Protein mobilization in germinating mung bean seeds involves vacuolar sorting receptors and multivesicular bodies

Plants accumulate and store proteins in protein storage vacuoles (PSVs) during seed development and maturation. Upon seed germination, these storage proteins are mobilized to provide nutrients for seedling growth. However, little is known about the molecular mechanisms of protein degradation during seed germination. Here we test the hypothesis that vacuolar sorting receptor (VSR) proteins play a role in mediating protein degradation in germinating seeds. We demonstrate that both VSR proteins and hydrolytic enzymes are synthesized de novo during mung bean (Vigna radiata) seed germination. Immunogold electron microscopy with VSR antibodies demonstrate that VSRs mainly locate to the peripheral membrane of multivesicular bodies (MVBs), presumably as recycling receptors in day 1 germinating seeds, but become internalized to the MVB lumen, presumably for degradation at day 3 germination. Chemical cross-linking and immunoprecipitation with VSR antibodies have identified the cysteine protease aleurain as a specific VSR-interacting protein in germinating seeds. Further confocal immunofluorescence and immunogold electron microscopy studies demonstrate that VSR and aleurain colocalize to MVBs as well as PSVs in germinating seeds. Thus, MVBs in germinating seeds exercise dual functions: as a storage compartment for proteases that are physically separated from PSVs in the mature seed and as an intermediate compartment for VSR-mediated delivery of proteases from the Golgi apparatus to the PSV for protein degradation during seed germination.     

Stimulation of nucleic acid and protein synthesis in mungbean (Vigna radiata L.) seeds by uv irradiation.

The effect of a range of ultraviolet (uv) irradiation doses on nucleic acid and protein synthesis has been studied during seed germination and seedling growth in mungbean (Vigna radiata L). The treatment of seeds with low dose irradiation were stimulative for the synthesis of these molecules.     

A mechanism for promoting the germination of Zinnia elegans seeds by hydrogen peroxide

H(2)O(2) promotes seed germination of cereal plants such as barley, wheat and rice, and several mechanisms have been proposed for its action [Naredo et al. (1998) Seed Sci. Technol. 26: 675-689]. We investigated the role of H(2)O(2) in the germination of Zinnia elegans seeds. H(2)O(2) promoted seed germination in a dose-dependent manner as did respiratory inhibitors, indicating that H(2)O(2) itself possibly promotes seed germination rather than O(2). Seed germination was promoted by removal of pericarp from seeds or by removal of ethanol-soluble compounds from the seeds with pericarp. The ethanol-soluble compounds suppressed the germination of seeds having no pericarp, and this effect was reversed by H(2)O(2). These findings indicate that oxidation of the germination inhibitor(s) present in the pericarp by H(2)O(2) promotes seed germination. Antioxidants which are derivatives of well-known germination inhibitors suppressed seed germination in a dose-dependent manner, suggesting that, to initiate seed germination, a germination inhibitor(s) should be decomposed by an oxidant such as H(2)O(2).     

The proteolysis of trypsin inhibitors in legume seeds

The seeds of plants often contain large amounts of proteins, which are subjected to extensive proteolytic processing during seed development and subsequent germination. One class of legume seed proteins, the Bowman-Birk-type trypsin inhibitors, has proved especially useful as a subject in studying these events. Sequence studies of the trypsin inhibitors from a number of legume species suggest that many of the inhibitors undergo a limited shortening at the amino terminus during seed development. However, during germination, the inhibitors appear to function as storage proteins. As such, they are subjected to extensive proteolysis, ultimately leading to their destruction. This degradative process has been studied extensively in the mung bean (Vigna radiata [L.] Wilczek). Proteolysis of the mung bean trypsin inhibitor involves, at least initially, an ordered sequence of limited proteolytic cleavages. The two proteases involved in the initial phases of this degradation have been identified and partially characterized.     

Mechanisms of seed ageing under different storage conditions for Vigna radiata (L.) Wilczek: lipid peroxidation,sugar hydrolysis,Maillard reactions and their relationship to glass state transition

Two primary biochemical reactions in seed ageing (lipid peroxidation and non-enzymatic protein glycosylation with reducing sugars) have been studied under different seed water contents and storage temperatures, and the role of the glassy state in retarding biochemical deterioration examined. The viability loss of Vigna radiata seeds during storage is associated with Maillard reactions; however, the contribution of primary biochemical reactions varies under different storage conditions. Biochemical deterioration and viability loss are greatly retarded in seeds stored below a high critical temperature (approximately 40 degrees C above glass transition temperature). This high critical temperature corresponds to the cross-over temperature (T(c)) of glass transition where molecular dynamics changes from a solid-like system to a normal liquid system. The data show that seed ageing slows down significantly, even before seed tissue enters into the glassy state.     

Starch Mobilization in Ultradried Seed of Maize (Zea mays L.) During Germination

The effects of ultradry storage on the starch mobilization in maize (Zea mays L.) seed after aging were investigated. The results indicated that there were no significant differences in the content of ATP,starch, and soluble sugar, as well as the activity of amylase, between ultradried seeds and seeds stored at -20 ℃ during germination. These results were consistent with the higher level of vigor of the ultradried seed. Sieve tube introduction of a fluorescence dye (carboxyl fluoresceindiacetate) and laser confocal microscopy were used to study the development of plasmodesmata in the ultradried seeds. The results indicated that plasmodesmata developed well in ultradried seeds. Fluorescence analysis also showed that the fluorescence intensity in the radicle of ultradried seeds was stronger than that in seeds with a higher moisture content. This suggests that ultradry treatment has no adverse effects on the seeds. After seed imbibition, cell orgaelles could be resumed. It is concluded that ultradry seed storage is beneficial for maintaining seed vigor and that starchy mobilization proceeds regularly during germination.     

Starch Mobilization in Ultradried Seed of Maize （Zea mays L.） During Germination

The effects of ultradry storage on the starch mobilization in maize (Zea mays L.) seed after aging were investigated. The results indicated that there were no significant differences in the content of ATP, starch, and soluble sugar, as well as the activity of amylase, between ultradried seeds and seeds stored at -20℃ during germination. These results were consistent with the higher level of vigor of the ultradried seed. Sieve tube introduction of a fluorescence dye (carboxyl fluoresceindiacetate) and laser confocal microscopy were used to study the development of plasmodesmata in the ultradried seeds. The results indicated that plasmodesmata developed well in ultradried seeds. Fluorescence analysis also showed that the fluorescence intensity in the radicle of ultradried seeds was stronger than that in seeds with a higher moisture content. This suggests that ultradry treatment has no adverse effects on the seeds. After seed imbibition, cell orgaelles could be resumed. It is concluded that ultradry seed storage is beneficial for maintaining seed vigor and that starchy mobilization proceeds regularly during germination.     

Dissecting the proteome of pea mature seeds reveals the phenotypic plasticity of seed protein composition

Pea (Pisum sativum L.) is the most cultivated European pulse crop and the pea seeds mainly serve as a protein source for monogastric animals. Because the seed protein composition impacts on seed nutritional value, we aimed at identifying the determinants of its variability. This paper presents the first pea mature seed proteome reference map, which includes 156 identified proteins (http://www.inra.fr/legumbase/peaseedmap/). This map provides a fine dissection of the pea seed storage protein composition revealing a large diversity of storage proteins resulting both from gene diversity and post‐translational processing. It gives new insights into the pea storage protein processing (especially 7S globulins) as a possible adaptation towards progressive mobilization of the proteins during germination. The nonstorage seed proteome revealed the presence of proteins involved in seed defense together with proteins preparing germination. The plasticity of the seed proteome was revealed for seeds produced in three successive years of cultivation, and 30% of the spots were affected by environmental variations. This work pinpoints seed proteins most affected by environment, highlighting new targets to stabilize storage protein composition that should be further analyzed.     

Mobilization of seed protein reserves

The mobilization of seed storage proteins upon seed imbibition and germination is a crucial process in the establishment of the seedling. Storage proteins fold compactly, presenting only a few vulnerable regions for initial proteolytic digestion. Evolutionarily related storage proteins have similar three-dimensional structure, and thus tend to be initially cleaved at similar sites. The initial cleavage makes possible subsequent rapid and extensive breakdown catalyzed by endo- and exopeptidases. The proteolytic enzymes that degrade the storage proteins during mobilization identified so far are mostly cysteine proteases, but also include serine, aspartic and metalloproteases. Plants often ensure early initiation of storage protein mobilization by depositing active proteases during seed maturation, in the very compartments where storage proteins are sequestered. Various means are used in such cases to prevent proteolytic attack until after imbibition of the seed with water. This constraint, however, is not always enforced as the dry seeds of some plant species contain proteolytic intermediates as a result of limited proteolysis of some storage proteins. Besides addressing fundamental questions in plant protein metabolism, studies of the mobilization of storage proteins will point out proteolytic events to avoid in large-scale production of cloned products in seeds. Conversely, proteolytic enzymes may be applied toward reduction of food allergens, many of which are seed storage proteins.     

Promoter regions of cysteine endopeptidase genes from legumes confer germination-specific expression in transgenic tobacco seeds

Cysteine endopeptidases, SH-EP from Vigna mungo and EP-C1 from Phaseolus vulgaris, act to degrade seed storage protein during seed germination. Using transgenic tobacco plants, expression of SH-EP and promoter activity of the EP-C1 gene were analyzed in transgenic tobacco plants. The promoters of the two genes in tobacco seeds showed germination-specific activation, although post-translational processing of SH-EP and regulatory regions of promoter of the gene for EP-C1 were found to differ between leguminous seeds and transgenic tobacco seeds.     

Water relations in germinating seeds

Life strategy of plants depends on successful seed germination in the available environment, and sufficient soil water is the most important external factor. Taking into account a broad spectrum of roles played by water in seed viability and its maintenance during germination, the review embraces early germination events in seeds different in their water status. Two seed types are compared, namely orthodox and recalcitrant seeds, in terms of water content in the embryonic axes, vacuole biogenesis, and participation of water channels in membrane water transport. Mature orthodox seeds desiccate to low water content and remain viable during storage, whereas mature recalcitrant seeds are shed while well hydrated but die during desiccation and cannot be stored. In orthodox Vicia faba minor air-dry seeds remaining viable at 8–10% water content in embryonic axes, the vacuoles in hypocotyl are preserved as protein storage vacuoles, then restored to vacuoles in imbibing seeds in the course of protein mobilization. However, in newly produced meristematic root cells, the vacuoles are formed de novo from provacuoles. In recalcitrant Aesculus hippocastanum seeds, embryonic axes have a water content of 63–64% at shedding and they lack protein storage vacuoles but preserve vacuoles preformed in maturing seeds. Independent of the vacuolar biogenetic patterns, their further trend is similar; they expand and fuse, thus producing an osmotic compartment, which precedes and becomes an obligatory step for the initiation of cell elongation. Prior to this, water moves in imbibing seeds through the membranes by diffusion, although the aquaporins forming water channels are present. In both seed types, water channels are opened and actively participate in water transport only after growth initiation. Aquaporin gene expression and their composition change in broad bean embryonic axes after growth initiation. This is the way how a mass water flow into growing seedling cells is achieved, independent of differences in seed water content and vacuole biogenesis patterns.     

小麦种子成熟和萌发过程中的假萌发素活性

用SDS-PAGE方法研究了假萌发素(ψG)在小麦种子成熟和萌发过程中活性的变化.结果表明:在种子成熟过程中只有ψG表达,扬花后10 d,在颖壳、内外桴、种皮和果皮中皆可检测到ψG的草酸氧化酶活性,随着发育进程的推进,ψG的活性增大.在种子萌发过程中,在小麦品种中育5号的维管束过渡区中除了萌发素G和G'外,还可检测到ψG的草酸氧化酶活性.由于ψG在种子成熟过程中主要存在于颖壳、内外桴、果皮及种皮这些保护组织中,且开始大量表达的时间正是生长接近停止时,于是推测ψG很可能通过降解草酸产生H2O2而推动这些组织细胞壁的木质化.     

Examination of the Possibility of the Involvement of the Alternative Respiration in Seed Germination

The possibility that the alternative respiration (AR) is involvedin seed germination was followed during and after imbibitionby noting the changes in the sensitivity to the respiratoryinhibitors, KCN and salicylhydroxamic acid (SHAM). This wascarried out by measuring oxygen uptake of mitochondrial preparationsfrom storage cotyledons and of the whole axis in a selectionof legumes (Vigna mungo, V. radiata, V. angularis, V. sinensisPhaseolus vulgaris, Pisum sativum and Dolichos lablab). Theextent of participation of AR in the respiration of cotyledonarymitochondria was variable according to species and time afterimbibition, and varied even within the same genus ( Vigna).In some species, AR was not observed. As to the axis, SHAM treatmentby itself had little effect on the rate of respiration, but,when applied with KCN, reduced oxygen uptake to an extent greaterthan KCN treatment alone. It was suggested that AR was not necessarilyimportant for seed germination. Key words: Alternative respiration, mitochondria, seed germination     

Cytoplasmic and Apoplastic Location of Phenolic Compounds in the Covering Tissue of the Brassica napus Radicle Between Embryogenesis and Germination

The localization and intensity of cytoplasmic and apoplasticdeposits of phenolic compounds in Brassica napus L. change betweenembryogenesis and 36 h after seed germination. In the late stageof embryogenesis there were no phenolic compounds that wouldbe precipitated with caffeine, located either in the cytoplasmor outside the plasmalemma. Seeds collected at this stage rotduring germination. During seed maturation phenolic compoundswere localized in small vesicles which correspond to vesicular-shapedendoplasmic reticulum (ER) characteristic of this stage. Thiswas followed by slightly larger deposits in vacuoles, and inmature seed dark deposits accumulated outside the plasmalemma.In these dormant seeds the deposits were thus mostly betweenthe plasmalemma and the cell wall. After 3 h in water such darkdeposits appeared outside the cell wall on the embryo surface.After 6 h the cytoplasmic deposits were very few, and after24 h deposits reappeared in the round vesicles and long ER cisternae.After 36 h, when the emerging radicle and hypocotyl were 3 mmlong, there were large deposits of phenolic compounds in thevacuoles of various sizes. The occurrence of these depositsparalleled the previously demonstrated waves of embryo activityat the same stages of development, such as mitoses, synthesisof DNA, RNA, and protein, and mobilization of storage material. Embryogenesis, phenolic compounds, germination, seedling     

Expression of an expansin is associated with endosperm weakening during tomato seed germination

Expansins are extracellular proteins that facilitate cell wall extension, possibly by disrupting hydrogen bonding between hemicellulosic wall components and cellulose microfibrils. In addition, some expansins are expressed in non-growing tissues such as ripening fruits, where they may contribute to cell wall disassembly associated with tissue softening. We have identified at least three expansin genes that are expressed in tomato (Lycopersicon esculentum Mill.) seeds during germination. Among these, LeEXP4 mRNA is specifically localized to the micropylar endosperm cap region, suggesting that the protein might contribute to tissue weakening that is required for radicle emergence. In gibberellin (GA)-deficient (gib-1) mutant seeds, which germinate only in the presence of exogenous GA, GA induces the expression of LeEXP4 within 12 hours of imbibition. When gib-1 seeds were imbibed in GA solution combined with 100 microM abscisic acid, the expression of LeEXP4 was not reduced, although radicle emergence was inhibited. In wild-type seeds, LeEXP4 mRNA accumulation was blocked by far-red light and decreased by low water potential but was not affected by abscisic acid. The presence of LeEXP4 mRNA during seed germination parallels endosperm cap weakening determined by puncture force analysis. We hypothesize that LeEXP4 is involved in the regulation of seed germination by contributing to cell wall disassembly associated with endosperm cap weakening.     

Pretreatment of seed with H2O2 improves salt tolerance of wheat seedlings by alleviation of oxidative damage and expression of stress proteins

Increased salinity is a stringent problem to crop production while seed pretreatment can effectively induce salt tolerance in plants. Hydrogen peroxide (H(2)O(2)), a stress signal molecule, was evaluated as seed treatment to produce the metabolic changes, which could lead to improved salt tolerance in wheat. Soaking in 1, 40, 80 and 120 microM H(2)O(2) revealed a low penetration, reaching maximum at 5h (2.58+/-0.23 micro mol g(-1) fresh seeds at 120 microM) and declining thereafter to the level of water control by 8h. This revealed the activation of antioxidants and H(2)O(2) scavenging in seed after 5h. Seeds treated with 1-120 microM H(2)O(2) for 8h and germinated in saline (150 mM NaCl) medium curtailed the mean germination time (MGT) being even less than water controls. Level of H(2)O(2) in seedlings arising from H(2)O(2)-treated seeds grown under salinity was markedly lower than salinized controls, suggesting the operation of antioxidant system in them. These seedlings exhibited better photosynthetic capacity, particularly the stomatal conductance (gs), thus improving the leaf gas exchange due to stomatal component of photosynthesis. Moreover, H(2)O(2) treatment improved leaf water relations and maintained turgor. Although Na(+) and Cl(-) content increased due to salinity, H(2)O(2)-treated seedlings displayed greater tissue K(+), Ca(2+), NO(3)(-) PO(4)(3-) levels and improved K(+):Na(+) ratio. H(2)O(2) treatment enhanced the membrane properties, as revealed from greatly reduced relative membrane permeability (RMP) and less altered ion leakage pattern (comparable to water controls). Seedlings exhibited the expression of two heat-stable (stress) proteins with apparent molecular masses of 32 and 52 kDa. Results suggest that H(2)O(2) signals the activation of antioxidants in seed, which persists in the seedlings to offset the ion-induced oxidative damage. These changes led to the expression of stress proteins and improved physiological attributes, which supported the seedling growth under salinity.     

Distinct lytic vacuolar compartments are embedded inside the protein storage vacuole of dry and germinating Arabidopsis thaliana seeds

Plant cell vacuoles are diverse and dynamic structures. In particular, during seed germination, the protein storage vacuoles are rapidly replaced by a central lytic vacuole enabling rapid elongation of embryo cells. In this study, we investigate the dynamic remodeling of vacuolar compartments during Arabidopsis seed germination using immunocytochemistry with antibodies against tonoplast intrinsic protein (TIP) isoforms as well as proteins involved in nutrient mobilization and vacuolar acidification. Our results confirm the existence of a lytic compartment embedded in the protein storage vacuole of dry seeds, decorated by γ-TIP, the vacuolar proton pumping pyrophosphatase (V-PPase) and the metal transporter NRAMP4. They further indicate that this compartment disappears after stratification. It is then replaced by a newly formed lytic compartment, labeled by γ-TIP and V-PPase but not AtNRAMP4, which occupies a larger volume as germination progresses. Altogether, our results indicate the successive occurrence of two different lytic compartments in the protein storage vacuoles of germinating Arabidopsis cells. We propose that the first one corresponds to globoids specialized in mineral storage and the second one is at the origin of the central lytic vacuole in these cells.     

Osmoconditioning as a Means of Counteracting the Ageing of Pepper Seeds during High-temperature Storage

Sweet pepper seeds were osmotically conditioned in 0.4 M mannitolsolution for 4 d (at 25 °C, in darkness) before or afterstorage at 35 °C for up to six months, and their germinationand viability was compared with that of untreated seeds storedunder the same conditions. Seeds that had been osmoconditionedprior to storage retained a high rate of germination and germinatedto a high final percentage (from 80 to 50 per cent) at both15 and 25 °C throughout the storage period. By contrast,both the rate and total level of germination of untreated pepperseeds declined rapidly at both germination temperatures, andby three months of storage the total level of seed viabilitywas already less than 10 per cent. Seeds that were first storedat 35 °C, and then osmoconditioned just prior to germination,showed a decline in germinability which when tested at 25 °Cwas the same as for untreated seeds, while tested at 15 °Coccurred at a slightly slower rate than for untreated seeds. It is evident that osmoconditioning prior to storage, in additionto the acceleration of germination, resulted in a dramatic delayof the ageing rate, thus increasing considerably the longevityof seeds. On the other hand, osmoconditioning after storagedid not seem to have any significant effect on seed viability,though it enhanced the germination rate. Capsicum annuum, sweet pepper, seed, germination, osmoconditioning, priming, storage, viability, ageing, longevity     

Nitric oxide enhances desiccation tolerance of recalcitrant Antiaris toxicaria seeds via protein S-nitrosylation and carbonylation

The viability of recalcitrant seeds is lost following stress from either drying or freezing. Reactive oxygen species (ROS) resulting from uncontrolled metabolic activity are likely responsible for seed sensitivity to drying. Nitric oxide (NO) and the ascorbate-glutathione cycle can be used for the detoxification of ROS, but their roles in the seed response to desiccation remain poorly understood. Here, we report that desiccation induces rapid accumulation of H(2)O(2), which blocks recalcitrant Antiaris toxicaria seed germination; however, pretreatment with NO increases the activity of antioxidant ascorbate-glutathione pathway enzymes and metabolites, diminishes H(2)O(2) production and assuages the inhibitory effects of desiccation on seed germination. Desiccation increases the protein carbonylation levels and reduces protein S-nitrosylation of these antioxidant enzymes; these effects can be reversed with NO treatment. Antioxidant protein S-nitrosylation levels can be further increased by the application of S-nitrosoglutathione reductase inhibitors, which further enhances NO-induced seed germination rates after desiccation and reduces desiccation-induced H(2)O(2) accumulation. These findings suggest that NO reinforces recalcitrant seed desiccation tolerance by regulating antioxidant enzyme activities to stabilize H(2)O(2) accumulation at an appropriate concentration. During this process, protein carbonylation and S-nitrosylation patterns are used as a specific molecular switch to control antioxidant enzyme activities.