Mobilization Of Storage Proteins Research Articles

Functional Association between Storage Protein Mobilization and Redox Signaling in Narrow-Leafed Lupin (Lupinus angustifolius L.) Seed Germination and Seedling Development.

(1) Background: Seed storage mobilization, together with oxidative metabolism, with the ascorbate-glutathione (AsA-GSH) cycle as a crucial signaling and metabolic functional crossroad, is one of the main regulators of the control of cell morphogenesis and division, a fundamental physiological process driving seed germination and seedling growth. This study aims to characterize the cellular changes, composition, and patterns of the protein mobilization and ROS-dependent gene expression of redox metabolism in Lupinus angustifolius L. (narrow-leafed lupin, NLL) cotyledons during seed germination. (2) Methods: We performed gene expression analyses via RT-qPCR for conglutins α (1, 2, and 3), β (1, 2, and 5), γ (1, 2), and δ (2 and 4), including a ubiquitin gene as a control, and for redox metabolism-related genes; GADPH was used as a control gene. A microscopic study was developed on cotyledon samples from different germination stages, including as IMB (imbibition), and 2-5, 7, 9, and 11 DAI (days after imbibition), which were processed for light microscopy. SDS-PAGE and immunocytochemistry assays were performed using an anti-β-conglutin antibody (Agrisera), and an anti-rabbit IgG Daylight 488-conjugated secondary antibody. The controls were made while omitting primary Ab. (3) Results and Discussion: Our results showed that a large amount of seed storage protein (SSP) accumulates in protein bodies (PBs) and mobilizes during germination. Families of conglutins (β and γ) may play important roles as functional and signaling molecules, beyond the storage function, at intermediate steps of the seed germination process. In this regard, metabolic activities are closely associated with the regulation of oxidative homeostasis through AsA-GSH activities (γ-L-Glutamyl-L-cysteine synthetase, NOS, Catalase, Cu/Zn-SOD, GPx, GR, GS, GsT) after the imbibition of NLL mature seeds, metabolism activation, and dormancy breakage, which are key molecular and regulatory signaling pathways with particular importance in morphogenesis and developmental processes. (4) Conclusions: The knowledge generated in this study provides evidence for the functional changes and cellular tightly regulated events occurring in the NLL seed cotyledon, orchestrated by the oxidative-related metabolic machinery involved in seed germination advancement.

Wild soybean salt tolerance metabolic model: Assessment of storage protein mobilization in cotyledons and C/N balance in the hypocotyl/root axis.

Salt stress has become one of the main factors limiting crop yield in recent years. The post-germinative growth is most sensitive to salt stress in soybean. In this study, cultivated and wild soybeans were used for an integrated metabonomics and transcriptomics analysis to determine whether wild soybean can resist salt stress by maintaining the mobilization of stored substances in cotyledons and the balance of carbon and nitrogen in the hypocotyl/root axis (HRA). Compared with wild soybean, the growth of cultivated soybean was significantly inhibited during the post-germinative growth period under salt stress. Integrating analysis found that the breakdown products of proteins, such as glutamate, glutamic acid, aspartic acid, and asparagine, increased significantly in wild soybean cotyledons. Asparagine synthase and fumarate hydratase genes and genes encoding HSP20 family proteins were specifically upregulated. In wild soybean HRA, levels of glutamic acid, aspartic acid, asparagine, citric acid, and succinic acid increased significantly, and the glutamate decarboxylase gene and the gene encoding carbonic anhydrase in nitrogen metabolism were significantly upregulated. The metabolic model indicated that wild soybean enhanced the decomposition of stored proteins and the transport of amino acids to the HRA in cotyledons and the GABA shunt to maintain carbon and nitrogen balance in the HRA to resist salt stress. This study provided a theoretical basis for cultivating salt-tolerant soybean varieties and opened opportunities for the development of sustainable agricultural practices.

Identification of Proteases and Protease Inhibitors in Seeds of the Recalcitrant Forest Tree Species Quercus ilex.

Proteases and protease inhibitors have been identified in the recalcitrant species Quercus ilex using in silico and wet methods, with focus on those present in seeds during germination. In silico analyses showed that the Q. ilex transcriptome database contained 2,240 and 97 transcripts annotated as proteases and protease inhibitors, respectively. They belonged to the different families according to MEROPS,1 being the serine and metallo ones the most represented. The data were compared with those previously reported for other Quercus species, including Q. suber, Q. lobata, and Q. robur. Changes in proteases and protease inhibitors alongside seed germination in cotyledon and embryo axis tissues were assessed using proteomics and in vitro and in gel activity assays. Shotgun (LC–MSMS) analysis of embryo axes and cotyledons in nonviable (NV), mature (T1) and germinated (T3) seeds allowed the identification of 177 proteases and 12 protease inhibitors, mostly represented by serine and metallo types. Total protease activity, as determined by in vitro assays using azocasein as substrate, was higher in cotyledons than in embryo axes. There were not differences in activity among cotyledon samples, while embryo axis peaked at germinated T4 stage. Gel assays revealed the presence of protease activities in at least 10 resolved bands, in the Mr range of 60–260 kDa, being some of them common to cotyledons and embryo axes in either nonviable, mature, and germinated seeds. Bands showing quantitative or qualitative changes upon germination were observed in embryo axes but not in cotyledons at Mr values of 60–140 kDa. Proteomics shotgun analysis of the 10 bands with protease activity supported the results obtained in the overall proteome analysis, with 227 proteases and 3 protease inhibitors identified mostly represented by the serine, cysteine, and metallo families. The combined use of shotgun proteomics and protease activity measurements allowed the identification of tissue-specific (e.g., cysteine protease inhibitors in embryo axes of mature acorns) and stage-specific proteins (e.g., those associated with mobilization of storage proteins accumulated in T3 stage). Those proteins showing differences between nonviable and viable seeds could be related to viability, and those variables between mature and germinated could be associated with the germination process. These differences are observed mostly in embryo axes but not in cotyledons. Among them, those implicated in mobilization of reserve proteins, such as the cathepsin H cysteine protease and Clp proteases, and also the large number of subunits of the CNS and 26S proteasome complex differentially identified in embryos of the several stages suggests that protein degradation via CNS/26S plays a major role early in germination. Conversely, aspartic proteases such as nepenthesins were exclusively identified in NV seeds, so their presence could be used as indicator of nonviability.

Storage protein composition during germination and its association with physiological seed quality in common bean

Seed reserve mobilization is considered a post-germination process; however, seed storage protein mobilization occurs during germination. Thus, the knowledge of seed protein composition is important to understand various processes during germination, and it be associated with seed vigor. Therefore, this study aimed to characterize landrace genotypes of common bean (Phaseolus vulgaris) through fractionation of seed storage proteins during germination and to verify the association between seed protein composition and seed vigor. Genotypes of the highest (55 and 81) and lowest (23 and 50) physiological quality were selected. Protein content analysis, profiling, and characterization were performed by combining different hydration times. Mass spectrometry involving in-gel digestion and MALDI-ToF analysis was employed for the qualitative identification of proteins. Glutelin extraction detected the enzyme lipoxygenase in genotypes 55 and 81 alone. In plants, this enzyme may be involved in diverse physiological processes, including growth and development, pest resistance, senescence, and response to wounding. In the process of germination, lipoxygenase removes reactive oxygen species during reserve mobilization; therefore, lipoxygenase may be a candidate biochemical marker for high-vigor genotypes.

Seed coat lignification level is crucial in Capsicum spp seed longevity.

Capsicum (pepper) is known for its poor seed germination, particularly seed longevity is usually much shorter than other Solanaceae. However, the molecular mechanisms involved are mostly unknown in these species. The present study examines the differences in seed longevity among Capsicum species and varietal types. Feral or less domesticated species, such as Capsicum chinense and particularly Capsicum frutescens, showed higher germination rates than the more domesticated Capsicum annuum after accelerated seed aging treatments. In addition, variability was detected in the expression of genes involved in the response to seed deterioration. The differences observed in ASPG1 expression led us to study the seed protein profile in dry and germinating seeds. Seed storage protein mobilization during germination was faster in seed aging-resistant genotypes. Similarly, the transcriptional change observed for the orthologous gene of the trans-species regulator AtHB25 prompted us to study the structure and molecular components of the seed coat in peppers. All the Capsicum pepper accessions analyzed presented very lignified testa and we observed a positive correlation between the amount of lignin and seed viability. Our results provide essential information to explain the poor germination observed in pepper seeds and provide an experimental framework for future improvements in this important character.

Tissue-Specific Proteome and Subcellular Microscopic Analyses Reveal the Effect of High Salt Concentration on Actin Cytoskeleton and Vacuolization in Aleurone Cells during Early Germination of Barley.

Cereal grain germination provides the basis for crop production and requires a tissue-specific interplay between the embryo and endosperm during heterotrophic germination involving signalling, protein secretion, and nutrient uptake until autotrophic growth is possible. High salt concentrations in soil are one of the most severe constraints limiting the germination of crop plants, affecting the metabolism and redox status within the tissues of germinating seed. However, little is known about the effect of salt on seed storage protein mobilization, the endomembrane system, and protein trafficking within and between these tissues. Here, we used mass spectrometry analyses to investigate the protein dynamics of the embryo and endosperm of barley (Hordeum vulgare, L.) at five different early points during germination (0, 12, 24, 48, and 72 h after imbibition) in germinated grains subjected to salt stress. The expression of proteins in the embryo as well as in the endosperm was temporally regulated. Seed storage proteins (SSPs), peptidases, and starch-digesting enzymes were affected by salt. Additionally, microscopic analyses revealed an altered assembly of actin bundles and morphology of protein storage vacuoles (PSVs) in the aleurone layer. Our results suggest that besides the salt-induced protein expression, intracellular trafficking and actin cytoskeleton assembly are responsible for germination delay under salt stress conditions.

Seed storage protein changes and mobilization pattern in Bambaranut (Vigna subterranea) (L.) Verdc. during germination

The germination of seeds involves series of events during which mobilization and utilization of seed storage proteins occur. This study is aimed at determining the changes in total and fractions of seed storage protein in six bambaranut landraces during 96 hrs germination period. The study assessed the changes in seed storage protein content, storage protein profile, endopeptidase activity, free amino acids and gibberellic acid levels. Significant (p<0.05) decrease in total storage protein after 24 hrs, albumin from 48 hrs and globulin and glutelin after 72 hrs germination period were observed in the studied landraces. Prolamin showed significant (p<0.05) decrease after 48 hrs in all the landraces. Five peptide bands were detected in the six landraces with molecular weights corresponding to 97.4 kDa, 45 kDa, 29 kDa, 20.1 kDa and 18 kDa. Peptide bands with molecular weight of 97 kDa and 29 kDa decreased in intensity after 48 hrs of germination in four landraces. Free amino acids content significantly (p<0.05) increased following 24 hrs germination period in all the landraces. The activity of endopeptidase increased significantly (p<0.05), reaching maximum after 96 hrs germination. Significant (p<0.05) increase in gibberellic acid level throughout germination period was also observed. Although slow degradation rate of storage proteins was observed, there was variation in the rate at which storage protein and its fractions decreased among the bambaranut landraces during germination. This variation could be utilized towards obtaining improved bambaranut genotypes with better germination characteristics.

Soybean Mutants Lacking Abundant Seed Storage Proteins Are Impaired in Mobilization of Storage Reserves and Germination.

Spontaneous and radiation-induced mutants of soybean, despite loss of abundant seed proteins, have been reported to grow and reproduce normally without any apparent physiological abnormalities. Here, we report the development and characterization of a soybean line (BSH-2) that lacks several abundant seed storage proteins. One-dimensional and high-resolution two-dimensional gel electrophoresis revealed the absence of the α′ and α subunits of β-conglycinin and G1, G2, G4, and G5 glycinin in the newly developed mutant line (BSH-2). Like our earlier developed soybean mutant line (BSH-3), the seeds of BSH-2 also accumulated high levels of free amino acids as compared with wild-type DN47 seeds. An examination of the germination rates revealed that both BSH-2 and BSH-3 had significantly lower germination rates compared with the parent line DN47. Two-dimensional gel electrophoresis analysis demonstrated that these mutants had slower rates of mobilization of seed storage proteins. The delayed mobilization of storage proteins in BSH-2 and BSH-3 seeds was also correlated with a delayed induction of proteolytic activity in the mutants when compared to DN47. Similarly, qRT-PCR analysis revealed distinct expression pattern of genes involved in proteolytic pathway in the mutants when compared to DN47. Transmission electron microscopy observation of soybean seeds at two germination stages revealed striking differences in the breakdown of protein storage vacuoles and lipid bodies in the mutants. Our study demonstrates that BSH-2 and BSH-3 are compromised in mobilization of storage reserves and the absence of abundant storage proteins may affect the seed germination efficiency and post-germinative seedling establishment.

Seed priming: An emerging technology to impart abiotic stress tolerance in rice

Plants are exposed to any number of potentially adverse environmental conditions such as water deficit, high salinity, extreme temperature, submergence, etc. These abiotic stresses adversely affect the plant growth and productivity. Seed priming is an easy, low cost and low risk method for improving growth and development of plants especially under adverse environmental conditions. Seed priming has been developed as an indispensable method to produce tolerant plants against various stresses in rice. The beneficial effects of seed priming include faster emergence, better stands, and lower incidence of re-sowing, more vigorous plants, better drought tolerance, earlier flowering, earlier harvest and higher grain yield. These beneficial effects of seed priming are due to several reasons such as activation of enzymes associated with endosperm utilization and seed germination, mobilization of storage proteins, changes in hormonal balance and synthesis of proteins that play an important role during seed germination. Seed priming emerges as a promising technology for combating abiotic stress in crops and alleviating the detrimental effects of abiotic stress without much affecting its fitness. Seed priming methods are widely used as an emerging technology to produce tolerant crop varieties against abiotic stresses.

Narrow‐leafed lupin (Lupinus angustifolius L.) β‐conglutin: A multifunctional family of proteins with roles in plant defence, human health benefits, and potential uses as functional food

AbstractLupinus angustifolius or narrow‐leafed lupin (NLL) is an important Pulse world‐wide and an outstanding alternative source of high quality proteins. NLL β‐conglutin (vicilin or 7S acid globulin), a multigene family of proteins, is the main representative protein in NLL seeds exhibiting multifunctional properties and benefits recently discovered. These functions may result from specific structural features of NLL β‐conglutins in comparison with most legume vicilin proteins. From the agricultural side, NLL β‐conglutin proteins (1a) exert antifungal properties promoting enhanced plant resistance to necrotrophic oomycete pathogens and (1b) displaying functional interplay between storage protein mobilization and seed oxidative metabolism as a functional regulatory and signalling crossroad for the control of physiological underlying processes of the seed dormancy breakage and release, during germination, and seedling growth. NLL β‐conglutins benefits go beyond nutritional properties because displayed nutraceutical functions make them an outstanding source of innovative ingredients for functional food. They have antioxidant activity and anti‐inflammatory properties, which are able to improve inflammatory‐related diseases such as type 2 diabetes through (a) regulation of insulin signalling pathway, (b) improving insulin sensitivity, (c) facilitating glucose uptake by cells, (d) together with their capacity for balancing the oxidative homeostasis, metabolic, and signalling pathways (e) while reducing reactive oxygen specie production and increase cellular oxidative defence capacity. Interestingly, NLL β‐conglutins also display anti‐nutritional properties as proteins capable to elicit allergic responses in sensitized individuals. This review outlines newly discovered knowledge of the narrow‐leafed lupin β‐conglutin proteins drawing on information gained from structure‐functional properties, agriculture benefits, as well as nutraceutics and molecular allergy point of views.

Plant Proteases: From Key Enzymes in Germination to Allies for Fighting Human Gluten-Related Disorders.

Plant proteases play a crucial role in many different biological processes along the plant life cycle. One of the most determinant stages in which proteases are key protagonists is the plant germination through the hydrolysis and mobilization of other proteins accumulated in seeds and cereal grains. The most represented proteases in charge of this are the cysteine proteases group, including the C1A family known as papain-like and the C13 family also called legumains. In cereal species such as wheat, oat or rye, gluten is a very complex mixture of grain storage proteins, which may affect the health of sensitive consumers like celiac patients. Since gluten proteins are suitable targets for plant proteases, the knowledge of the proteases involved in storage protein mobilization could be employed to manipulate the amount of gluten in the grain. Some proteases have been previously found to exhibit promising properties for their application in the degradation of known toxic peptides from gluten. To explore the variability in gluten-degrading capacities, we have now analyzed the degradation of gluten from different wheat cultivars using several cysteine proteases from barley. The wide variability showed highlights the possibility to select the protease with the highest potential to alter grain composition reducing the gluten content. Consequently, new avenues could be explored combining genetic manipulation of proteolytic processes with silencing techniques to be used as biotechnological tools against gluten-related disorders.

The Major Storage Protein in Potato Tuber Is Mobilized by a Mechanism Dependent on Its Phosphorylation Status

The role of the protein phosphorylation mechanism in the mobilization of vegetative storage proteins (VSPs) is totally unknown. Patatin is the major VSP of the potato (Solanum tuberosum L.) tuber that encompasses multiple differentially phosphorylated isoforms. In this study, temporal changes in the phosphorylation status of patatin isoforms and their involvement in patatin mobilization are investigated using phosphoproteomic methods based on targeted two-dimensional electrophoresis (2-DE). High-resolution 2-DE profiles of patatin isoforms were obtained in four sequential tuber life cycle stages of Kennebec cultivar: endodormancy, bud break, sprouting and plant growth. In-gel multiplex identification of phosphorylated isoforms with Pro-Q Diamond phosphoprotein-specific stain revealed an increase in the number of phosphorylated isoforms after the tuber endodormancy stage. In addition, we found that the phosphorylation status of patatin isoforms significantly changed throughout the tuber life cycle (P < 0.05) using the chemical method of protein dephosphorylation with hydrogen fluoride-pyridine (HF-P) coupled to 2-DE. More specifically, patatin phosphorylation increased by 32% from endodormancy to the tuber sprouting stage and subsequently decreased together with patatin degradation. Patatin isoforms were not randomly mobilized because highly phosphorylated Kuras-isoforms were preferably degraded in comparison to less phosphorylated non-Kuras isoforms. These results lead us to conclude that patatin is mobilized by a mechanism dependent on the phosphorylation status of specific isoforms.

Subtilase activity and gene expression during germination and seedling growth in barley

Proteases play a main role in the mobilization of storage proteins during seed germination. Until today, there is little information about the involvement of serine proteases, particularly subtilases, in the germination of barley grains. The aims of the present work were to study the contribution of serine proteases to the total proteolytic activity induced during germination of barley grains and evaluate the specific involvement of subtilases in this process. Proteolytic activity assayed against azocasein in the presence of specific inhibitors, showed that serine proteases contributed between 10 and 20% of total activity along germination. Subtilase activity increased from day 1 after imbibition with a peak between days 4–5. Moreover, in vivo determination of subtilase activity in germinating grains revealed increasing activity along germination mainly localized in the seed endosperm and developing rootlets. Finally, the expression of 19 barley genes encoding subtilases was measured by real time PCR during germination. Three of the analyzed genes increased their expression along germination, five showed a transient induction, one was down-regulated, nine remained unchanged and one was not expressed. The present work demonstrates the involvement of subtilases in germination of barley grains and describes the positive association of eight subtilase genes to this process.

Toward characterizing germination and early growth in the non-orthodox forest tree species Quercus ilex through complementary gel and gel-free proteomic analysis of embryo and seedlings

By using two complementary proteomics, gel-based and gel-free (shotgun) approaches, the protein profiles of the non-orthodox forest tree species Quercus ilex seeds during germination and early seedling growth have been compared. Proteins were extracted from embryo axis, radicle and shoot tissues at different developmental stages. Proteins were subjected to one- and two-dimensional gel electrophoresis. A multivariate analysis (PCA) revealed that SDS-PAGE clearly separated germination (0–24 h post-imbibition), postgermination (72–216 h post-imbibition) and early seedling growth stages (2 weeks post-imbibition). Image analysis of the two-dimensional gels revealed a total of 732 spots, 103 of which were significantly variable among developmental stages. After MALDI-TOF/TOF MS analysis, 90 spots were identified, belonging to six main functional categories: carbohydrate, amino acids, energy, and protein metabolism, biosynthesis of secondary metabolites, and redox processes. The gel-based approach disclosed important metabolic changes that occurred in the holm oak seed after the germination. However, few proteins were significantly altered during the germination period (from 0 h to 24 h post imbibition) and, because of that, a further shotgun analysis was therefore used to analyse changes in the protein profile during seed germination. Up to 1250 proteins could be confidently identified, with 153 being variable. They belonged to the main functional categories of carbohydrate, amino acids and secondary metabolism, protein degradation, and responses to abiotic stress. The accumulation of proteases and amino acids metabolism proteins in mature seeds can be reflecting the production of energy from the mobilization of storage proteins to start germination. These results, therefore, corroborate the hypothesis that the mature non-orthodox seeds of Q. ilex have all the machinery necessary for rapidly resuming metabolic activities and starting the germination process, in contrast to that occurs in orthodox seeds, which metabolic activity ceases in mature dry seeds.The use of a genus-specific database combined with the public Viridiplantae database improved the quality and quantity of protein identification in this orphan species. In addition, both proteomics approaches (gel-based and shotgun) were complementary, with shotgun increasing by over two-fold the coverage of the proteome analysed. Both approaches provided similar results and supported the same conclusions on the metabolic switch experienced by the seed upon germination. SignificanceThe optimal seed germination is a prerequisite for successful seedling establishment and plant vigour, being of great relevance in the case of crops and commercial woody plants. By using a complementary gel-based and gel-free proteomic strategy we have study the protein profiles of the non-orthodox forest tree species Quercus ilex seeds during germination and early seedling growth. The contribution of this work is of great importance, due to the complemented proteomic approaches giving similar clues to the metabolic state of the mature Q. ilex seed before the germination starts, and the metabolic switch experienced by the imbibed acorn until the seedling is established.

The influence of chosen fungicides on the activity of aminopeptidases in winter oilseed rape during pods development

Cultivation of oilseed rape requires application of specific fungicides. Besides their protective role, they can potentially influence the expression and activity of crucial enzymes in the plant. Among the large number of enzymes expressed in plants, aminopeptidases play a key role in all crucial physiological processes during the whole life cycle (e.g. storage protein mobilization and thus supplying plant with needed amino acids, as well as plant aging, protection and defense responses). In the present paper, we evaluate for the first time, the influence of the treatment of winter oilseed rape with commercially available fungicides (Pictor 400 SC, Propulse 250 SE and Symetra 325 SC), on the activity of aminopeptidases expressed in each plant organ (flowers, leaves, stems and pods separately). Fungicides were applied once, at one of the three stages of oilseed rape development (BBCH 59–61, BBCH 63–65 and BBCH 67–69). The aminopeptidase activity was determined using six different amino acid p-nitroanilides as substrates. The results have shown, that in control plants, at the beginning of intensive pods development and seeds production, hydrophobic amino acids with bulky side chains (Phe, Leu) were preferentially hydrolysed. In control plants, the activity was ~3.5 times higher in stems and pods, compared to leaves. The treatment with all pesticides caused significant increase in aminopeptidases hydrolytic activity toward small amino acids Gly, Ala as well as proline, mostly in flowers and leaves. These amino acids are proven to be crucial in the mechanisms of delaying of plant aging, development of better resistance to stress and plant defense. It can be suggested, that studied fungicides enhance such mechanisms, by activating the expression of genes coding for aminopeptidases, which are active in hydrolysis of N-terminal amino acids such as Gly, Ala, Pro from storage peptides and proteins. Depending on fungicide, the major increase of aminopeptidase activity was observed after application at BBCH 67–69 (Pictor 400 SC and Symetra 325 SC) and BBCH 63–65 (Propulse 250 SE) stages of development. Our study revealed, that agrochemical treatment and time of application, influenced the expression and activity of aminopeptidases, even though they were not molecular targets of applied fungicides. Since aminopeptidases are widely distributed throughout all organisms and are crucial in many key physiological processes, it can be expected, that factors influencing their expression and activity in plants, can also influence these enzymes in other organisms, especially humans and other mammals.

Mobilization and synthesis of seed storage and LEA proteins during natural priming of Buddleja cordata and Opuntia tomentosa

Background: In crop plants, the induction of seed storage protein mobilization and synthesis of LEA family proteins has been extensively described.Question: Natural priming promotes mobilization and synthesis of seed storage and LEA proteins in wild plants?Species studied: Buddleja cordata and Opuntia tomentosa. Study site: Reserva Ecológica del Pedregal de San Angel (REPSA).Methods: Natural priming treatments were applied to B. cordata and O. tomentosa seeds during one and six months respectively. Phosphorylated proteins fractions were obtained by affinity chromatography from control and treated seeds. Differences in protein pattern between control and treated seeds were determined by electrophoresis and the treated seed proteins were identified by LS/LS/MALDITOF. Results: The phosphorylated fraction of B. cordata treated seeds presented two proteins that were absent in the control protein fraction. These two proteins were identified as two different 11S globulins. The phosphorylated fraction of O. tomentosa treated seeds also presented two proteins that were absent in the control protein fraction. These two proteins were identified as a 12S globulin and a LEA protein.Conclusion: Seeds are subjected to changes in soil water content during their permanence in soil; this natural priming process promotes mobilization and synthesis of storage and LEA proteins that are involved in stress resistance.

Modulation of Reserve Mobilization by Sucrose, Glutamine, and Abscisic Acid During Seedling Establishment in Sunflower

We carried out in vitro feeding experiments using sunflower as a model to differentiate the modulatory effects of metabolites (sucrose and glutamine) and hormones (gibberellic acid and abscisic acid) on reserve mobilization, metabolite partitioning, and key enzyme activities. Exogenous sucrose negatively not only modulated the mobilization of carbon reserves (oils and starch), but it also delayed the degradation of nitrogen reserves (storage proteins) in the cotyledons. Similarly, exogenous glutamine negatively not only modulated storage protein hydrolysis, but it also retarded oil and starch degradation. Different from the metabolites, exogenous abscisic acid affected only the mobilization of oils and storage proteins. Sucrose and glutamine caused non-reducing sugar accumulation in the cotyledons and axis, but abscisic acid did not change the content of these compounds in both seedling parts. Curiously, glutamine failed to cause amino acid accumulation in the cotyledons and abscisic acid increased the amino acid content in both cotyledons and axis. Gibberellic acid did not stimulate reserve mobilization and metabolite consumption. Although the mobilization of oils, storage proteins, and starch has been delayed by sucrose and glutamine, these metabolites augmented the activity of isocitrate lyase, acid proteases, and amylases. Only abscisic acid reduced amylase activity and increased glutamine synthetase activity. Accordingly, sucrose and glutamine exert a “crossed effect” on reserve mobilization, that is, sucrose delays storage protein hydrolysis and glutamine retards oil and starch degradation. These effects may be mediated by non-reducing sugars and they are, at least in part, different from those exerted by abscisic acid.

HvPap-1 C1A Protease and HvCPI-2 Cystatin Contribute to Barley Grain Filling and Germination

Proteolysis is an essential process throughout the mobilization of storage proteins in barley (Hordeum vulgare) grains during germination. It involves numerous types of enzymes, with C1A Cys proteases the most abundant key players. Manipulation of the proteolytic machinery is a potential way to enhance grain yield and quality, and it could influence the mobilization of storage compounds along germination. Transgenic barley plants silencing or over-expressing the cathepsin F-like HvPap-1 Cys protease show differential accumulation of storage molecules such as starch, proteins, and free amino acids in the grain. It is particularly striking that the HvPap-1 artificial microRNA lines phenotype show a drastic delay in the grain germination process. Alterations to the proteolytic activities in the over-expressing and knock-down grains associated with changes in the level of expression of several C1A peptidases were also detected. Similarly, down-regulating cystatin Icy-2, one of the proteinaceous inhibitors of the cathepsin F-like protease, also has important effects on grain filling. However, the ultimate physiological influence of manipulating a peptidase or an inhibitor cannot be always predicted, since the plant tries to compensate the modified proteolytic effects by modulating the expression of some other peptidases or their inhibitors.

Analysis of expression and inhibitory activity of a TrcC-6 phytocystatin present in developing and germinating seeds of triticale (×Triticosecale Wittm.)

Storage proteins of cereal seeds are processed during accumulation and degraded during germination primarily by cysteine proteinases. One of the mechanisms controlling the activity of these enzymes is the synthesis of specific inhibitors named phytocystatins. Here we present the complete gene sequence of a triticale ( × Triticosecale Wittm.) phytocystatin, TrcC-6, which encodes a 152-amino acid protein with a putative 25-amino acid signal peptide. This protein has a calculated molecular mass of 16.2 kDa, and was assigned to phylogenetic group B of phytocystatins. Because TrcC-6 transcripts are present in triticale seeds, we hypothesized that this phytocystatin regulates storage protein accumulation and degradation. Therefore, changes in gene expression during the entire period of seed development and germination were examined. TrcC-6 transcripts and TrcC-6 protein levels increased during the maturation of seeds and remained high during the first hours of germination. This enabled us to conclude that TrcC-6 likely regulates seed germination by the regulation of storage protein hydrolysis. For the analysis of TrcC-6 inhibitory activity, recombinant protein was expressed in Escherichia coli BL21 (DE3) and purified. Recombinant TrcC-6 proved to be a potent inhibitor of cysteine proteinases. It inhibited the in vitro activity of papain (EC 3.4.22.2) and ficin (EC 3.4.22.3). Furthermore, native PAGE analysis revealed that recombinant TrcC-6 inhibits the activity of endogenous cysteine proteinases present in germinating seeds of triticale. Based on these results, TrcC-6 is likely one of the important factors that regulate cysteine proteinase activity during the accumulation and mobilization of storage proteins.

Identification and expression analysis of a novel phytocystatin in developing and germinating seeds of triticale (×Triticosecale Wittm.)

In this paper the complete cDNA sequence of a newly identified triticale phytocystatin, TrcC-7, was analyzed. Because &lt;em&gt;TrcC-7&lt;/em&gt; transcripts were present in seeds, we hypothesized that it may regulate storage protein accumulation and degradation. Therefore, changes in mRNA and protein levels during the entire period of seed development and germination were examined. Expression of &lt;em&gt;TrcC-7&lt;/em&gt; increased during development and decreased at the end of maturation and subsequently increased during seed germination. Based on these results, TrcC-7 likely regulates cysteine proteinase activity during the accumulation and mobilization of storage proteins.