Degradation of storage proteins in germinating seeds

The criteria for the participation of proteases in the mobilization of storage proteins during seed germination are formulated. The proteases that satisfy these criteria, namely the acid cysteine endopeptidases, serine carboxypeptidases and neutral aminopeptidases, are reviewed. The possibility of other seed proteases participating in storage protein degradation is discussed. The course of 11S and 7S storage protein degradation through the action of endogenous and exogenous proteases is described. The 11S and 7S proteins are modified during the early stages of proteolysis and the effects of these modifications on the regulation of breakdown are examined. A scheme for 11S protein degradation in germinating seeds is presented.     

Proteolysis in the quiescent seed

The importance of the proteolytic system of the quiescent seed in regard to mobilization of storage protein was assessed by identification of proteases already present at this stage. Extracts of quiescent cotyledons of white lupin ( Lupinus albus ) submitted to 45 h autolysis in vitro displayed a higher degree of protein degradation than that observed in vivo after two days of seed imbibition. Differences in the susceptibility to proteolysis were verified by densitometric analysis of the polypeptides after electrophoretic separation. The pH dependence of the proteolytic activities and the responses to specific protease inhibitors showed that the proteolytic systems vary from quiescent to 1 - to 3-day-imbibed cotyledons. By labelling an endogenous globulin with ¹²⁵I a sensitive radiometric assay allowed the identification of both an acidic and a neutral proteolytic system in the quiescent cotyledon. Within the quiescent seed there already exists a high potential for initiating proteolysis, so that the requirement for proteolysis by specific endopeptidases synthesized de novo upon imbibition only applies to part of the reserve proteins.     

Comparative Proteomics Reveals the Mechanisms Underlying Variations in Seed Vigor Based on Maize (Zea mays L.) Ear Positions

Seed vigor is influenced by seed position in plant. However, current understanding of its underlying mechanism is limited. In this study, we used isobaric tags for relative and absolute quantitation technique to study the comparative proteomes between middle seeds (with higher vigor) and top seeds of maize (Zea mays L.) ears at 0 h, 24 h, and 48 h of imbibition. A total of 159 differentially accumulated proteins were identified. Among these, the largest number of proteins was from the functional categories of Disease/Defense and Metabolism. Compared with top seeds, most of the differentially accumulated proteins of Protein Synthesis and Energy showed higher accumulation in middle seeds at 0 h and 24 h of imbibition, but lower accumulation at 48 h of imbibition. Seed water absorption activates metabolic processes. The water content of middle seeds was significantly lower than that of top seeds at between 12 h and 30 h of imbibition, but energy production would be higher in the middle seeds at 24 h of imbibition. Meanwhile, tonoplast intrinsic proteins 3.1 and 3.2, which mediate water inflow into protein storage vacuoles, then activating enzymes involved in reserve mobilization, showed higher accumulation in middle seeds at 24 h of imbibition. In addition, our data also showed middle seeds may suffer less fungal damages. Our results contribute to understanding the mechanisms underlying the effects of growth position on seed vigor.

     

Proteases and proteolytic cleavage of storage proteins in developing and germinating dicotyledonous seeds

Proteolytic cleavage plays an important role in storage proteindeposition and reactivation in seeds. Precursor polypeptidesare processed by limited proteolysis to mature subunits of reserveproteins in storage tissue cells of developing seeds. Stepsof proteolytic processing are closely related to steps in intracellularprotein transfer through the endomembrane system and to thedeposition in the storage vacuole. In germinating seeds specialendopeptidases trigger storage protein breakdown by limitedproteolysis. The induced conformation changes of storage proteinsopen them to attack by additional endo- and exopeptidases whichdegrade the protein reserves completely. Proteases that catalyselimited cleavage or complete degradation are synthesized asprecursors which also undergo stepwise limited proteolysis whenthey are formed in cotyledons of developing or germinating seeds.In general, this processing transforms enzymatically inactiveproenzymes into active proteases. Different compartments participatein the processing steps. Many of the proteases are encoded bysmall multigene families. Different members of the correspondingprotease families seem to act during seed development and germination.Proteolytic processes that contribute to the molecular maturationand to the reactivation of storage proteins in dicotyledonousseeds seem to be controlled by (1) differential expression ofmembers of the protease-encoding gene families; (2) stepwiseprocessing and activation of protease precursor polypeptides;(3) transient differential compartmentation of precursors andmature polypeptides of proteases and storage proteins, respectively;and (4) interacting changes in storage protein structure andprotease action. The present knowledge on these processes isreviewed. Key words: Dicotyledons, seeds, storage proteins, proteolytic cleavage, proteases     

Redox-sensitive proteome and antioxidant strategies in wheat seed dormancy control

Oxidative signalling by ROS has been demonstrated to play a role in seed dormancy alleviation, but the detailed molecular mechanisms underlying this process remain largely unknown. Here, we show dynamic differences in redox-sensitive proteome upon wheat seed dormancy release. Using thiol-specific fluorescent labelling, solubility-based protein fractionation, 2-D IEF PAGE, and MS analysis in conjunction with wheat EST sequence libraries, proteins with reversible oxidoreductive changes were characterized. Altogether, 193 reactive Cys were found in 79 unique proteins responding differentially in dormant, non-dormant, abscisic, or gibberellic acid-treated seed protein extracts from RL4137, a wheat cultivar with extreme dormancy. The identified proteins included groups that are redox-, stress-, and pathogen-responsive, involved in protein synthesis and storage, are enzymes of carbohydrate metabolism, proteases, and those involved in transport and signal transduction. Two types of redox response could be detected: (i) a dramatic increase in protein thiol redox state in seeds during imbibition and hormonal treatment; (ii) higher antioxidant capacity related to sensing of a threshold redox potential and balancing the existing redox pools, in dry dormant versus non-dormant seeds. These results highlight occurrence of the antioxidant defence mechanisms required for the protection of seed during a dormancy stage.     

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.     

Mobilization of Reserve Proteins During Early Stages of Seed Germination in Fagopyrum Esculentum

Hydrolysis of 13S globulin, the main storage protein in grains of common buckwheat (Fagopyrum esculentum Moench), proceeds in at least two phases during germination. The first stage, involving a limited proteolytic cleavage of the protein, is associated with increased activity of proteases having maximum activity at pH 7.6. The second stage, involving further hydrolysis of the partially cleaved protein, starts after 12 h of imbibition. During this phase, activity of proteases increased and activity maximum shifted to pH 5.6. Nevertheless, 13S globulin retains its antigenic identity till the emergence of radicle and plumule. Thus, it may not be the major source of amino acids utilized by the germinating seed during the initial stages of imbibition. This revised version was published online in July 2006 with corrections to the Cover Date.     

Stored proteinases and the initiation of storage protein mobilization in seeds during germination and seedling growth

Though endopeptidases and carboxypeptidases are present in protein bodies of dry quiescent seeds the function of these proteases during germination is still a matter of debate. In some plants it was demonstrated that endopeptidases of dry protein bodies degrade storage proteins of these organelles. Other studies describe cases where this did not happen. The role that stored proteinases play in the initiation of storage protein breakdown in germinating seeds thus remains unclear. Numerous reviews state that the initiation of reserve protein mobilization is attributed to de novo formed endopeptidases which together with stored carboxypeptidases degrade the bulk of proteins in storage organs and tissues after seeds have germinated. The evidence that the small amounts of endopeptidases in protein bodies of embryonic axes and cotyledons of dry seeds from dicotyledonous plants play an important role in the initiation of storage protein mobilization during early germination is summarized here.     

Storage protein accumulation in the absence of the vacuolar processing enzyme family of cysteine proteases

The role(s) of specific proteases in seed protein processing is only vaguely understood; indeed, the overall role of processing in stable protein deposition has been the subject of more speculation than direct investigation. Seed-type members of the vacuolar processing enzyme (VPE) family were hypothesized to perform a unique function in seed protein processing, but we demonstrated previously that Asn-specific protein processing in developing Arabidopsis seeds occurs independently of this VPE activity. Here, we describe the unexpected expression of vegetative-type VPEs in developing seeds and test the role(s) of all VPEs in seed storage protein accumulation by systematically stacking knockout mutant alleles of all four members (alphaVPE, betaVPE, gammaVPE, and deltaVPE) of the VPE gene family in Arabidopsis. The complete removal of VPE function in the alphavpe betavpe gammavpe deltavpe quadruple mutant resulted in a total shift of storage protein accumulation from wild-type processed polypeptides to a finite number of prominent alternatively processed polypeptides cleaved at sites other than the conserved Asn residues targeted by VPE. Although alternatively proteolyzed legumin-type globulin polypeptides largely accumulated as intrasubunit disulfide-linked polypeptides with apparent molecular masses similar to those of VPE-processed legumin polypeptides, they showed markedly altered solubility and protein assembly characteristics. Instead of forming 11S hexamers, alternatively processed legumin polypeptides were deposited primarily as 9S complexes. However, despite the impact on seed protein processing, plants devoid of all known functional VPE genes appeared unchanged with regard to protein content in mature seeds, relative mobilization rates of protein reserves during germination, and vegetative growth. These findings indicate that VPE-mediated Asn-specific proteolytic processing, and the physiochemical property changes attributed to this specific processing step, are not required for the successful deposition and mobilization of seed storage protein in the protein storage vacuoles of Arabidopsis seeds.     

Proteomic analysis of rice (Oryza sativa) seeds during germination

Although seed germination is a major subject in plant physiological research, there is still a long way to go to elucidate the mechanism of seed germination. Recently, functional genomic strategies have been applied to study the germination of plant seeds. Here, we conducted a proteomic analysis of seed germination in rice (Oryza sativa indica cv. 9311) - a model monocot. Comparison of 2-DE maps showed that there were 148 proteins displayed differently in the germination process of rice seeds. Among the changed proteins, 63 were down-regulated, 69 were up-regulated (including 20 induced proteins). The down-regulated proteins were mainly storage proteins, such as globulin and glutelin, and proteins associated with seed maturation, such as "early embryogenesis protein" and "late embryogenesis abundant protein", and proteins related to desiccation, such as "abscisic acid-induced protein" and "cold-regulated protein". The degradation of storage proteins mainly happened at the late stage of germination phase II (48 h imbibition), while that of seed maturation and desiccation associated proteins occurred at the early stage of phase II (24 h imbibition). In addition to alpha-amylase, the up-regulated proteins were mainly those involved in glycolysis such as UDP-glucose dehydrogenase, fructokinase, phosphoglucomutase, and pyruvate decarboxylase. The results reflected the possible biochemical and physiological processes of germination of rice seeds.     

A proteomic analysis of rice seed germination as affected by high temperature and ABA treatment

Seed germination is a critical phase in the plant life cycle, but the specific events associated with seed germination are still not fully understood. In this study, we used two‐dimensional gel electrophoresis followed by mass spectrometry to investigate the changes in the proteome during imbibition of Oryza sativa seeds at optimal temperature with or without abscisic acid (ABA) and high temperature (germination thermoinhibition) to further identify and quantify key proteins required for seed germination. A total of 121 protein spots showed a significant change in abundance (1.5‐fold increase/decrease) during germination under all conditions. Among these proteins, we found seven proteins specifically associated with seed germination including glycosyl hydrolases family 38 protein, granule‐bound starch synthase 1, Os03g0842900 (putative steroleosin‐B), N‐carbamoylputrescine amidase, spermidine synthase 1, tubulin α‐1 chain and glutelin type‐A; and a total of 20 imbibition response proteins involved in energy metabolism, cell growth, cell defense and storage proteins. High temperature inhibited seed germination by decreasing the abundance of proteins involved in methionine metabolism, amino acid biosynthesis, energy metabolism, reserve degradation, protein folding and stress responses. ABA treatment inhibited germination and decreased the abundance of proteins associated with methionine metabolism, energy production and cell division. Our results show that changes in many biological processes including energy metabolism, protein synthesis and cell defense and rescue occurred as a result of all treatments, while enzymes involved in methionine metabolism and weakening of cell wall specifically accumulated when the seeds germinated at the optimal temperature.     

Characterization of asparaginyl endopeptidase activity in endosperm of developing and germinating castor oil seeds

A spectrophotometric assay was devised to characterize the asparaginyl (Asn) endopeptidase activity from the endosperm of castor oil seeds. (Ricinus communis L. var. Baker 296). The assay measures the release of p-nitroaniline from the hydrolysis of benzoyl-l-Asn-p-nitroanilide. Assay sensitivity was improved through diazotization of the reaction product with N(]-napthy])-ethylenediamine dihydrochloride: diazotized p-nitroaniline was determined spectrophotometrically at 548 nm (?₅₄₈= 1.64 × 10^?1M^?1 cm^?2). By using this assay. Asn endopeptidase activity was detected in endosperm extracts of developing, mature and germinating castor seeds. Comparison of the Asn endopeptidase activities of developing and germinating castor endosperms revealed that they: 1) have identical pH-activity profiles with optimal activity occuring at pH 5.4: 2) are heat-labile proteins displaying comparable thermal stability profiles, and 3) are activated and inhibited by dithiothreitol and thiol modifying reagents, respectively. Thus, the Asn endopeptidases of developing and germinating castor seeds are very similar, if not identical, cysteine proteases. The most significant increase in the activity of endosperm Asn endopeptidase occurs during the full coryledon to maturation stage of seed development, this period coincides with the most active phase of reserve protein accumulation by ripening castor oil seeds. Asn endopeptidase activity of fully mature (dry) castor seeds was about 2-fold lower than that of muturation stage ripening castor oil seed. Asn endopeptidase activity showed a slight reduction over the inicial 2-day period following seed imbibition, and then rapidly decreased over the next several days of germination. The results are compatible with the proposal that Asn endopeptidase functions both to process storage preproteins following their import into protein bodies of developing seeds, as well as to participate in the mobilization of storage proteins during the early phase of seed germination.     

Mobilization of storage proteins in soybean seed (Glycine max L.) during germination and seedling growth

During germination and early growth of the seedling, storage proteins are degraded by proteases. Currently, limited information is available on the degradation of storage proteins in the soybean during germination. In this study, a combined two-dimensional gel electrophoresis and mass spectrometry approach was utilized to determine the proteome profile of soybean seeds (Glycine max L.; Eunhakong). Comparative analysis showed that the temporal profiles of protein expression are dramatically changed during the seed germination and seedling growth. More than 80% of the proteins identified were subunits of glycinin and β-conglycinin, two major storage proteins. Most subunits of these proteins were degraded almost completely at a different rate by 120h, and the degradation products were accumulated or degraded further. Interestingly, the acidic subunits of glycinin were rapidly degraded, but no obvious change in the basic chains. Of the five acidic subunits, the degradation of G2 subunit was not apparently affected by at least 96h but the levels decreased rapidly after that, while no newly appearing intermediate was detected upon the degradation of G4 subunit. On the other hand, the degradation of β-conglycinin during storage protein mobilization appeared to be similar to that of glycinin but at a faster rate. Both α and α' subunits of β-conglycinin largely disappeared by 96h, while the β subunits degraded at the slowest rate. These results suggest that mobilization of subunits of the storage proteins is differentially regulated for seed germination and seedling growth. The present proteomic analysis will facilitate future studies addressing the complex biochemical events taking place during soybean seed germination.     

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.     

Role of H2O2 and cell wall monoamine oxidases in germination of Vigna radiata seeds

Plant cell wall expresses monoamine oxidases (MAOs) that catalyze oxidation of secreted amines and produce H2O2 in the process. The H2O2, so produced is used by cell wall peroxidases for lignification of cell wall or for plant defense. The natural substrates for these MAOs are elusive, but polyamines and certain catecholamines have been proposed as candidates. Reactive oxygen species are also known to act as signaling molecules controlling plant metabolism. Mungbean (Vigna radiata) has long served as the plant model of choice while studying molecular programs followed during germination and seed development. In this study, we tested the effect of externally added MAO substrates epinephrine and H2O2 on storage protein mobilization in germinating seeds of Vigna radiata. The seeds were imbibed in the presence of 50 microM epinephrine and 10 microM H2O2. These low concentrations of the two compounds were used to exclude direct effects on proteolysis and were arrived at after testing a range of the two and choosing the most effective concentration. These seeds showed 11% and 7% decrease in fresh weight respectively, indicating greater storage mobilization and a corresponding 19% and 46% increase in axis length as compared to untreated seeds. Soluble protein in seeds treated with epinephrine and H2O2 decreased significantly by 34% and 33% as compared to untreated seeds. Electrophoretic analysis of seed proteins revealed a startling and selective depletion of storage proteins in treated seeds. The results indicated a clear involvement of H2O2 in storage protein mobilization in the cotyledons. We propose that H2O2 generated within cell walls of seeds serves as a signaling molecule guiding germination events, including protein reserve mobilization.     

Gene Expression Profile Changes in Germinating Rice

Water absorption is a prerequisite for seed germination. During imbibition, water influx causes the resumption of many physiological and metabolic processes in growing seed. In order to obtain more complete knowledge about the mechanism of seed germination, two‐dimensional gel electrophoresis was applied to investigate the protein profile changes of rice seed during the first 48 h of imbibition. Thirty‐nine differentially expressed proteins were identified, including 19 down‐regulated and 20 up‐regulated proteins. Storage proteins and some seed development‐ and desiccation‐associated proteins were down regulated. The changed patterns of these proteins indicated extensive mobilization of seed reserves. By contrast, catabolism‐associated proteins were up regulated upon imbibition. Semi‐quantitative real time polymerase chain reaction analysis showed that most of the genes encoding the down‐ or up‐regulated proteins were also down or up regulated at mRNA level. The expression of these genes was largely consistent at mRNA and protein levels. In providing additional information concerning gene regulation in early plant life, this study will facilitate understanding of the molecular mechanisms of seed germination.