The outermost layers of embryo cells of seeds without testae of all four species absorbed and sequestered the nonpermeating pigment Evan's blue after 5 to 15 minutes imbibition, indicating that membranes had ruptured. This occurred to a much lesser extent in seeds with intact testae. Both soybean and bean embryos without testae were observed to disintegrate during imbibition, whereas those of pea and peanut did not. These data indicate that seeds of certain legumes are susceptible to cellular rupture during imbibition when seed coats are damaged or missing.     

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.     

蓝花丹种子生物学特性及其采集、贮藏技术研究

蓝花丹（Plumbago auriculata Lam.）原产南非,由于其存在自交不亲和现象,导致自然结实率极低。本文研究了蓝花丹种子的形态结构、吸水特性及最适萌发温度,根据形态观测以及发芽率等指标筛选出优质种子,并对优质种子不同的干燥方法、贮藏温度等进行探讨。结果表明：蓝花丹种子呈长椭圆状,长8.00~13.80 mm,长宽比约为5.3：1。种皮薄而柔软,有网状褶皱。外种皮表面有附着物,大面积分布有腺体,利于种子传播。胚芽靠近合点,萌发率较高。种子播种前的浸种时间应保持在11 h以上,最适发芽温度为25~30℃。当种皮颜色为深褐色,种子长度大于11 mm且发芽率大于75%时,可判断为优质种子,发生此性状时即为最佳采种时间。种子适宜的干燥方式为烘箱内30℃干燥1 d,干燥种子最佳贮藏温度为-86℃,且贮藏时间越短发芽率越高,而新鲜种子在此条件下不宜贮藏超过15 d。     

Phytochrome A-mediated inhibition of seed germination in tomato

Far-red light (FR) inhibition of seed germination of tomato (Solanum lycopersicum L.) was studied with the phytochrome (phy)-hypersensitive mutants, hp-1w, hp-1w,fri1, a phyA-deficient double mutant, and hp-1w,tri1, a phyB1-deficient double mutant. Seeds of all mutants germinated readily in the dark at 25 degrees C, and the germination was retarded by a single 100-s FR pulse given 1-3 h after sowing. The effect of an FR pulse was red-light reversible in all mutants used. After 24 h where a single FR pulse was no longer effective, prolonged FR exposure or hourly FR pulses suppressed germination in hp-1w and hp-1w,tri1, whereas in hp-1w,fri1 the suppressive effect of FR was almost absent. The effect of the prolonged FR was greater than that of the hourly 3-min FR pulses having equal photon fluence, and was fluencerate dependent. Thus we conclude that the germination inhibition by FR in tomato seed consists of a low-fluence response and a high irradiance response (HIR); the latter is controlled by phyA, but not phyB1. This is the first indication of phyA being involved in the HIR of seed germination inhibition.     

Characterization of tt15, a novel transparent testa mutant of Arabidopsis thaliana (L.) Heynh.

The Arabidopsis thaliana seed coat typically has a brown color due to the accumulation of flavonoid pigments in the testa. Mutants of A. thaliana with defects in pigment biosynthesis often produce seeds that are olive brown or even yellow in appearence, and the responsible genetic loci are referred to as TRANSPARENT TESTA (TT). Large-scale screening for mutants affected in seed development and complementation analysis of a candidate mutant line with all published A. thalianatt mutants identified a new tt locus designated tt15. The tt15 mutation maps to the lower part of chromosome 1. Mutant plants produced pale greenish-brown seeds whose dormancy was slightly reduced. The phenotype was consistent with the maternal origin of the testa. Analysis of pigment accumulation and the study of expression patterns of genes involved in flavonoid biosynthesis in tt15 plants and seeds indicated a seed-specific phenotype. Most notable was a reduction of the cyanidin and quercetin content of tt15 seeds. Received: 2 October 1998 / Accepted: 10 October 1998     

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.     

The influence of the testa, damage and seed dressing on the emergence of groundnut (Arachis hypogea)

The effects of damaging the testa and the application of seed dressings were examined in field trials on several short-and long-season cultivars of groundnut (Arachis hypogea) with differently-pigmented testas at Samaru, Nigeria, in 1967. There was a high correlation between the number of seedlings which emerged in these trials and the resistance or susceptibility of the seed to invasion by the fungus Aspergillus flavus, as assessed by laboratory tests. When the testa was undamaged the emergence of white (susceptible) seed was only 50% while that of coloured (resistant) seed was between 95 and 98%. Damage to the testa by scratching greatly decreased emergence. The application of seed dressing increased the emergence of susceptible seed and also restored the emergence of scratched seeds to the level of undamaged seeds. The effect of the complete removal of the testa was not counteracted by seed dressing; naked seeds, with and without dressing, gave c. 10% emergence. The importance of pigment in the testa, the condition of the seed and the effects of seed dressing are discussed.     

The 7B-1 mutant in tomato shows blue-light-specific resistance to osmotic stress and abscisic acid

Germination of wild-type (WT) tomato ( Lycopersicon esculentum Mill.) seed is inhibited by mannitol (100-140 mM) in light, but not in darkness, suggesting that light amplifies the responsiveness of the seed to osmotic stress (M. Fellner, V.K. Sawhney (2001) Theor Appl Genet 102:215-221). Here we report that white light (W) and especially blue light (B) strongly enhance the mannitol-induced inhibition of seed germination, and that the effect of red light (R) is weak or nil. The inhibitory effect of mannitol could be completely overcome by fluridone, an inhibitor of abscisic acid (ABA) biosynthesis, indicating that mannitol inhibits seed germination via ABA accumulation in seeds. The inhibition of WT seed germination by exogenous ABA was also amplified by W or B, but not by R. In a recessive, ABA-overproducing, 7B-1 mutant of tomato, seed germination and hypocotyl growth were resistant to inhibition by mannitol or exogenous ABA, both in W or B. Experiments with fluridone suggested that inhibition of hypocotyl growth by W or B is also partially via ABA accumulation. De-etiolation in the mutant was especially less in B compared to the WT, and there was no difference in hypocotyl growth between the two genotypes in R. Our data suggest that B amplifies the responsiveness of tomato seeds and hypocotyls to mannitol and ABA, and that W- or B-specific resistance of the 7B-1 mutant to osmotic stress or ABA is a consequence of a defect in B perception or signal transduction.