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.     

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.     

Effects of desiccation on peanut (Arachis hypogaea L.) seed protein composition

Response of peanut to desiccation was studied by monitoring changes in the seed protein content and composition during a 14 day desiccation period using a combination of electrophoretic and immunochemical techniques. Following desiccation, the protein content of ‘white’ (most immature) and ‘orange’ seed increased, while that of the ‘brown’ (mature) seed was not affected. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed no major qualitative differences in the protein composition during desiccation of the samples. However, immunoblotting with anti-dehydrin antisera revealed the presence of several new proteins in the desiccated samples compared with the controls. One of the dehydrin-like proteins, band ‘c’ was found to be related to water-stress, while the other proteins appeared to be the storage proteins accumulated as the seed matured in vitro. Capillary electrophoresis (CE) showed major changes in the protein quantity and quality of ‘white’ seed during the 0–14 days of desiccation. In contrast, in the ‘orange’ and ‘brown’ seeds changes in protein composition were less significant. Results indicated that there are several dehydrin-like proteins expressed in peanuts, however, not all of them are related to water stress.     

Free Amino Acid, Protein and Water Content Changes Associated with Seed Development in Araucaria angustifolia

The free amino acid, protein, water and dry matter contents were determined during the seed development of Araucaria angustifolia. Soluble and insoluble proteins in the mature seed represent 4.2 % of the fresh matter. The embryonic axis stored the greatest amount of soluble proteins, while cotyledons both with the embryonic axis showed the largest quantities of insoluble proteins in the mature seed. The greatest concentration of free amino acids was detected during the stage when cotyledons start to develop. Glutamic acid, aspartic acid, alanine and serine were predominant in the whole seed while arginine, lysine and γ-aminobutyric acid were present in great amounts only in cotyledons and embryonic axis. Although megagametophyte was important as a source of free amino acids, it was not the major protein storage organ in the mature seed. In the embryogenetic process, the rise of cotyledons is closely related to physiological and biochemical changes. This revised version was published online in July 2006 with corrections to the Cover Date.     

A comparative study of water distribution and dehydrin protein localization in maturing pea seeds

In this study, the distribution of water in pea seeds after harvesting at different seed stages was traced by magnetic resonance imaging (MRI). MRI visualized the process of water loss in maturing pea seeds. MR images showed local inhomogeneities of water distribution inside seeds. The intensity of the signal coming from water declined from the inner to the outer part of cotyledon tissue. This spatial inhomogeneity of water signals inside cotyledons may be correlated with the gradient of storage substances accumulation within cotyledons. Tissue localization of dehydrins showed the presence of dehydrin protein in the area of protovascular tissue of both the embryo axis and cotyledons. The temporal accumulation of two dehydrin proteins with molecular masses of 30 and 35kDa correlated well with seed desiccation. The pattern of dehydrin localization reflected the pattern of water distribution in the protovascular bundles region of maturing pea embryos, suggesting the involvement of these proteins in promoting water influx into the vascular bundles.     

种子活力与生物膜的研究现状

种子活力与生物膜的结构和功能密切相关.活力高的种子,膜结构比较完整,在吸水时,膜系统恢复的速度较快,并且修复的较完善.研究表明,超干和引发处理可使膜结构得到保持与修复,很多种类的物质参与了膜结构的保护,例如可溶性糖、蛋白质(包括酶)、两性分子、Ca2 、多胺及其他非酶促自由基清除系统等,保护物质协同作用,稳定膜脂及膜蛋白的结构,保持膜系统的完整性,使膜功能得以正常发挥,强化了种子活力.     

种子活力与生物膜的研究现状

种子活力与生物膜的结构和功能密切相关。活力高的种子,膜结构比较完整,在吸水时,膜系统恢复的速度较快,并且修复的较完善。研究表明,超干和引发处理可使膜结构得到保持与修复,很多种类的物质参与了膜结构的保护,例如可溶性糖、蛋白质(包括酶)、两性分子、Ca2+、多胺及其他非酶促自由基清除系统等,保护物质协同作用,稳定膜脂及膜蛋白的结构,保持膜系统的完整性,使膜功能得以正常发挥,强化了种子活力。     

Aquaporins and unloading of phloem-imported water in coats of developing bean seeds

Nutrients are imported into developing legume seeds by mass flow through the phloem, and reach developing embryos following secretion from their symplasmically isolated coats. To sustain homeostasis of seed coat water relations, phloem-delivered nutrients and water must exit seed coats at rates commensurate with those of import through the phloem. In this context, coats of developing French bean seeds were screened for expression of aquaporin genes resulting in cloning PvPIP1;1, PvPIP2;2 and PvPIP2;3. These genes were differentially expressed in all vegetative organs, but exhibited their strongest expression in seed coats. In seed coats, expression was localized to cells of the nutrient-unloading pathway. Transport properties of the PvPIPs were characterized by expression in Xenopus oocytes. Only PvPIP2;3 showed significant water channel activity (Pos = 150-200 microm s(-1)) even when the plasma membrane intrinsic proteins (PIPs) were co-expressed in various combinations. Permeability increases to glycerol, methylamine and urea were not detected in oocytes expressing PvPIPs. Transport active aquaporins in native plasma membranes of seed coats were demonstrated by measuring rates of osmotic shrinkage of membrane vesicles in the presence and absence of mercuric chloride and silver nitrate. The functional significance of aquaporins in nutrient and water transport in developing seeds is discussed.     

Transition from hormonal to nonhormonal regulation as exemplified by seed dormancy release and germination triggering

It is generally believed that seed dormancy release is terminated by germination and that this process is controlled by phytohormones. Most attention was paid to gibberellins (GAs) because treatment with GAs is most frequently applied for seed dormancy breaking. The review characterizes the hormonal regulation of seed dormancy and its release, as exemplified by arabidopsis seeds possessing non-deep physiological dormancy. Dormancy release occurs under the influence of low temperature and/or illumination with red light. Two main trends are typical of this process: (1) a decrease in ABA content and blocking of signal transduction from ABA, and (2) GA synthesis and activation of GA signaling pathway. Dormancy release ends with the GA-induced syntheses of some proteins, enzymes in particular, required for the start of germination. Quiescent seeds are capable of realizing the germination program without hormonal induction, due to nothing but seed hydration. In imbibing seeds, the triggering role of water lies in the successive activation of basic metabolic systems after attaining the water content thresholds characteristic of these systems and in preparing cells of embryo axial organs for germination. Thus, seed dormancy release is controlled by phytohormones, whereas subsequent germination manifesting itself as the initiation of cell elongation in embryo axes is controlled by water inflow.     

Studies onMadhuca butyraceae seed proteins

DefattedMadhuca butyraceae seeds contain 24% of crude protein and 10.4% of saponins. The solubility ofMadhuca seed proteins was determined in water and NaCl as a function of pH and minimum solubility occurred at pH 4.0. The proteins consist of three components with S_20,w values of 2.2, 9.8 and 15.4. On gel filtration the proteins gave three peaks and on diethylaminoethyl cellulose chromatography they resolved into two components. Thein vitro digestibility ofMadhuca seed protein was found to be 69% when assayed with a pepsin-pancreatin system.     

The solubilization and degradation of pumpkin seed globulin during germination

Pumpkin seed globulin decreased 88% during the first 4 daysof germination. This decrease was concomitant with a 2.5 foldincrease in water soluble protein which arose directly fromthe water insoluble globulin. The sequence of solubilizationand breakdown of the globulin was followed through 12 days ofgermination. Pumpkin seed globulin was determined to have subunitsof 56,000 daltons, while the new water soluble protein consistedof two proteins, as determined by sodium dodecyl sulfate polyacrylamideelectrophoresis; one having a molecular weight of 42,000 daltonsand the other 28,000 daltons. Trypsin mimicked the first stepof the breakdown by solubilizing pumpkin seed globulin to yieldidentical digestion products as were obtained in vitro. Maximumproteolytic acitvity, as measured by the release of ninhydrinpositive, materials occurred at 6 days of germination, at whichtime both the concentration of free amino acids and the incorporationof ¹⁴C into amino acids increased rapidly. A second proteolyticenzyme system which solubilized the pumpkin seed globulin butdid not act on hemoglobin, casein, or bovine serum albumin reachedits maximum activity at two days of germination. (Received April 17, 1978; )     

Seed coat composition in black and white soybean seeds with differential water permeability

• The seed coat composition of white (JS 335) and black (Bhatt) soybean (Glycine max (L.) Merr) having different water permeability was studied.
• Phenols, tannins and proteins were measured, as well as trace elements and metabolites in the seed coats.
• The seed coat of Bhatt was impermeable and imposed dormancy, while that of JS 335 was permeable and seeds exhibited imbibitional injury. Bhatt seed coats contained comparatively higher concentrations of phenols, tannins, proteins, Fe and Cu than those of JS 335. Metabolites of seed coats of both genotypes contained 164 compounds, among which only 14 were common to both cultivars, while the remaining 79 and 71 compounds were unique to JS 331 and Bhatt, respectively.
• Phenols are the main compounds responsible for seed coat impermeability and accumulate in palisade cells of Bhatt, providing impermeability and strength to the seed coat. JS 335 had more cracked seed coats, mainly due to their lower tannin content. Alkanes, esters, carboxylic acids and alcohols were common to both genotypes, while cyclic thiocarbamate (1.07%), monoterpene alcohols (1.07%), nitric esters (1.07%), phenoxazine (1.07%) and sulphoxide (1.07%) compounds were unique to the JS 335 seed coat, while aldehydes (2.35%), amides (1.17%), azoles (1.17%) and sugar moieties (1.17%) were unique to Bhatt seed coats. This study provides a platform for isolation and understanding of each identified compound for its function in seed coat permeability.

     

Novel clues on abiotic stress tolerance emerge from embryo proteome analyses of rice varieties with contrasting stress adaptation

Cereal embryos sustain severe water deficit at the final stage of seed maturation. The molecular mechanisms underlying the acquisition of desiccation tolerance in seed embryos are similar to those displayed during water deficit in vegetative tissues. The genetic variation among six rice genotypes adapted to diverse environmental conditions was analysed at the proteome level to get further clues on the mechanisms leading to water-stress tolerance. MS analysis allowed the identification of 28 proteins involved in stress tolerance (late embryogenesis abundant proteins), nutrient reservoir activity, among other proteins implicated in diverse cellular processes potentially related to the stress response (e.g., mitochondrial import translocase). Hierarchical clustering and multidimensional scaling analyses revealed a close relationship between the stress-sensitive genotypes, whereas the stress-tolerant varieties were more distantly related. Besides qualitative and significant quantitative changes in embryo proteins across the distinct varieties, we also found differences at post-translational level. The results indicated that late embryogenesis abundant Rab21 was more strongly phosphorylated in the embryos of the sensitive varieties than in the embryos of the tolerant ones. We propose that the differences found in the phosphorylation status of Rab21 are related to stress tolerance.