Analysis of proteome profile in germinating soybean seed, and its comparison with rice showing the styles of reserves mobilization in different crops.
ABSTRACT: BACKGROUND: Seed germination is a complex physiological process during which mobilization of nutrient reserves happens. In different crops, this event might be mediated by different regulatory and metabolic pathways. Proteome profiling has been proved to be an efficient way that can help us to construct these pathways. However, no such studies have been performed in soybean germinating seeds up to date. RESULTS: Proteome profiling was conducted through one-dimensional gel electrophoresis followed by liquid chromatography and tandem mass spectrometry strategy in the germinating seeds of soybean (glycine max). Comprehensive comparisons were also carried out between rice and soybean germinating seeds. 764 proteins belonging to 14 functional groups were identified and metabolism related proteins were the largest group. Deep analyses of the proteins and pathways showed that lipids were degraded through lipoxygenase dependent pathway and proteins were degraded through both protease and 26S proteosome system, and the lipoxygenase could also help to remove the reactive oxygen species during the rapid mobilization of reserves of soybean germinating seeds. The differences between rice and soybean germinating seeds proteome profiles indicate that each crop species has distinct mechanism for reserves mobilization during germination. Different reserves could be converted into starches before they are totally utilized during the germination in different crops seeds. CONCLUSIONS: This study is the first comprehensive analysis of proteome profile in germinating soybean seeds to date. The data presented in this paper will improve our understanding of the physiological and biochemical status in the imbibed soybean seeds just prior to germination. Comparison of the protein profile with that of germinating rice seeds gives us new insights on mobilization of nutrient reserves during the germination of crops seeds.
Project description:Background and Aims:Palm seeds are interesting models for studying seed reserve mobilization at the tissue level due to the abundance and complexity of reserves stored in their living endosperm cells and the development of a highly specialized haustorium. We studied structural and physiological aspects of the initial phases of reserve mobilization in seeds of a neotropical palm, Butia capitata, and sought to characterize the interactions between the different developmental pathways of the haustorium and endosperm. Methods:Morphological and histochemical evaluations of the haustorium, the endosperm adjacent to the embryo, and the peripheral endosperm of dry, imbibed, dormant seeds and seeds geminating for 2, 5 and 10 d were performed. Biochemical analyses included determinations of endo-β-mannanase activity, hormonal profiling (20 hormones belonging to eight classes) and H2O2 quantification in various tissues. Key Results:The mobilization of haustorium reserves was associated with germination and involved distinct hormonal alterations in the endosperm related to H2O2 production. The mobilization of endosperm reserves occurred as a post-germination event controlled by the seedling and involved major structural changes in the haustorium, including growth (which increased contact with, and pressure on, the endosperm) and the formation of an aerenchyma (thus facilitating O2 diffusion). The flow of O2 to the endosperm and changes in endogenous contents of H2O2 and hormones (cytokinins, auxins, brassinosteroids and ethylene) induced the establishment of an endosperm digestion zone and the translocation of reserves to the haustorium. Conclusions:The haustorium-endosperm relationship during reserve mobilization plays a pivotal role in signal integration between growth and degradation pathways in germinating seeds of Butia capitata.
Project description: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 ?' and ? subunits of ?-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.
Project description:Salt stress is an important abiotic stressor affecting crop growth and productivity. Of the 20 percent of the terrestrial earth's surface available as agricultural land, 50 percent is estimated by the United Nations Environment Program to be salinized to the level that crops growing on it will be salt-stressed. Increased soil salinity has profound effects on seed germination and germinating seedlings as they are frequently confronted with much higher salinities than vigorously growing plants, because germination usually occurs in surface soils, the site of greatest soluble salt accumulation. The growth of soybean exposed to 40 mM NaCl is negatively affected, while an exposure to 80 mM NaCl is often lethal. When treated with the bacterial signal compounds lipo-chitooligosaccharide (LCO) and thuricin 17 (Th17), soybean seeds (variety Absolute RR) responded positively at salt stress of up to 150 mM NaCl. Shotgun proteomics of unstressed and 100 mM NaCl stressed seeds (48 h) in combination with the LCO and Th17 revealed many known, predicted, hypothetical and unknown proteins. In all, carbon, nitrogen and energy metabolic pathways were affected under both unstressed and salt stressed conditions when treated with signals. PEP carboxylase, Rubisco oxygenase large subunit, pyruvate kinase, and isocitrate lyase were some of the noteworthy proteins enhanced by the signals, along with antioxidant glutathione-S-transferase and other stress related proteins. These findings suggest that the germinating seeds alter their proteome based on bacterial signals and on stress, the specificity of this response plays a crucial role in organ maturation and transition from one stage to another in the plants' life cycle; understanding this response is of fundamental importance in agriculture and, as a result, global food security. The mass spectrometry proteomics data have been deposited to the ProteomeXchange with identifier PXD004106.
Project description:Seed germination is a complex trait determined by both quantitative trait loci (QTLs) and environmental factors and also their interactions. In this study, we mapped one major QTLqSE3 for seed germination and seedling establishment under salinity stress in rice. To understand the molecular basis of this QTL, we isolated qSE3 by map-based cloning and found that it encodes a K+ transporter gene, OsHAK21. The expression of qSE3 was significantly upregulated by salinity stress in germinating seeds. Physiological analysis suggested that qSE3 significantly increased K+ and Na+ uptake in germinating seeds under salinity stress, resulting in increased abscisic acid (ABA) biosynthesis and activated ABA signaling responses. Furthermore, qSE3 significantly decreased the H2 O2 level in germinating seeds under salinity stress. All of these seed physiological changes modulated by qSE3 might contribute to seed germination and seedling establishment under salinity stress. Based on analysis of single-nucleotide polymorphism data of rice accessions, we identified a HAP3 haplotype of qSE3 that was positively correlated with seed germination under salinity stress. This study provides important insights into the roles of qSE3 in seed germination and seedling establishment under salinity stress and facilitates the practical use of qSE3 in rice breeding.
Project description:Since seed reserves can influence seed germination, the quantitative and qualitative differences in seed reserves may relate to the germination characteristics of species. The purpose of our study was to evaluate the correlation between germination and seed reserves, as well as their mobilization during germination of six grassland species (Chloris virgata, Kochia scoparia, Lespedeza hedysaroides, Astragalus adsurgens, Leonurus artemisia, and Dracocephalum moldavica) and compare the results with domesticated species. We measured starch, protein, and fat content in dry seeds and the initial absorption of water during imbibition. Starch, soluble protein, fat, and soluble sugar content also were determined at five stages during germination. Starch, protein, and fat reserves in dry seeds were not significantly correlated with germination percentage and rate (speed), but soluble sugar and soluble protein contents at different germination stages were positively significantly correlated with germination rate for the six species. Starch was mainly used during seed imbibition, and soluble protein was used from the imbibition stage to the highest germination stage. Fat content for all species remained relatively constant throughout germination for six species, regardless of the proportion of other seed reserves in the seeds. Our results for fat utilization differ from those obtained for cultivated grasses and legumes. These results provide new insight on the role of seed reserves as energy resources in germination for wild species.
Project description:Application of nanomaterials for agriculture is relatively new as compared to their use in biomedical and industrial sectors. In order to promote sustainable nanoagriculture, biocompatible silver nanoparticles (AgNPs) have been synthesized through green route using kaffir lime leaf extract for use as nanopriming agent for enhancing seed germination of rice aged seeds. Results of various characterization techniques showed the successful formation of AgNPs which were capped with phytochemicals present in the plant extract. Rice aged seeds primed with phytosynthesized AgNPs at 5 and 10 ppm significantly improved germination performance and seedling vigor compared to unprimed control, AgNO3 priming, and conventional hydropriming. Nanopriming could enhance ?-amylase activity, resulting in higher soluble sugar content for supporting seedlings growth. Furthermore, nanopriming stimulated the up-regulation of aquaporin genes in germinating seeds. Meanwhile, more ROS production was observed in germinating seeds of nanopriming treatment compared to unprimed control and other priming treatments, suggesting that both ROS and aquaporins play important roles in enhancing seed germination. Different mechanisms underlying nanopriming-induced seed germination were proposed, including creation of nanopores for enhanced water uptake, rebooting ROS/antioxidant systems in seeds, generation of hydroxyl radicals for cell wall loosening, and nanocatalyst for fastening starch hydrolysis.
Project description:Rice seeds germinating in flooded soils encounter hypoxia or even anoxia leading to poor seed germination and crop establishment. Introgression of AG1 and AG2 QTLs associated with tolerance of flooding during germination, together with seed pre-treatment via hydro-priming or presoaking can enhance germination and seedling growth in anaerobic soils. This study assessed the performance of elite lines incorporating AG1, AG2 and their combination when directly seeded in flooded soils using dry seeds. The QTLs were in the background of two popular varieties PSB Rc82 and Ciherang-Sub1, evaluated along with the donors Kho Hlan On (AG1) and Ma-Zhan Red (AG2) and recipient parents PSB Rc82 and Ciherang-Sub1. In one set of experiments conducted in the greenhouse, seedling emergence, growth, and carbohydrate mobilization from seeds were assessed. Metabolites associated with reactive oxygen species (ROS) scavenging including malondialdehyde (MDA) as a measure of lipid peroxidation, ascorbate, total phenolic concentration (TPC), and activities of ROS scavenging enzymes were quantified in seeds germinating under control (saturated) and flooded (10?cm) soils. In another set of experiments conducted in a natural field with 3-5?cm flooding depths, control and pretreated seeds of Ciherang-Sub1 introgression lines and checks were used. Flooding reduced seedling emergence of all genotypes, though emergence of AG1?+?AG2 introgression lines was greater than the other AG lines. Soluble sugars increased, while starch concentration decreased gradually under flooding especially in the tolerant checks and in AG1?+?AG2 introgression lines. Less lipid peroxidation and higher ?-amylase activity, higher ascorbate (RAsA) and TPC were observed in the tolerant checks and in the AG1?+?AG2 introgression lines. Lipid peroxidation correlated negatively with ascorbate, TPC, and with ROS scavengers. Seed hydro-priming or pre-soaking increased emergence by 7-10% over that of dry seeds. Introgression of AG2 and AG1?+?AG2 QTLs with seed pretreatment showed 101-153% higher emergence over dry seeds of intolerant genotypes in the field. Lines carrying AG1?+?AG2 QTLs showed higher ?-amylase activity, leading to rapid starch degradation and increase in soluble sugars, ascorbate, and TPC, together leading to higher germination and seedling growth in flooded soils. Seed hydro-priming or pre-soaking for 24?h also improved traits associated with flooding tolerance. Combining tolerance with seed management could therefore, improve crop establishment in flooded soils and encourage large-scale adoption of direct seeded rice system.
Project description:Salinity stress is an important environmental constraint limiting the productivity of many crops worldwide. In this report, experiments were conducted to investigate the effects of seed presoaking by bovine hemoglobin, an inducer of heme oxygenase-1 (HO-1), on salinity tolerance in rice (Oryza sativa) plants. The results showed that different concentrations of the hemoglobin (0.01, 0.05, 0.2, 1.0, and 5.0 g/L) differentially alleviated the inhibition of rice seed germination and thereafter seedling shoot growth caused by 100 mM NaCl stress, and the responses of 1.0 g/L hemoglobin was the most obvious. Further analyses showed that application of hemoglobin not only increased the HO-1 gene expression, but also differentially induced catalase (CAT), ascorbate peroxidase (APX), and superoxide dismutase (SOD) activities or transcripts, thus decreasing the lipid peroxidation in germinating rice seeds subjected to salt stress. Compared with non-hemoglobin treatment, hemoglobin presoaking also increased the potassium (K) to sodium (Na) ratio both in the root and shoot parts after salinity stress. The effect is specific for HO-1 since the potent HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX) blocked the positive actions of hemoglobin on seed germination and seedling shoot growth. Overall, these results suggested that hemoglobin performs an advantageous role in enhancement of salinity tolerance during rice seed germination.
Project description:Germinating oilseeds convert stored lipids into sugars and thereafter in metabolic energy that is used in seedling growth and establishment. During germination, the induced lipolysis linked to the glyoxylate pathway and gluconeogenesis produces sucrose, which is then transported to the embryo and driven through catabolic routes. Herein, we report that the sunflower transcription factor HaWRKY10 regulates carbon partitioning by reducing carbohydrate catabolism and increasing lipolysis and gluconeogenesis. HaWRKY10 was regulated by abscisic acid and gibberellins in the embryo leaves and highly expressed during sunflower seed germination and seedling growth, concomitantly with lipid mobilization. Sunflower leaf discs overexpressing HaWRKY10 showed repressed the expression of genes related to sucrose cleavage and glycolysis compared to controls. Moreover, HaWRKY10 constitutive expression in Arabidopsis seeds produced higher decrease in lipid reserves, whereas starch and sucrose were more preserved compared to wild type. Gene transcripts abundance and enzyme activities involved in stored lipid mobilization and gluconeogenesis increased more in transgenic than in wild type seeds 36 hours after imbibition whereas the negative regulator of lipid mobilization, ABI4, was repressed. Altogether, the results point out a functional parallelism between tissues and plant species, and reveal HaWRKY10 as a positive regulator of storage reserve mobilization in sunflower. Overall design: Transient sunflower discs (Manavella and Chan, Nature Protocols 2009) 35S Promoter::GUS vs. 35S Promoter::HaHB11. Three biological replicates each.
Project description:Phosphoenolpyruvate carboxylase (PEPC; E.C. 188.8.131.52) was characterized in developing and germinating sorghum seeds, focusing on the transcript and polypeptide abundance of multiple plant-type phosphoenolpyruvate carboxylase (PTPC) genes, and the post-translational modification of each isoenzyme by phosphorylation versus monoubiquitination during germination. We observed high levels of SbPPC4 (Sb07g014960) transcripts during early development (stage I), and extensive transcript abundance of SbPPC2 (Sb02g021090) and SbPPC3 (Sb04g008720) throughout the entire life cycle of the seed. Although tandem mass spectrometry (MS) analysis of immunopurified PTPC indicated that four different PTPC isoenzymes were expressed in the developing and germinating seeds, SbPPC3 was the most abundant isozyme of the developing seed, and of the embryo and the aleurone layer of germinating seeds. In vivo phosphorylation of the different PTPC isoenzymes at their conserved N-terminal seryl phosphorylation site during germination was also established by MS/MS analysis. Furthermore, three of the four isoenzymes were partially monoubiquitinated, with MS/MS pinpointing SbPPC2 and SbPPC3 monoubiquitination at the conserved Lys-630 and Lys-624 residues, respectively. Our results demonstrate that monoubiquitination and phosphorylation simultaneously occur in vivo with different PTPC isozymes during seed germination. In addition, we show that PTPC monoubiquitination in germinating sorghum seeds always increases at stage II (emergence of the radicle), is maintained during the aerobic period of rapid cell division and reserve mobilization, and remains relatively constant until stage IV-V when coleoptiles initiate the formation of the photosynthetic tissues.