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.     

蔷薇种子的休眠及解除方法

分析了蔷薇(Rosa L.)种子休眠原因、解除休眠方法以及环境条件对休眠与萌发的影响.蔷薇种子休眠的主要原因有瘦果果皮和种皮的限制作用,胚生理休眠以及果肉、瘦果果皮、种皮和胚中的抑制物质.解除休眠的方法包括去除瘦果果皮限制、解除胚的生理休眠、去除抑制物质等.种子发育过程中及成熟后,环境因子,如温度、水分和光照,对种子休眠和萌发有影响.此外,微生物、果实采集时间也对种子休眠及萌发有较大影响.蔷薇种子的休眠机制复杂,且种间差异很大.     

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 Arabidopsis aleurone layer responds to nitric oxide, gibberellin, and abscisic acid and is sufficient and necessary for seed dormancy

Seed dormancy is a common phase of the plant life cycle, and several parts of the seed can contribute to dormancy. Whole seeds, seeds lacking the testa, embryos, and isolated aleurone layers of Arabidopsis (Arabidopsis thaliana) were used in experiments designed to identify components of the Arabidopsis seed that contribute to seed dormancy and to learn more about how dormancy and germination are regulated in this species. The aleurone layer was found to be the primary determinant of seed dormancy. Embryos from dormant seeds, however, had a lesser growth potential than those from nondormant seeds. Arabidopsis aleurone cells were examined by light and electron microscopy, and cell ultrastructure was similar to that of cereal aleurone cells. Arabidopsis aleurone cells responded to nitric oxide (NO), gibberellin (GA), and abscisic acid, with NO being upstream of GA in a signaling pathway that leads to vacuolation of protein storage vacuoles and abscisic acid inhibiting vacuolation. Molecular changes that occurred in embryos and aleurone layers prior to germination were measured, and these data show that both the aleurone layer and the embryo expressed the NO-associated gene AtNOS1, but only the embryo expressed genes for the GA biosynthetic enzyme GA3 oxidase.     

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 Role of the Lens in Controlling Heat-induced Breakdown of Testa-imposed Dormancy in Native Australian Legumes

A high proportion of seeds of most species of the Fabaceae havetesta-imposed dormancy, which prevents them from imbibing watereven under favourable environmental conditions. This allowsseeds to accumulate in a persistent soil seed bank. Dormancyis usually considered to be broken only when the testa is disrupted,particularly at the lens. In Mediterranean-type ecosystems,the primary natural mechanism causing the breakdown of thisdormancy is the disruption of testas in the soil seed bank byheat from fires that warms the soil in which the seed bank occurs.We examined experimentally the role of the lens in controllingthis heat-induced breakdown of dormancy in 16 native Australianspecies of the Faboideae and Mimosoideae. Two groups of patternsare recognized: species of the Mimosoideae: Acacieae and Faboideae:Mirbelieae, where water permeability is regulated by the lens;and species of the Faboideae: Bossiaeeae, where water permeabilityapparently occurs at non-localized regions of the testa. Thespecies in these two groups differ in the structure of theirtestas. Thus, testa-imposed dormancy does not represent a singledormancy mechanism in legumes, as is often assumed when dormancyis broken artificially.Copyright 1998 Annals of Botany Company Leguminosae, Fabaceae, seed dormancy, hard-seededness, germination, lens, strophiole, Australia.     

Communication between the maternal testa and the embryo and/or endosperm affect testa attributes in tomato

Two tomato (Lycopersicon esculentum) mutants with dark testae displaying poor germination rate and percentage on both water and 100 microM gibberellin(4 + 7) were recovered. The mutants were allelic (black seed1-1; bks1-1 and bks1-2), inherited in Mendelian fashion as a recessive gene residing on chromosome 11. They are not allelic to bs (brown seed) -1, -2, or -4, which impair seed germination and possess dark testae. The bks/bs mutants accumulated dark pigment in the cell layers of the testa above the endothelium, which itself accumulated proanthocyanidins similar to wild type. The poor germination performance of bks mutant seeds was because of impediment of the mutant testae to radicle egress. Imbibition on gibberellin(4 + 7) did not ameliorate germination percentage or rate. The toughening of the bks testa and associated poor germination were partially overcome when seeds were not dried before germination or were dried under N(2). The seeds of the bks mutant have elevated activity of at least one enzyme responsible for the detoxification of reactive oxygen species. The bks mutant is epistatic to 12 anthocyaninless mutants of tomato. Bio- and physicochemical analysis of the bks testa determined that it accumulated a melanic substance. Inheritance of bks/bs mutations contrasts with that of the anthocyaninless mutants, which are inherited according to the genotype of the maternally derived testa. This suggests that the testa manufactures components before its demise that can maximize testa strength, whereas the endosperm/embryo produces factors that are conveyed to the testa, mitigating this process.     

新疆干旱区植物藜的种子异型性及其萌发机理

新疆干旱区分布的植物藜(Chenopodium album)的种子有黑色和褐色两种类型。对藜的异型性种子从形态结构、不同环境因素及激素或化学物质对萌发的影响以及同工酶谱等方面进行了研究,并对其萌发及适应异质环境的机理进行了讨论。结果表明:(1)藜的异型性种子在形态结构、萌发休眠特性等方面都存在明显差异:黑色种子种皮厚且硬,休眠,萌发慢,萌发率低;褐色种子种皮薄而软,不休眠,萌发快且萌发率高;(2)黑色种子的休眠可通过切除胚根外缘种皮得以完全解除;(3)赤霉素、乙烯利对黑色种子的萌发无明显促进作用;KNO3可较显著促进黑色种子的萌发;协同使用乙烯利和KNO3时,可显著提高黑种子萌发率,完全打破休眠;(4)黑色种子和褐色种子的酯酶、过氧化物酶及过氧化氢酶同工酶谱带存在差异;(5)黑色种子的萌发需要光照,而褐色种子则对光不敏感;低温贮藏对二者的萌发均无显著影响,尽管黑色种子的萌发率有波动。研究结果初步显示黑色种子的休眠是内源(胚)和外源(种皮)因素共同所致。藜的种子异型性及其萌发机理的形成是其对新疆干旱区异质化环境的高度适应。     

Analyses to determine the role of the megagametophyte and other seed tissues in dormancy maintenance of yellow cedar (Chamaecyparis nootkatensis) seeds; morphological, cellular and physiological changes following moist chilling and during germination

Yellow cedar (Chamaecyparis nootkatensis) seeds exhibit prolonged dormancy following their dispersal from the parent plant. Embryos excised fully from their enclosing seed tissues exhibit 100% germination, indicating that the seed tissues enclosing the embryo (the testa, remnants of the nucellus and the megagametophyte) play an inhibitory role and prevent radicle emergence. As part of an assessment of the role of seed tissues in the dormancy mechanism of yellow cedar seeds, light microscopy was used to examine changes within the major structures of the seed following a 90 d war (26C)/cold (4C) moist treatment ('stratification') and during germination. In the micropylar tip of the seed, the nucellus forms a hard nucellar cap covering the radicle. The nucellar cap is composed primarily of degenerated cells; histological staining with ruthenium red revealed a predominance of pectins. There were no obvious cellular or morphological differences (detected by light microscopy) between mature seeds subjected to a 3 d soak and seeds subjected to a 3 d soak and the 90 d dormancy-breaking treatment. However, just prior to germination there was an outward projection of the nucellar cap through the micropyle, which appeared to be caused by the extension of highly folded proteinaceous strands lying immediately in front of the radicle. When the testa was removed, the embryo enclosed within the intact megagametophyte was incapable of germination. If, however, the megagametophyte surrounding the embryo was slit or the embryo surrounded by an intact megagametophyte was subjected to a 3d rinse in water, some germination occurred, perhaps as a result of an enhanced release of inhibitors from the megagametophyte. After stratification, dormancy of yellow cedar seeds is broken; concurrent with dormancy breakage, there was a mechanical weakening of the megagametophyte. The embryo also underwent changes that included an increase in turgor and a reduced sensitivity to highly negative osmotic potential. It is concluded that coat-imposed dormancy of yellow cedar seeds is enforced by mechanical restraint of the megagametophyte as well as a leachable chemical inhibitor (most probably ABA).     

Leaching of Ions, Organic Molecules, and Enzymes from Seeds of Peanut (Arachis hypogea L.) Imbibing without Testas or with Intact Testas

Reducing sugars, phosphates, potassium ions, total electricallyconducting material, proteins, and phenolics leached from twovarieties of peanut (Arachis hypogea L. ) seeds during 24 himbibition in distilled water. The same substances leached fromthe seeds when testas were removed before imbibition. The quantitiesof substances leached from seeds without testas after 24 h,except protein and phenolics, were much greater than from seedswith intact testas. Time courses of leaching of sugars, phosphates,potassium ions, and of total electrically conducting materialshowed fastest leaching rates in the first hour of imbibition.In the first 4 h of imbibition more sugars leached from intactseeds than from seeds without testas. Peroxidase was presentin leachates from intact seeds but was undetectable in leachatesfrom seeds without testas. Hydrogen peroxide-reducing activitywas detected in 24 h leachates from intact seeds and from seedswithout testas. The activity was partly inhibited by 1 mM ascorbate.It was concluded that catalase was present in the leachates. The peanut testas were thin. Their presence around the embryodid not reduce the rate of imbibition as compared with thatof seeds without testas. Thus the greater leaching of some substancesin the absence of the testa could not be ascribed to the absenceof a physical barrier to water uptake.     

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.     

红松种子休眠与种皮的关系

本文探讨红松(Pinus koraiensis)种子休眠与其种皮之间的关系。夹破中种皮后,种子萌发率很低。在离体胚培养基中外加 ABA 及经 ABA 溶液浸泡种子的萌发实验表明,ABA也不是导致休眠的关键因素。试验确认红松种子存在透气障碍,即中、内种皮对氧气的进入都有阻碍作用。经低温砂藏后,种皮的阻碍作用明显减小。种皮的透气性障碍可能是诱导休限的主导因素。     

Genetic Regulation of Seed Dormancy in Purshia tridentata(Rosaceae)

Seeds of Purshia tridentata, a shrub of semi-arid North America,require chilling to become non-dormant. Using seeds producedfrom controlled crosses in a common garden, we examined effectsof ovule parent, pollen parent, and year of production on germinationpercentage in response to chilling for 2 weeks at 2°C. Differencesamong ovule parents accounted for most of the variance in chillingresponse, and these differences (2 to 83% germination) wereconsistent across years. Differences among pollen parents werealso significant, producing a two- to five-fold difference inmean germination percentage. Differences among years were significantbut small. Ovule parent by year interactions showed that among-yearvariation in ripening environment did not affect ovule parentsequally. There was no significant pollen parent by year interaction,suggesting that the effect of maturation environment was mediatedthrough maternal tissues. In reciprocal crosses, two plantsthat showed contrasting dormancy levels as ovule parents producedseeds with similar dormancy as pollen parents, indicating thatthe genetic difference between them was at the testa level.Two plants that produced seeds with contrasting dormancy aspollen parents showed a similar but stronger pattern of contrastas ovule parents, showing that the genetic difference betweenthem was at both embryo and endosperm or testa level. Testaor endosperm genotype was primarily responsible for chillingresponse of intact seeds, while embryo genotype affected chillingresponse and also exercised primary control over low temperaturegermination rate, whether of excised embryos or of intact seeds.Copyright 2000 Annals of Botany Company Antelope bitterbrush, bitterbrush, common garden study, germination     

桃儿七种子解剖结构及其萌发生长期形态特征

为探明种皮和胚乳是否是限制桃儿七种子萌发的主要因素,利用组织切片和显微技术,对桃儿七种子及其不同萌发期（1、7、14、21、28 d）解剖结构和播种后一定时期内（7~210 d）的植株生长形态进行观察。桃儿七种子由种皮、胚乳和胚构成。种皮包括外种皮和内种皮,外种皮致密规整,由外至内分别为栅状石细胞和表皮层细胞,内种皮由5~6层海绵细胞组成。胚乳占种子体积的绝大部分,包括珠孔胚乳和外胚乳。胚由胚根、胚轴和子叶组成,被致密种皮、多层珠孔胚乳和外胚乳包围。萌发期1~7 d胚根和胚轴开始伸长,7~14 d两片子叶分离,14~21 d胚根突破珠孔胚乳和种皮,21~28 d胚根、胚轴和子叶继续扩张伸长。种子播种210 d后可平均形成3片功能真叶和5条不定根。致密种皮（物理休眠）和多层胚乳（机械休眠）是限制桃儿七种子萌发的两个主要因素。     

The Role of ABA in the Control of Apple Seed Dormancy Re-appraised by Combined Gas Chromatography-Mass Spectrometry

Measured by GC—MS²—SIR³, endogenous ABA⁴ in embryonicaxes of seeds of Malus pumila L. cv. Golden Delicious decreased8-fold and cotyledon ABA by only 60%, during 10–50 d ofstratification at 5 ?C, after ABA leaching during an initial24 h soaking. During stratification, the percentage germinationof embryos transferred to 17?C showed a significant linear dependenceon log_e of ABA levels in the axes at transfer. Between 50 and70 d, ABA levels increased markedly in axes and testa both ofstratified seeds and seeds allowed to re-dry at 17 ?C afterinitial soaking. The ability of fully stratified axes with elevatedendogenous ABA to germinate indicated that stratification haddecreased their ABA sensitivity. Changes in cotyledon ABA couldnot account for the promotory effect of cotyledons on germinationduring the first 30 d of stratification. Loss of testa inhibitionof germination during stratification was not linked with changesin testa ABA. Stratification markedly increased the sequestrationin the axes of exogenous ABA supplied via the cotyledons. Changesboth in axis ABA levels and sensitivity were thus correlatedwith dormancy release, but subject to modifying control by thecotyledons and testa not involving ABA. Rehydration of driedseeds affected axis ABA later during storage via mechanismsunconnected with dormancy. Key words: ABA, seed dormancy, stratification     

Sequential temperature control of multi-phasic dormancy release and germination of Paeonia corsica seeds

Aims The physiological responses during dormancy removal and multi-phasic germination were investigated in seeds of Paeonia corsica (Paeoniaceae).Methods Seeds of P. corsica were incubated in the light at a range of temperatures (10–25 and 25/10°C), without any pre-treatment, after W (3 months at 25°C), C (3 months at 5°C) and W + C (3 months at 25°C followed by 3 months at 5°C) stratification, and a GA 3 treatment (250 mg·l^-1 in the germination substrate). Embryo growth, time from testa to endosperm rupture and radicle emergence were assessed as separate phases. Epicotyl–plumule emergence was evaluated incubating the germinated seeds at 15°C for 2 weeks, at 5 and 25°C for 2 months on agar water before transplanting to the soil substrate at 10, 15 and 20°C and at 15°C for 2 months on the surface agar water with GA 3 .Important findings Embryo growth, testa rupture, endosperm rupture (radicle emergence) and growth of the epicotyl were identified as four sequential steps in seeds of P. corsica. Gibberellic acid alone and warm stratification followed by 15°C promoted embryo growth and subsequent seed germination. Cold stratification induced secondary dormancy, even when applied after warm stratification. After radicle emergence, epicotyl–plumule emergence was delayed for ca. 3 months. Mean time of epicotyl–plumule emergence was positively affected by cold stratification (2 months at 5°C) and GA 3. P. corsica seeds exhibited differential temperature sensitivity for the four sequential steps in the removal of dormancy and germination processes that resulted in the precise and optimal timing of seedling emergence.