Osmoconditioning prevents the onset of microtubular cytoskeleton and activation of cell cycle and is detrimental for germination of Jatropha curcas L. seeds

• Jatropha curcas is an oilseed crop renowned for its tolerance to a diverse range of environmental stresses. In Brazil, this species is grown in semiarid regions where crop establishment requires a better understanding of the mechanisms underlying appropriate seed, seedling and plant behaviour under water restriction conditions. In this context, the objective of this study was to investigate the physiological and cytological profiles of J. curcas seeds in response to imbibition in water (control) and in polyethylene glycol solution (osmoticum).
• Seed germinability and reactivation of cell cycle events were assessed by means of different germination parameters and immunohistochemical detection of tubulin and microtubules, i.e. tubulin accumulation and microtubular cytoskeleton configurations in water imbibed seeds (control) and in seeds imbibed in the osmoticum.
• Immunohistochemical analysis revealed increasing accumulation of tubulin and appearance of microtubular cytoskeleton in seed embryo radicles imbibed in water from 48 h onwards. Mitotic microtubules were only visible in seeds imbibed in water, after radicle protrusion, as an indication of cell cycle reactivation and cell proliferation, with subsequent root development. Imbibition in osmoticum prevented accumulation of microtubules, i.e. activation of cell cycle, therefore germination could not be resumed.
• Osmoconditioned seeds were able to survive re‐drying and could resume germination after re‐imbibition in water, however, with lower germination performance, possibly due to acquisition of secondary dormancy. This study provides important insights into understanding of the physiological aspects of J. curcas seed germination in response to water restriction conditions.

     

Changes in Some ATP-Dependent Activities in Seeds during Treatment with Polyethyleneglycol and during the Redrying Process

During the imbibition of seeds in polyethyleneglycol (PEG),increasing amounts of ATP accumulated up to 24 h. Similar amountsaccumulated in the seeds during 4–5 h of imbibition inwater. Radioactive amino acids were increasingly incorporatedin the acid-insoluble fraction up to 24 h imbibition in PEG,as well as in water, after which a sharp decrease occurred upto 5 d of imbibition. If seeds were imbibed in PEG or waterin the presence of radioactive acetate, water-insoluble radioactivityincreased linearly in seeds during 5 d of imbibition. The amountsof incorporated amino acids or acetate were about double inPEG-imbibed as compared with in water-imbibed seed. The incorporationof AMP into the acid-insoluble fraction in seeds imbibed inPEG in the presence of radioactive AMP levelled off after 24h followed by a sharp decrease of up to 10% of the peak 5 dafter the start of imbibition. In water-imbibed seeds the incorporationof AMP continued to increase during at least 5 d of imbibition.During redrying of PEG-treated seeds (24 h), at least 80% ofthe accumulated ATP decreased during 18 d. The total radioactiveamino acids and nucleotide decreased during 3 d of redryingby 20% and 60%, respectively. At that time, the acid-insolubleincorporates increased by 20% and 50%, respectively. Some ofthe AMP was released as CO₂. Key words: AMP, Germination, Nucleic acid synthesis, Osmoconditioning, PEG, Protein synthesis     

Effects of the pericarp on imbibition,seed germination,and seedling establishment in seeds of <Emphasis Type="Italic">Hedysarum scoparium</Emphasis> Fisch. et Mey

Four independent experiments were designed to investigate the effects of the pericarp on seed imbibition, dehydration, germination, seedling establishment, and seed longevity in the field in seeds of Hedysarum scoparium Fisch. et Mey. The results showed that the presence of the pericarp decreased seed imbibition rates in the first 6 h, but the seeds attained significantly higher final water content after 24 h of soaking. The pericarp caused seed dormancy, and removal of the pericarp improved the germination percentage to 90 from 44%. In the pot experiment, where the level of moisture was maintained at field capacity (control), seeds with the pericarp removed had significantly improved seedling establishment. However, no statistical differences were observed in seedling establishment when the experiment was repeated under dry conditions at 40% of the field water capacity. The seedling biomass derived from seeds without the pericarp was much higher in the control but the trend was reversed under dry conditions. For seed longevity, 2 months burial in the field killed almost all seeds without the pericarp, while more than 70% of the seeds with the pericarp intact remained viable. These results indicated that the pericarp was beneficial for seedling establishment and seed longevity in arid environments. The results of this study may have practical application in grassland restoration in dry areas, especially for aerial seeding, which has been extensively used in the northern part of China.     

Soaking injury and its reversal with polyethylene glycol in relation to respiratory metabolism in high and low vigor soybean seeds

Germination and growth of unaged (high vigor) and accelerated aged (41°C and 100% RH for 4 days; low vigor) soybean [ Glycine max (L.) Merr. cv. Essex] seeds were severely reduced by soaking the seeds under water at 25°C during the first 1 to 8 h of imbibition. Soaking in 30% polyethylene glycol (PEG) did not injure high vigor seeds and improved the performance of low vigor seeds. Soaking in water increased subsequent RQ values and acetaldehyde and ethanol levels. In high vigor seeds, the increase in RQ lasted only 1 h after transfer to aerobic conditions, whereas in low vigor seeds the increase persisted into the second h. Increases in ethanol and acetaldehyde after soaking were more pronounced in low than in high vigor seeds. The data indicate that relatively short periods under water can cause water uptake injury to imbibing soybean seeds and that this injury can be avoided by osmotically reducing the initial rate of water uptake. Seeds whose vigor has declined during accelerated aging show an additional injury which increases with soaking duration and involves alterations in respiratory metabolism including accumulation of acetaldehyde and ethanol to high, and possibly toxic, levels during imbibition.     

菜用大豆种子活力与DNA,RNA及蛋白质合成的关系

菜用大豆种子随着其活力的下降,对DNA,RNA和蛋白质前体的吸收,以及合成这些大分子的能力都明显下降,已丧失合成DNA和蛋白质能力的失活种子,仍能进行微弱的RNA合成。高活力种子在吸胀初期DNA合成速率较低,然后增加,至16h达高峰;RNA的合成速率在吸胀一开始就很高,在整个吸胀过程中均保持较高水平;蛋白质的合成速率则在开始较高,并随着吸胀过程呈增强趋势。     

Imbibition period as the critical temperature sensitive stage in germination of lima bean seeds

Lima bean seeds (Phaseolus lunatus L.) and excised embryonic axes can be injured during imbibition at temperatures below 25°. The early imbibitional stage is critical; imbibition at 25° followed by low temperature exposure does not cause injury. Sensitivity to chilling injury is conditioned by the pre-harvest seed history. Low vigor (bleached) seeds are most sensitive to injury, the effects of which can be intensified by restricted oxygen supply during early axis growth. The seed coat, by preventing water uptake, can permit the seed to avoid injury. This protective mechanism is most effective at low temperature and high moisture stress. Immediately following low temperature imbibition, injured axes lose organic materials, probably nucleotides. This organic leachate is a potential influence on soil microorganisms and, together with the temperature sensitivity, vigor, and seed coat effect undoubtedly is important in controlling the potential variability in germination shown by a seed population.     

Germination and Food Reserves in Bambarra Groundnut Seeds (Voandzeia subterranea Thouars) after Different Periods of Storage

Studies were made on bambarranut seeds (Voandzeia subterraneaThouars) after 0, 6, 12, 18 and 24 months of storage in gunnybags under laboratory conditions (25–35 °C). Seeddeterioration during storage was indicated by delayed germination,reduced germinability, reduced growth of seedlings and increasednumber of stunted seedlings culminating in a total failure ofgermination after two years. Slight depletion of food reserves occurred during seed storage.The loss in fat was higher than starch or protein. Total solublesugars decreased while the content of total fatty acids andamino acids and soluble protein increased. Total nitrogen (N)remained unaffected while soluble-N and amino-N increased. Allthese components showed a rapid change (increase or decrease)from 12 months to 18 months of storage which was associatedwith commencement of rapid decline in germinability of the seedsand growth of the seedlings. Initial rapid imbibition of water was observed in viable aswell as non-viable seeds, though at a higher rate in the latterand followed by a lag period in both. At the end of 24 h ofimbibition, water content in non-viable seeds was less thanthat in viable ones. Key words: Voandzeia subterranea, Seed germination, Seed storage     

Protein Synthesis During Rehydration, Germination and Seedling Growth of Naturally and Precociously Matured Soybean Seeds (Glycine max)

Soybean seeds [Glycine max (L.) Merr.] synthesize de novo andaccumulate several non-storage, soluble polypeptides duringnatural and precocious seed maturation. These polypeptides havepreviously been coined ‘maturation polypeptides’.The objective of this study was to determine the fate of maturationpolypeptides in naturally and precociously matured soybean seedsduring rehydration, germination, and seedling growth. Developingsoybean seeds harvested 35 d after flowering (mid-development)were precociously matured through controlled dehydration, whereasnaturally matured soybean seeds were harvested directly fromthe plant. Seeds were rehydrated with water for various timesbetween 5 and 120 h. Total soluble proteins and proteins radio-labelledin vivo were extracted from the cotyledons and embryonic axesof precociously and naturally matured and rehydrated seed tissuesand analyzed by one-dimensional PAGE and fluorography. The resultsindicated that three of the maturation polypeptides (21, 31and 128 kDa) that had accumulated in the maturing seeds (maturationpolypeptides) continued to be synthesized during early stagesof seed rehydration and germination (5–30 h after imbibition).However, the progression from seed germination into seedlinggrowth (between 30 and 72 h after imbibition) was marked bythe cessation of synthesis of the maturation polypeptides followedby the hydrolysis of storage polypeptides that had been synthesizedand accumulated during seed development. This implied a drasticredirection in seed metabolism for the precociously maturedseeds as these seeds, if not matured early, would have continuedto synthesize storage protein reserves. Glycine max (L.) Merr, soybean, cotyledons, maturation, germination/seedling growth     

苦马豆和披针叶黄华种子硬实特性与活力关系

将苦马豆和披针叶黄华种子在恒温25℃下吸胀,每24 h取出吸胀种子,16 d后未吸胀的种子为硬实种子（H）,硬实种子用硫酸处理后恒温吸胀24 h,与非硬实种子进行发芽试验和各项活力指标测定。结果显示每日内吸胀的种子数量随时间推移以一定比列下降,苦马豆非硬实种子第3天后吸胀率下降到1%,第13～16天突然上升后又下降到1%,披针叶黄华非硬实种子第3天后下降到1%,第9、10天突然上升后又下降到1%。两种豆类都显示出硬实种子发芽率、发芽指数、活力指数、脱氢酶活性、呼吸速率和超氧化物歧化酶（SOD）活性均高于非硬实种子,而电导率、浸出液可溶性糖和丙二醛（MDA）含量低于非硬实种子,缓慢吸胀的硬实种子活力指标高于快速吸胀的硬实种子,这表明硬实种子活力高于非硬实种子,硬实种子吸胀过程中存在吸胀损伤。而在非硬实种子中,根据以上活力指标判断,晚吸胀的种子比早吸胀的种子活力高。     

Effect of high temperature on moisture loss,imbibition and germination of seeds ofTrianthema Govindia Buch.-Ham. ex DC

Summary Seeds of erect and prostrate plants ofTrianthema govindia Buch. ham. ex DC., growing in shade and open respectively, differed significantly in seed weight and percentage germination. Effect of high temperature exposure to these seeds has been studied in view of water depletion, imbibition and seed germination. The seeds of both the types were subjected to temperatures of 40, 50, 60, and 70° C for 24, 48, 96, and 144 hours. The three factors viz., loss of water, water imbibition and germination of seeds were positively correlated to the duration of treatment at different temperatures. A highly significant positive correlation was also observed between moisture depletion and imbibition, and between imbibition and germination. The percentage germination was favoured at 40° C in both the types of seeds and was increased with the increase of treatment duration. However, at higher temperatures (50 and 60° C) the percentage declined while at 70° C the seeds lost their vitality.     

beta]-Tubulin Accumulation and DNA Replication in Imbibing Tomato Seeds

The activation of the cell cycle in embryo root tips of imbibing tomato (Lycopersicon esculentum Mill. cv Lerica) seeds was studied by flow cytometric analyses of the nuclear DNA content and by immunodelection of [beta]-tubulin. With dry seeds, flow cytometric profiles indicated that the majority of the cells were arrested at the G1 phase of the cell cycle. In addition, [beta]-tubulin was not detectable on western blots. Upon imbibition of water, the number of cells in G2 started to increase after 24 h, and a 55-kD [beta]-tubulin signal was detected between 24 and 48 h. Two-dimensional immunoblots revealed at least three different [beta]-tubulin isotypes. Thus, [beta]-tubulin accumulation and DNA replication were induced during osmotic priming. These processes, as well as seed germination rate, were enhanced upon subsequent imbibition of water, compared with control seeds that imbibed but were not primed. By contrast, when aged seeds imbibed, DNA replication, [beta]-tubulin accumulation, and germination were delayed. In all cases studied, both DNA replication and [beta]-tubulin accumulation preceded visible germination. We suggest that activation of these cell-cycle-related processes is a prerequisite for tomato seed germination. Furthermore, [beta]-tubulin expression can be used as a parameter for following the initial processes that are activated during seed imbibition.     

Seeds of tomato (Lycopersicon esculentum Mill.) which develop in a fully hydrated environment in the fruit switch from a developmental to a germinative mode without a requirement for desiccation

Seed water content is high during early development of tomato seeds (10–30 d after pollination (DAP)), declines at 35 DAP, then increases slightly during fruit ripening (following 50 DAP). The seed does not undergo maturation drying. Protein content during seed development peaks at 35 DAP in the embryo, while in the endosperm it exhibits a triphasic accumulation pattern. Peaks in endosperm protein deposition correspond to changes in endosperm morphology (i.e. formation of the hard endosperm) and are largely the consequence of increases in storage proteins. Storage-protein deposition commences at 20 DAP in the embryo and endosperm; both tissues accumulate identical proteins. Embryo maturation is complete by 40 DAP, when maximum embryo protein content, size and seed dry weight are attained. Seeds are tolerant of premature drying (fast and slow drying) from 40 DAP.Thirty-and 35-DAP seeds when removed from the fruit tissue and imbibed on water, complete germination by 120 h after isolation. Only seeds which have developed to 35 DAP produce viable seedlings. The inability of isolated 30-DAP seed to form viable seedlings appears to be related to a lack of stored nutrients, since the germinability of excised embryos (20 DAP and onwards) placed on Murashige and Skoog (1962, Physiol. Plant. 15, 473–497) medium is high. The switch from a developmental to germinative mode in the excised 30- and 35-DAP imbibed seeds is reflected in the pattern of in-vivo protein synthesis. Developmental and germinative proteins are present in the embryo and endosperm of the 30- and 35-DAP seeds 12 h after their isolation from the fruit. The mature seed (60 DAP) exhibits germinative protein synthesis from the earliest time of imbibition. The fruit environment prevents precocious germination of developing seeds, since the switch from development to germination requires only their removal from the fruit tissue.Abbreviations DAP days after pollination - kDa kilodaltons - SP1-4 storage proteins 1–4 - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - HASI hours after seed isolation - MS medium Murashige and Skoog (1962) medium This work is supported by National Science and Engineering Research Council of Canada grant A2210 to J.D.B.     

The impact of different seed priming agents and priming durations on stand establishment and biochemical attributes of Stevia rebaudiana Bertoni

Stevia (Stevia rebaudiana Bertoni) is an alternative sugar crop currently gaining importance in several geographic regions of the world. Generally, crop is propagated by seeds; however, low seed germination hinders the adaptability of the crop in different cropping systems. Seed priming with different compounds improves germination of several arable crops under stressful and benign environmental conditions. This study evaluated the role of different seed priming agents and priming durations on stand establishment, allometric and yield-related traits, and steviol glycosides contents of Stevia in two different experiments. The first experiment consisted of five different seed priming agents (i.e., hydropriming with distilled water, ascorbic acid, potassium chloride, benzyl amine purine and unprimed seeds) and five priming durations (0, 8, 16, 24 and 32 h). Seed priming with potassium chloride (KCl) and benzyl amine purine (BAP) for 32 h improved stand establishment and biochemical attributes of Stevia seeds. Therefore, these two seed priming agents along with unprimed seeds were included in the second experiment to quantify their impact on allometric and yield-related traits and steviol glycosides contents. Seed priming with both KCl and BAP resulted in better allometric traits (plant height, number of leaves per plant, leaf area and chlorophyll index) compared with unprimed seeds. Similarly, seed priming with KCl resulted in higher fresh and dry biomass production of stem and leaves. Nonetheless, higher stevioside and rebaudioside A contents were recorded for the seeds primed with KCl, whereas unprimed seeds resulted in the lowest values. It is concluded that Stevia seeds must be primed with KCl for 32 h before sowing to get higher seed germination, leaf yield and steviol glycosides contents. Nonetheless, the role of KCl priming in improving abiotic stress tolerance of Stevia must be explored in the future studies.     

Protein Patterns, Characterized by Computer Image Analysis, of Lentil Embryo Axes Germinating Under Salt Stress

Following 16, 40 and 64 h exposure to 0.33 M NaCl given after 8 h water imbibition, lentil seeds showed a gradual decrease of germination upon their transfer to water. These salt related changes were accompanied by modifications in the protein patterns of embryo axes as revealed by two-dimensional electrophoresis separation and by the computer image analysis of protein spots. In comparison with 8 h water imbibed seeds, prominent proteins comprised between the 5.1 – 7.6 pH isoelectric point in the first dimension and 75 – 50 kDa molecular mass in the second dimension showed a significant increase in their abundance as salt exposure increased. On transfer to water to complete germination, the content of many of these proteins decreased at 24h in 2 – 3 cm length embryo axes in comparison with the corresponding embryo axes of seeds continuously imbibed in water for 24 h. Some groups of proteins ranging between 15.5 – 17.3 kDa, already present after 8 h water imbibition, were not detectable after 24 h but were expressed in seeds exposed to NaCl and transferred to water for 24 h. Up- and down-regulated proteins in lentil embryo axes, imbibed under non-lethal salt stress conditions, have been tentatively identified by comparison with the protein map of germinating seeds of the model plant Arabidopsis. This revised version was published online in July 2006 with corrections to the Cover Date.     

大豆种子萌发过程中的差异蛋白质组研究

运用蛋白质组学技术对大豆(Glycinemax)N2899种子萌发0h、8h、36h、60h4个时期蛋白质的差异表达情况进行了研究.结果发现,在考马斯亮蓝染色的双向电泳pH3～10胶上,PDQuest图像分析软件可识别的点约350个,其中表达量变化2.5倍以上的蛋白质点有24个,而绝大部分大豆种子贮藏蛋白在萌发期尚未降解.在萌发的第一阶段,24个差异表达蛋白中有10个蛋白质的丰度发生变化.第二阶段,差异表达蛋白的种类和量增加,其中15个蛋白质是动态变化的,14个蛋白质在胚根突破种皮时表达量达到峰值,表明吸胀后种子内的生命活动越来越强.对这24个蛋白质点进行胶内酶解,用基质辅助激光解析电离飞行时间质谱测定均获得肽质量指纹图谱.搜索大豆的UniGene库初步鉴定出6个蛋白质,分别是核苷二磷酸激酶、热激蛋白、硫氧还蛋白、35ku种子成熟蛋白及种子成熟蛋白PM36.对这些蛋白质在种子萌发过程中可能的作用进行了讨论.     

Prevention of imbibitional injury in low vigor soybean embryonic axes by osmotic control of water uptake

Deterioration of soybean [ Glycine max (L.) Merr. cv. Essex] seeds during accelerated aging at 41°C and 100% relative humidity predisposes the embryonic axis to injury during the initial period of imbibition. This injury was prevented or greatly reduced in severity when excised axes were imbibed on blotters containing 30% polyethylene glycol which slowed the rate of water uptake and when axes were pre-equilibrated to a high moisture level. Rates of water uptake by "high"(no treatment) and "low vigor"(accelerated aged) excised axes were identical. However, high vigor axes tolerated rapid water uptake during early imbition, whereas low vigor axes did not. Leakage of electrolytes during early imbibition was nearly six times greater in low than in high vigor axes. Polyethylene glycol significantly reduced the leakage of electrolytes from both low and high vigor axes. The data are in agreement with the hypothesis that seed deterioration in soybeans involves membrane changes which may predispose embryonic tissues to injury during imbibition. Reduction of the rate of water uptake during the initial period of imbibition would allow extra time for membrane repair or rearrangement, thus permitting the tissues to develop in a more orderly manner. The data indicate that deterioration in soybean seeds involves, at least in part, a decrease in ability of seed axes to tolerate rapid water uptake at the start of imbibition and that this weakness may be compensated by osmotic control of water uptake.     

Weakening of muskmelon perisperm envelope tissue 4

Muskmelon (Cucumis melo L.) embryos are enclosed in an envelopeof tissue consisting of a layer of endosperm and a multi-cell-layeredperisperm that the radicle must penetrate for germination tooccur. The force and energy required to penetrate the perispermenvelope tissue were measured using an Instron universal testingmachine at a crosshead speed of 5 mm min^–1 after 0, 10,15, 22, 23, and 25 h of imbibition at 25C. The cellular structureof perisperm envelope tissue surrounding the radicle was observedafter 10, 15, 20, 25, and 48 h of imbibition using scanningelectron microscopy. The force required to puncture 5-mm-long,micropylar seed pieces declined steadily from 1.65 N in driedseeds to 0.65 N after 21 h of imbibition. The penetration energydeclined from 3.0 N mm in dry seeds to 1.1 N mm at 21 h afterthe start of imbibition when the first seeds germinated. Theforce and energy required to penetrate germinated seed pieceswere 0.55 N and 0.9 N mm, respectively, so the net punctureforce and energy needed to rupture the micropylar region ofthe perisperm envelope was roughly 0.10 N and 0.2 N mm at radicleemergence, respectively. Instron measurements of penetrationforce and energy decreased dramatically at crosshead speedsless than the 5 mm min^–1. Crosshead speeds greater than5 mm min^–1 may overestimate the pressure needed to ruptureperisperm and endosperm tissues. Intracellular cracks were firstobserved in SEM images 15 h after the start of imbibition, andafter 20 h cracking was apparent throughout the micropylar regionof the perisperm envelope. The perisperm envelope ruptured inone of two ways, coincident with radicle emergence. In approximately85% of muskmelon seeds, a large crack formed in the perispermenvelope adjacent to the radicle, while in roughly 15 % a circulararea of the perisperm envelope detached during radicle emergence.In dead seeds, the penetration force remained constant from10–24 h after the start of imbibition, and there wereno visible signs of tissue degradation. Cellular degradationand weakening of the perisperm envelope tissue precedes radicleemergence in muskmelon seeds. Key words: Seed, Instron, turgor, cell wall, electron microscopy, Cucumis melo     

超干对种子活力及水分热力学特性的影响

以耐干的菜心种子和不耐干的豇豆种子为材料,研究了超干对其活力和水分吸附等温线及热力学参数（△S、△H、△G）的影响。超干使红豇豆种子活力下降,SOD、POD活性降低,膜结构受损,这些变化即发生在水分降低的干燥过程中又发生在眼干状态下的贮藏过程中,菜心种子的活力和SOD,POD的活性受超干的影响小,超干对细胞膜的轻微损伤可在萌发吸胀时得到修复,超干种子水分吸附等温线上区段1和区段2的含水量明显下降;超干种子除－△S和－△H在区段1略有所升高外,－△S、－△H在区段和2和区段3和－△G在各区段均有程度不同的下降;这些改变尤以豇豆种子较显著,说明豇豆种子的水分束缚能力低,较易失去水分吸附等温线上的强吸附水,继而引起大分子结构的改变,降低种子活力;而菜心种子则相反,能在超干状态下较好地保持酶的活性和细胞膜的结构,故适宜超干贮藏。     

Imbibition temperature sensitivity of lima bean seeds controlled by initial seed moisture

Excised embryonic axes and whole seeds of Phaseolus lunatus L. were previously shown to be injured if exposed to low (5°-15°) temperature during the initial stages of imbibition. Present data show that this chilling injury during imbibition of liquid water can be prevented if axes are first allowed to absorb water vapor. The increase of initial water content to 20% increases growth even of unchilled axes, and reduces leaching of 264 mμ absorbing compounds. Protection resulting from increased water content is at first independent of the temperature at which water vapor was absorbed. However, longer exposure of high moisture axes to low temperature results in typical chilling injury. The response to initial seed moisture is repeatedly reversible with changes in water content. Because the same response occurs in intact seeds, it may be possible both to protect them against low temperature injury and to increase vigor by increasing seed water-content prior to planting.