Proteome-wide characterization of seed aging in Arabidopsis: a comparison between artificial and natural aging protocols

A variety of mechanisms have been proposed to account for the extension of life span in seeds (seed longevity). In this work, we used Arabidopsis (Arabidopsis thaliana) seeds as a model and carried out differential proteomics to investigate this trait, which is of both ecological and agricultural importance. In our system based on a controlled deterioration treatment (CDT), we compared seed samples treated for different periods of time up to 7 d. Germination tests showed a progressive decrease of germination vigor depending on the duration of CDT. Proteomic analyses revealed that this loss in seed vigor can be accounted for by protein changes in the dry seeds and by an inability of the low-vigor seeds to display a normal proteome during germination. Furthermore, CDT strongly increased the extent of protein oxidation (carbonylation), which might induce a loss of functional properties of seed proteins and enzymes and/or enhance their susceptibility toward proteolysis. These results revealed essential mechanisms for seed vigor, such as translational capacity, mobilization of seed storage reserves, and detoxification efficiency. Finally, this work shows that similar molecular events accompany artificial and natural seed aging.     

Reversal of the effects of aging in soybean seeds

Accelerated aging predisposed seeds to imbibition injury. Slowing the rate of hydration prevented the loss of germinability due to imbibition injury. Germinability of accelerated aged seeds (50 hours) was increased from 10 to 90% by controlling the rate of imbibition. Slow hydration also prevented seed electrolyte leakage. This may indicate that cell membrane permeability or rupture was a major factor contributing to the loss of germinability after aging.

Reversal of the effects of aging (repair) was accomplished by slowly inbibing and then redrying seeds (priming). This treatment lowered steep water conductivity by a factor of 2 to 5. Priming also increased the per cent germination of low vigor seeds. The mechanism of this reversal was probably metabolic because it depended on temperature, seed moisture, and treatment duration.

Priming doubled the survival of seeds in the accelerated aging vigor test. The `rejuvenation' was accepted as evidence for metabolic repair. Since the `vigor' of seeds was increased by priming, metabolic repair probably included other subcellular components as well as the plasma membrane.

     

Toward characterizing seed vigor in alfalfa through proteomic analysis of germination and priming

Alfalfa, the most widely grown leguminous crop in the world, is generally exposed to severe salinity stress in Tunisia, notably affecting its germination performance. Toward a better understanding of alfalfa seed vigor, we have used proteomics to characterize protein changes occurring during germination and osmopriming, a pretreatment that accelerates germination and improves seedling uniformity particularly under stress conditions. The data revealed that germination was accompanied by dynamic changes of 79 proteins, which are mainly involved in protein metabolism, cell structure, metabolism, and defense. Comparative proteomic analysis also revealed 63 proteins specific to osmopriming, 65 proteins preferentially varying during germination, and 14 proteins common to both conditions. Thus, the present study unveiled the unexpected finding that osmopriming cannot simply be considered as an advance of germination-related processes but involves other mechanisms improving germination such as the mounting of defense mechanisms enabling osmoprimed seeds to surmount environmental stresses potentially occurring during germination. The present results therefore provide novel avenues toward understanding the mechanisms of invigoration of low vigor seeds by priming treatments that are widely used both in commercial applications and in developing countries (on farm seed priming) to better control crop yields.     

<Emphasis Type="Italic">NnHSP17.5</Emphasis>, a cytosolic class II small heat shock protein gene from <Emphasis Type="Italic">Nelumbo nucifera</Emphasis>, contributes to seed germination vigor and seedling thermotolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

In plants, small heat shock proteins (sHSPs) are unusually abundant and diverse proteins involved in various abiotic stresses, but their functions in seed vigor remain to be fully explored. In this study, we report the isolation and functional characterization of a sHSP gene, NnHSP17.5, from sacred lotus (Nelumbo nucifera Gaertn.) in seed germination vigor and seedling thermotolerance. Sequence alignment and phylogenetic analysis indicate that NnHSP17.5 is a cytosolic class II sHSP, which was further supported by the cytosolic localization of the NnHSP17.5-YFP fusion protein. NnHSP17.5 was specifically expressed in seeds under normal conditions, and was strongly up-regulated in germinating seeds upon heat and oxidative stresses. Transgenic Arabidopsis seeds ectopically expressing NnHSP17.5 displayed enhanced seed germination vigor and exhibited increased superoxide dismutase activity after accelerated aging treatment. In addition, improved basal thermotolerance was also observed in the transgenic seedlings. Taken together, this work highlights the importance of a plant cytosolic class II sHSP both in seed germination vigor and seedling thermotolerance.     

种子老化的生理生化与分子机理研究进展

种子作为植物遗传资源的有效保存体以及重要的种质创新原料,其老化或者劣变将直接导致发芽率、活力、生活力降低,抑制种胚正常发育以及幼苗生长,由此造成植物生产水平及其品质大幅下降。这也将进一步涉及因种质资源匮乏、土壤种子库系统功能紊乱所引发的全球生物多样性减小、草地退化和荒漠化加剧等生态危机问题。对种子老化生理生化特性和分子机理等研究进行了综述。总结了近年来关于种子老化涉及的理化反应包括保护酶活性的改变、核酸以及蛋白质的分解、内源激素的消长、质膜完整性降低等相关研究;并从蛋白代谢、核酸代谢、种子含水量以及基因重组等多角度总结和阐述了与老化机理有关的最新研究观点,以期为种子老化、种子活力修复和种子寿命延长等机理研究提供基础理论参考。目前对种子老化的研究多集中于传统的生理生化过程和内外影响因子相对独立变化的片段性研究,缺乏系统综合的多层面体系研究。种子作为生命体,随着探讨生命衰老机理的生物技术日新月异,通过蛋白组学、酶学、基因工程技术、转录组测序等新技术的应用,必将对未来种子老化机理机制的揭示有突破性推进作用。     

Proteomic investigation of the effect of salicylic acid on Arabidopsis seed germination and establishment of early defense mechanisms

The influence of salicylic acid (SA) on elicitation of defense mechanisms in Arabidopsis (Arabidopsis thaliana) seeds and seedlings was assessed by physiological measurements combined with global expression profiling (proteomics). Parallel experiments were carried out using the NahG transgenic plants expressing the bacterial gene encoding SA hydroxylase, which cannot accumulate the active form of this plant defense elicitor. SA markedly improved germination under salt stress. Proteomic analyses disclosed a specific accumulation of protein spots regulated by SA as inferred by silver-nitrate staining of two-dimensional gels, detection of carbonylated (oxidized) proteins, and neosynthesized proteins with [35S]-methionine. The combined results revealed several processes potentially affected by SA. This molecule enhanced the reinduction of the late maturation program during early stages of germination, thereby allowing the germinating seeds to reinforce their capacity to mount adaptive responses in environmental water stress. Other processes affected by SA concerned the quality of protein translation, the priming of seed metabolism, the synthesis of antioxidant enzymes, and the mobilization of seed storage proteins. All the observed effects are likely to improve seed vigor. Another aspect revealed by this study concerned the oxidative stress entailed by SA in germinating seeds, as inferred from a characterization of the carbonylated (oxidized) proteome. Finally, the proteomic data revealed a close interplay between abscisic signaling and SA elicitation of seed vigor.     

Organization of lipid reserves in cotyledons of primed and aged sunflower seeds

Imbibing sunflower (Helianthus annuus L., cv. Briosol) seeds at water potentials between –2 MPa and –5 MPa leads to faster (priming) or slower (accelerated ageing) germination depending on the temperature and duration of treatment. Mobilization of food reserves may be associated with the changes in seed vigor. To study this, morphological, biochemical and phase properties of lipid, the major food reserve in sunflower, were compared in freshly harvested (i.e., control), primed and aged sunflower cotyledons using electron microscopy, biochemical analyses and differential scanning calorimetry, respectively. Lipid bodies became smaller and more dispersed throughout the cytoplasm during priming and ageing. Despite ultrastructural changes, there were few measured changes in biochemistry of the neutral lipid component; lipid content, proportion of saturated and unsaturated fatty acids and level of free fatty acids were unchanged in primed and slightly aged seeds, with only severely aged seeds showing a net decrease in polyunsaturated fatty acids and an increase in free fatty acids. Subtle changes in the calorimetric behavior of lipids within sunflower cotyledons were observed. Sunflower lipids exhibited polymorphic crystalline and amorphous solid phases when cooled to <–100°C, but priming decreased the rate of crystallization in vivo and ageing increased the rate of crystallization, but decreased percentage crystallinity. The observed changes in thermal behavior in vivo are consistent with losses and gains, respectively, of interacting non-lipid moieties in the triacylglycerol matrix.     

Catalase is a key enzyme in seed recovery from ageing during priming

Ageing induces seed deterioration expressed as the loss of seed vigour and/or viability. Priming treatment, which consists in soaking of seeds in a solution of low water potential, has been shown to reinvigorate aged seeds. We investigate the importance of catalase in oxidation protection during accelerated ageing and repair during subsequent priming treatment of sunflower (Helianthus annuus L.) seeds. Seeds equilibrated to 0.29 g H₂O g⁻¹ dry matter (DM) were aged at 35 °C for different durations and then primed by incubation for 7 days at 15 °C in a solution of polyethylene glycol 8000 at −2 MPa. Accelerated ageing affected seed germination and priming treatment reversed partially the ageing effect. The inhibition of catalase by the addition of aminotriazol during priming treatment reduced seed repair indicating that catalase plays a key role in protection and repair systems during ageing. Ageing was associated with H₂O₂ accumulation as showed by biochemical quantification and CeCl₃ staining. Catalase was reduced at the level of gene expression, protein content and affinity. Interestingly, priming induced catalase synthesis by activating expression and translation of the enzyme. Immunocytolocalization of catalase showed that the enzyme co-localized with H₂O₂ in the cytosol. These results clearly indicate that priming induce the synthesis of catalase which is involved in seed recovery during priming.     

The Maillard reaction and oxidative stress during aging of soybean seeds

The chemical reactions that may lead to the loss of seed viability were investigated both during the accelerated aging and natural aging of soybeans ( Glycine max Merrill cv. Chippewa 64). Under conditions of accelerated aging (36°C and 75% RH), fluorescence of soluble proteins accumulated, which was closely correlated with the loss of seed germinability and vigor. We were able to show this correlation by using partially purified proteins for the assay. Fluorescence also increased in seeds under good storage conditions (5°C for up to 21 years), although there was a less significant correlation between seed viability and the accumulation of fluorescent products during the time of natural aging. The rise in protein fluorescence is interpreted as an increase of Maillard products. The carbonyl content of soluble proteins (a measure of the oxidative damage) did not change significantly during either accelerated aging or natural aging: however the elimination of carbonyls during germination seemed to be hindered in seeds that had poor germination. The Maillard reaction may be a consequence of the formation of reducing sugars through a gradual hydrolysis of oligosaccharides during aging. Preliminary evidence from the natural aging study showed that, when seeds were in the glassy state, the sugar hydrolysis was inhibited. These results suggest that the Maillard reaction and oxidative reaction may play an important role in seed deterioration.     

人工老化处理对结球甘蓝种子生理生化特性的影响

以结球甘蓝品种‘冬升’种子为材料,研究高温(40℃)高湿(相对湿度100%)人工老化处理过程中种子的萌发特性、种子浸出液相对电导率和丙二醛、可溶性蛋白、可溶性糖含量以及抗氧化酶活性的变化,以揭示种子劣变的机理。结果表明:(1)人工老化处理甘蓝种子的含水量和不正常苗率均随着老化时间的延长逐渐增加,而种子发芽势、发芽率、发芽指数和活力指数的增加均逐渐降低。(2)随着处理天数的延长,老化处理甘蓝种子的浸出液电导率显著增大,浸出液可溶性糖含量逐渐升高,可溶性蛋白含量表现出显著下降趋势,而种子MDA含量呈先升高后逐渐下降的趋势。(3)在结球甘蓝种子老化进程中,其种子中SOD、POD、CAT活性变化的趋势相似,均随老化程度的加深而逐渐降低;而APX活性在老化处理的最初2d显著增加,第6天显著降低。研究发现,在结球甘蓝种子老化进程中,种子活力和萌发率显著降低,其种子浸出液电导率、可溶性蛋白含量、可溶性糖含量、保护酶活性变化与种子老化及劣变程度密切相关,膜脂过氧化作用可能是引起或加剧种子老化劣变的重要原因之一。     

Proteomic analysis of arabidopsis seed germination and priming

To better understand seed germination, a complex developmental process, we developed a proteome analysis of the model plant Arabidopsis for which complete genome sequence is now available. Among about 1,300 total seed proteins resolved in two-dimensional gels, changes in the abundance (up- and down-regulation) of 74 proteins were observed during germination sensu stricto (i.e. prior to radicle emergence) and the radicle protrusion step. This approach was also used to analyze protein changes occurring during industrial seed pretreatments such as priming that accelerate seed germination and improve seedling uniformity. Several proteins were identified by matrix-assisted laser-desorption ionization time of flight mass spectrometry. Some of them had previously been shown to play a role during germination and/or priming in several plant species, a finding that underlines the usefulness of using Arabidopsis as a model system for molecular analysis of seed quality. Furthermore, the present study, carried out at the protein level, validates previous results obtained at the level of gene expression (e.g. from quantitation of differentially expressed mRNAs or analyses of promoter/reporter constructs). Finally, this approach revealed new proteins associated with the different phases of seed germination and priming. Some of them are involved either in the imbibition process of the seeds (such as an actin isoform or a WD-40 repeat protein) or in the seed dehydration process (e.g. cytosolic glyceraldehyde-3-phosphate dehydrogenase). These facts highlight the power of proteomics to unravel specific features of complex developmental processes such as germination and to detect protein markers that can be used to characterize seed vigor of commercial seed lots and to develop and monitor priming treatments.     

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。     

Lipid peroxidation and peroxide-scavenging enzymes associated with accelerated aging and hydration of watermelon seeds differing in ploidy

Hydration offers an effective means for raising seed performance in many crop species. The objective of this study was to evaluate the effect of vermiculite hydration on germinability and several physiological activities related to vigor in artificially aged watermelon seeds differing in ploidy. Aging was achieved by incubating the seeds at 45°C and 79% relative humidity for 6 days, then the seeds were air-dried to their original moisture level (4.7%). Hydration was achieved by mixing the untreated and aged seeds with moist vermiculite No. 3 at 25°C for 24 h. The partially hydrated seeds were air-dried at 25°C for 36 h to 4.7% moisture level. Significant differences existed between unaged and aged seeds, with lower germination percentage and slower germination speed in the latter. Aging also increased lipid peroxidation and reduced the activity of peroxide-scavenging enzymes. The germinability of aged watermelon seed was restored partially by vermiculite hydration. The activities of protein synthesis and peroxide-scavenging enzymes in axis and cotyledon portions of the seeds were also increased by hydration treatment. The changes in germination and related physiological responses in relation to aging and hydration are similar in seeds differing in ploidy, despite differences in their germination performance, seed leakage, extent of lipid peroxidation and activities of peroxide-scavenging enzymes.     

Metabolic control of seed germination

We have used proteomics to better characterize germination and early seedling vigor in sugarbeet. Our strategy includes (1) construction of proteome reference maps for dry and germinating seeds of a high-vigor reference seed lot; (2) investigation of the specific tissue accumulation of proteins (root, cotyledon, perisperm); (3) investigation of changes in protein expression profiles detected in the reference seed lot subjected to different vigor-modifying treatments, e.g. aging and/or priming. More than 1 000 sugarbeet seed proteins have been identified by LC/MS-MS mass spectrometry (albumins, globulins and glutelins have been analyzed separately). Due to the conservation of protein sequences and the quality of MS sequencing (more than 10 000 peptide sequences have been obtained), the success rate of protein identification was on the average of 80%. This is to our knowledge the best detailed proteome analysis ever carried out in seeds. The data allowed us to build a detailed metabolic chart of the sugarbeet seed, generating new insights into the molecular mechanisms determining the development of a new seedling. Also, the proteome of a seed-storage tissue as the perisperm is described for the first time.     

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.     

Biochemical Changes During Osmopriming of Leek Seeds

Osmotic priming treatments reduced both the mean time to germinationand the spread of germination for two leek seed-lots of highviability but differing vigour. In addition the differencesin germination performance between these two seed-lots was abolishedby the priming treatments. In the unprimed seed-lots, differencesin germination performance were reflected in differences inrates of protein biosynthesis in leek embryo tissue during germination.Osmopriming treatments abolished these differences upon subsequentgermination of osmotically primed seed and furthermore inducedhigh levels of protein biosynthesis in embryo tissue. DNA synthesiswas detectable in leek embryos during the priming period inthe absence of any cell division and was followed by a five-foldincrease in the rate of DNA synthesis in embryo tissue upongermination following priming at which time the rates of DNAsynthesis in these leek embryos was significantly greater thanthat found at any time over the first 4 d of germination inembryos of unprimed leek seeds. The increases in rates of bothprotein and DNA synthesis observed upon germination of primedseed occurred only after a 6–12 h lag period during whichtime there is little increase in these rates above those foundat the end of priming Analysis of nucleotide and nucleotide sugar levels in leek embryosboth during and after priming showed that only traces of GTPand CTP and low levels of ATP and UTP were present in embryosduring priming. After a 6 h lag period following the end ofpriming these levels increased sharply, probably via de novosynthesis. A similar pattern was found for UDP glucose levelsduring priming and subsequent germination. These results indicatethat there is considerable biochemical activity during primingand that the significant benefits in germination performanceof primed leek seeds is accompanied by marked increases in protein,DNA and nucleotide biosynthesis after a lag period of 6–12h following the end of the priming period Allium porrum, leek, seed, osmopriming, germination, protein synthesis, nucleic acids, nucleotides, nucleotide sugars     

Changes in the Composition and Synthesis of Proteins in Cellular Membranes of Echinochloa crus-galli (L.) Beauv. Seeds during the Transition from Dormancy to Germination

The effect of alcohols which stimulate or have no effect on germination on the composition and synthetic pattern of proteins in the cellular membranes of Echinochloa crus-galli (L.) Beauv. seeds was studied. Imbibition of dry seeds was accompanied by an increase in the synthesis of proteins and by synthesis of new proteins in their intracellular membranes. The transition of the seeds from a dormant to a nondormant state was associated with synthesis of specific proteins and a decrease in content of others in the plasma membrane. The synthesis of a 23 kilodalton protein was strongly increased upon release from dormancy. The changes in the pattern of protein synthesis were not directly associated with the beginning of germination. The results suggest that the plasma membrane constitutes the first site in the seed cells, at which the stimulus from external factors affecting seed dormancy is detected.     

Natural priming as an important metabolic event in the life history of Wigandia urens (Hydrophyllaceae) seeds

Natural priming promotes fast and synchronic seed germination and enhances the establishment of seedlings from Wigandia urens seeds. In this study, seeds of this species were buried in the field in three different sites, and each site was divided into three different microsites (natural priming). They were then exhumed and air-dried at the beginning of the rainy season. The control and exhumed W. urens seeds were tested for differences in germination, and in the protein patterns derived from heat-stable and phospho-protein enriched fractions, sucrose concentrations, amylase isoenzyme patterns, as well as catalase activity and H₂O₂ levels. All the exhumed seeds germinated faster and more synchronically than the control seeds. Nevertheless, the germinative pattern showed a microsite effect related to the microenvironmental conditions. Most exhumed samples kept the germinative advantages acquired from the soil during natural priming for 2 years but seeds from the most heterogeneous site (a forest gap) did not. Natural priming also promoted mobilization of 7-S globulin-like proteins, solubilization of the 11-S globulin-like proteins as well as sucrose consumption before radicle protrusion. Although there were no changes in starch concentration during the germination of either the control or buried seeds, a different isoenzyme amylase pattern was observed. Catalase activity decreased and hydrogen peroxide levels were lower in exhumed seeds during germination. The changes in the protein and sucrose patterns were related to advances in the germinative process acquired during burial. Metabolic advantages were maintained systematically in all seed samples along the 2 years. The ecological significance of natural priming is discussed.