Transgenic rice established to express corn cystatin exhibits strong inhibitory activity against insect gut proteinases

Corn cystatin (CC), a phytocystatin, shows a wide inhibitory spectrum against various cysteine proteinases. We produced transgenic rice plants by introducing CC cDNA under CaMV 35S promoter as a first step to obtain a rice plant with insecticidal activity. This attempt was based on the observation that many insect pests, especially Coleoptera, have cysteine proteinases, probably digestive enzymes, and also that oryzacystatin, an intrinsic rice cystatin, shows a narrow inhibition spectrum and is present in ordinary rice seeds in insufficient amounts to inhibit the cysteine proteinases of rice insect pests. The transgenic rice plants generated contained high levels of CC mRNA and CC protein in both seeds and leaves, the CC protein content of the seed reaching ca. 2% of the total heat soluble protein. We also recovered CC activity from seeds and found that the CC fraction efficiently inhibited both papain and cathepsin H, whereas the corresponding fraction from non-transformed rice seeds showed much lower or undetectable inhibitory activities against these cysteine proteinases. Furthermore, CC prepared from transgenic rice plants showed potent inhibitory activity against proteinases that occur in the gut of the insect pest, Sitophilus zeamais.     

Purification and characterisation of a novel papain inhibitor from common bean (Phaseolus vulgaris) seed

A proteinaceous inhibitor of papain was purified to apparent homogeneity from mature seeds of common bean ( Phaseolus vulgaris L.). After four chromatographic steps, the papain inhibitor was purified 219‐fold with 12% recovery. On the basis of papain inhibitory activity, cystatins have been estimated to account for about 0.1% of the total protein content of mature common bean seeds. The purified protein, as other plant cystatins, is an acidic protein, heat stable and insensitive to reducing agents. Its molecular mass is about 37 kDa as judged by size exclusion chromatography and SDS‐PAGE. Moreover it is immunologically related to oryzacystatins, since it is recognised by a specific oryzacystatin I antiserum. Based on its biochemical properties the papain inhibitor described here belongs to the phytocystatin family. Papain inhibitory assays carried out during seed development showed that bean cystatin is active since early maturation stages. Our results suggest that, in common bean seed, cysteine proteinase inhibitors are important during seed development with a putative role in the control and regulation of endogenous thiol protease activity.     

Molecular cloning of a cysteine proteinase inhibitor of rice (oryzacystatin). Homology with animal cystatins and transient expression in the ripening process of rice seeds

A cDNA clone for a cysteine proteinase inhibitor of rice (oryzacystatin) was isolated from a lambda gt10 cDNA library of rice immature seeds by screening with synthesized oligonucleotide probes based on partial amino acid sequences of oryzacystatin. A nearly full-length cDNA clone was obtained which encoded 102-amino acid residues. The amino acid sequence of oryzacystatin deduced from the cDNA sequence was significantly homologous to those of mammalian cystatins, especially family 2 cystatins. Oryzacystatin contained the sequence Gln-Val-Val-Ala-Gly conserved among most members of the cystatin superfamily. The gene for oryzacystatin was transcribed into a single mRNA species of about 700 nucleotides. The content of mRNA reached its highest level 2 weeks after flowering and then gradually decreased to undetectable levels at 10 weeks. This feature of transient expression is coordinate with that of glutelin (a major storage protein), although the expression of oryzacystatin precedes that of glutelin by about 1 week.     

Gene organization of oryzacystatin-II, a new cystatin superfamily member of plant origin, is closely related to that of oryzacystatin-I but different from those of animal cystatins

The gene structure of oryzacystatin-II, a new cystatin superfamily member of rice seed origin, was determined. It spans approximately 2.5 kbp and comprises 3 exons. The number of exons and the intron-breakpoints coincide with those of oryzacystatin-I, the first well-defined plant cystatin. However, no similar sequences were observed between the two oryzacystatin genes in 5'-upstream regulatory regions, even though both are expressed specifically during the ripening stage of rice seeds. The gene organization of these two plant cystatins is generally different from that of animal cystatins.     

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.     

Hydrogen peroxide scavenging regulates germination ability during wheat (Triticum aestivum L.) seed maturation

Hydrogen peroxide (H₂O₂) promotes seed germination of cereal plants and ascorbic acid which acts as antioxidant suppresses the germination of wheat seeds, but the role of H₂O₂ scavenging on germination during seed maturation has not been demonstrated. We investigated relationship of germination, ascorbate, H₂O₂ scavenging enzymes and sensitivity to ascorbic acid (AsA) maturing seeds of two typical wheat (Triticum aestivum L.) cultivars, cvs. Shirogane-Komugi and Norin61. Shirogane-Komugi had marked high germination ability than Norin61 during seed maturation. Although the H₂O₂ content had no difference in the two culti-vars, sensitivity to AsA of Norin61 seeds was higher than that of Shirogane-Komugi seeds during seed maturation. The sensitivity to AsA closely correlated with germination characteristic in the two cultivars. Especially, at 28 days after pollination (DAP), sensitivity to AsA in Norin61 seeds was remarkably high. At that stage, no significant differences were observed in endogenous AsA level, ascorbate peroxidase (APX, EC 1.11.1.11) and dehydroascorbate reductase (DHAR, EC 1.8.5.1) activities in the two cultivars. However, catalase (CAT, EC 1.11.1.6) activity and CAT mRNA in Norin61 were remarkably higher than in Shirogane-Komugi. Sensitivity to AsA at 35 and 42 DAPs kept high levels in Norin61, and endogenous AsA and CAT activity in the seeds were significantly higher than in Shirogane-Komugi. These results revealed a direct correlation between germination and antioxidant sensitivity during the developmental stages of wheat seeds.Key words: ascorbic acid, germination, hydorogen peroxide, maturation, wheat seed     

Changes in sugars during rice seed desiccation

The correlation between desiccation tolerance and soluble sugars was investigated in seeds of a number of rice cultivars belonging to the Asian rice Oryza sativa L. They were dried or ultradried to various low moisture content and then imbibed for germination testing. Few or no changes on germination percentage and vigor index were found in Indian rice seeds even after their moisture content fell to 3.5%, indicating that Indian rice exhibited a strong desiccation tolerance. On the contrary, Japonica rice seed germination percentage rapidly decreased, after their moisture content fell to 4.5%. The capacity for desiccation tolerance in Japonica (cv. Chunjiang 15) and Indian (cv. Zhongzu 1) developing seeds increased on 23–40 and 15–25 days after pollination, respectively. Though the level of monosaccharides declined, the content of sucrose has increased during desiccation. These results suggest that desiccation tolerance might be associated with the increase in seed viability and the changes in sugar level, and that raffinose could be capable of contributing to the desiccation tolerance to ultradrying. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 2, pp. 220–226. The text was submitted by the authors in English.     

The inhibitory effects of the cysteine protease inhibitor, oryzacystatin, on digestive proteases and on larval survival and development of the southern corn rootworm (Diabrotica undecimpunctata howardi)

At least eight proteolytic activities have been identified in the midgut contents of larval Southern corn rootworm (Diabrotica undecimpunctata howardi). Around 70% of protease activity could be arrested by the cysteine protease inhibitors E-64 and chicken egg-white cystatin, while the aspartic acid protease inhibitor pepstatin caused 30% inhibition. The cysteine protease activity was found to be highly sensitive to inhibition by both chicken egg-white cystatin and the rice cystatin, oryzacystatin I. Oryzacystatin I, expressed as a fully functional fusion protein in E. coli, was found to strongly inhibit larval gut protease activity. This recombinant oryzacystatin, incorporated into artificial diet at concentrations of 10 mM and above, caused significant decreases in larval survival and weight gain. E-64 was also shown to cause a significant antimetabolic in vivo effect. These results demonstrate the great potential for cysteine protease inhibitors, such as oryzacystatin, as tools for exploitation in the control of the Southern corn rootworm.     

Two distinct cystatin species in rice seeds with different specificities against cysteine proteinases. Molecular cloning, expression, and biochemical studies on oryzacystatin-II.

Oryzacystatin (oryzacystatin-I) is a proteinaceous cysteine proteinase inhibitor (cystatin) in rice seeds and is the first well defined cystatin of plant origin. In this study we isolated cDNA clones for a new type of cystatin (oryzacystatin-II) in rice seeds by screening with the oryzacystatin-I cDNA probe. The newly isolated cDNA clone encodes 107 amino acid residues whose sequence is similar to that of oryzacystatin-I (approximately 55% of identity). These oryzacystatins have no disulfide bonds, and so could be classified as family-I cystatins; however, the amino acid sequences resemble those of family-II members more than family-I members. Oryzacystatin-I and -II are remarkably distinct in two respects: 1) their specificities against cysteine proteinases; and 2) the expression patterns of their mRNAs in the ripening stage of rice seeds. Oryzacystatin-I inhibits papain more effectively (Ki 3.0 x 10(-8) M) than cathepsin H (Ki 0.79 x 10(-6) M), while oryzacystatin-II inhibits cathepsin H (Ki 1.0 x 10(-8) M) better than papain (Ki 0.83 x 10(-6) M). The mRNA for oryzacystatin-I is expressed maximally at 2 weeks after flowering and is not detected in mature seeds, whereas the mRNA for oryzacystatin-II is constantly expressed throughout the maturation stages and is clearly detected in mature seeds. Western blotting analysis using antibody to oryzacystatin-II showed that, as is the case with oryzacystatin-I, oryzacystatin-II occurs in mature rice seeds. Thus, these two oryzacystatin species are believed to be involved in the regulation of proteolysis caused by different proteinases.     

Change of thiamin-binding protein and thiamin levels during seed maturation and germination in sesame

Thiamin-binding proteins (TBPs) occur in many types of plant seeds. The biochemical and structural properties such as subunit structure and affinity for thiamin of the proteins have been characterized. However, the change of TBP and thiamin during seed maturation and germination is little known. Sesame (Sesamum indicum L.) seeds have unique albumin TBPs, because the other TBPs from plant seeds are generally globulins. In this study, we studied the change of the TBP and thiamin levels in sesame seeds. The protein content and thiamin-binding activity of the seeds increased with seed development after flowering. Immunological analysis using an antibody against the TBP of sesame seeds showed that the protein was accumulated in seeds during maturation. The thiamin content of the seeds increased with seed development after flowering. On the other hand, the thiamin-binding activity decreased during seed germination when TBP was degraded. The thiamin content of the seeds decreased during the germination. However, the amount of thiamin phosphate in the seeds during germination was little changed. These results suggested that thiamin was accumulated and stored as a complex with TBP in sesame seeds.     

Structural and expression analysis of prolamin genes in <Emphasis Type="Italic">Oryza sativa</Emphasis> L.

Rice is a staple crop with a small genome of 389 Mb. Rice grain is a source of carbohydrates and proteins and has a relatively low protein content compared to other legume seeds. Glutelin and prolamin are the major storage proteins in rice. Prolamins are characterized by high glutamine and proline content and are generally soluble only in strong alcohol solutions. In this study, we obtained a total of 51,383 expressed sequence tags (ESTs) from Ilpumbyeo (Oryza sativa L.), of which 33,201 and 18,182 clones were obtained from immature and germinating seeds, respectively. From the EST clones, 15,148 unigenes were identified, and 2,590 genes were expressed in both immature and germinating seeds. Gene expression profiling of rice prolamins indicated that prolamin gene expression increased 5 days after heading and reached maximal expression after 30 days, suggesting a high demand for prolamins during seed development and germination. Phylogenetic analysis grouped 33 prolamin genes based on the abundance of sulfur-containing amino acids methionine and cysteine according to the deduced amino acid sequences. Our results enhance the understanding of the regulation of seed maturation and germination, which can result in improved agricultural traits for the seed industry.     

Better performance of germination in hyperosmotic solutions in conspecific weedy rice than cultivated rice

Weeds and crops that grow together often confront similar types of environmental stress, especially drought stress. Weedy rice (Oryza sativa f. spontanea) and cultivated rice (O. sativa L.) provide a unique pair consisting of a weed and a conspecific model crop that can be used to study the drought tolerance of plants across a large distributional range. The investigation on weedy rice's damage to paddy fields showed that it was more serious in dry direct seeding than water direct seeding. Compared with water direct seeding, the seeds of cultivated rice and weedy rice in dry direct seeding will absorb water and germinate under the condition of insufficient soil moisture. Our hypothesis is that weedy rice seeds have evolved stronger germination ability than coexisting cultivated rice under water stress, so that they can obtain more growth space in the early stage in dry direct seeding and thus obtain higher fitness. Seeds of weedy rice populations and coexisting rice cultivars were collected from 61 sites across China and were germinated with 20% polyethylene glycol‐6000 to simulate drought stress. Two drought response indices, which assessed germination rate and germination index, plus one germination stress tolerance index, indicated significantly greater drought tolerance in weedy rice populations than in coexisting rice cultivars (P < 0.01). Drought tolerance for the three indexes were indica weedy rice > indica rice cultivars, japonica weedy rice > japonica rice cultivars, and indica weedy rice > japonica rice cultivars. These results indicate that weedy rice populations show stronger drought stress tolerance than coexisting rice cultivars at various sites, specifically during the seed germination period. Furthermore, Pearson's correlation found that drought response of weedy rice populations and coexisting rice cultivars were significantly different with these environmental factors: latitude, altitude, annual mean precipitation, mean annual temperature, mean precipitation in the sowing month, mean temperature in the sowing month, and sowing methods. Weedy rice shows different patterns of drought tolerance variation across geographical (latitude and altitude) and environmental (precipitation) gradients compared to coexisting rice cultivars. This study suggests that weedy rice might have evolved new drought tolerance and could provide a useful source of genetic resources for improving drought tolerance of crop cultivars and breeding direct seeded cultivars to reduce the usage of seeds in direct seeding.     

Changes in Level and Activity of Phospholipid Transfer Protein during Maturation and Germination of Maize Seeds

The variations of the amounts of phospholipid transfer proteins (PLTP), determined by ELISA and immunoblotting methods, were followed during the maturation and germination of maize (Zea mays L.) seeds. Changes of the amounts of PLTP occur during seed maturation. The levels of PLTP, low in the first 3 weeks after fecondation, strongly raised 3 to 5 weeks after, then reached and maintained a high value (10% of total soluble proteins) during the last steps of maturation. These variations, determined by ELISA, are in accordance with the observations made by immunoblotting. Changes in phospholipid transfer activity were also found when protein extracts prepared from seeds at different stages of maturation were assayed for transfer activity. The levels of PLTP were also determined during the germination of maize seeds and the early growth of the plantlets, both in the endosperm and the aerial parts. While no major change was observed in the endosperm, a high increase in PLTP level was found in the aerial part of the plantlet, both by ELISA and immunoblotting. An enhancement of the phospholipid transfer activity was parallely observed in the protein extracts of plantlets at various stages of germination. These results are consistent with an in vivo correlation between the synthesis of phospholipid transfer protein, observed during the maturation and germination of maize seeds, and the biogenesis of membranes which involves intracellular movements of phospholipids.     

Differences in the Effects of Dehusking during Formation of Seeds on the Germination of Seeds of Indica and Japonica Rice (Oryza sativaL.)

Differences in germination of intact and dehusked seeds of two japonica and two indica cultivars of rice (Oryza sativaL.) were examined during the development and maturation of seeds both under high-temperature (30/23°C) and low-temperature (20/13°C) regimes and a 14-h photoperiod in growth cabinets. As described previously for seeds grown in the paddy fields, germination of freshly harvested japonica rice seeds that developed and matured in growth cabinets was inhibited by dehusking. We observed a roughly consistent triphasic pattern with respect to the germination of intact and dehusked seeds during the development and maturation of seeds of indica and japonica rice. The triphasic pattern consisted of: (a) an initial phase, during which germination was stimulated by dehusking both in indica and japonica rice; (b) a second phase during which almost no dehusked seeds of indica or japonica rice germinated; and (c) a third phase during which intrinsic differences between indica and japonica rice were observed, with dehusking stimulating germination of indica rice but inhibiting that of japonica. Temperature regimes did not affect this triphasic pattern, but the time from the day of anthesis to mass maturity was affected by temperature regimes, and the effect was more pronounced in indica than in japonica rice.     

Characterization of isoperoxidase activity in Arachis hypogaea cultivars

Isoperoxidases (both anodic and cathodic) from individual seeds of several peanut cultivars (Arachis hypogaea), after ammonium sulfate precipitation of the major storage protein, arachin, from the extracts were characterized by polyacrylamide gel electrophoresis with respect to seed development, maturity and germination and the geographic areas where grown. All cultivars had a major cathodic isozyme near the origin of the gels. Much anodic intra- and inter-varietal isozyme polymorphism was observed in mature seeds collected from different geographic areas. These polymorphic isozymes were consistently present during the earlier stages of seed development (the activities decreased quantitatively and became variable during the later stages of maturation), and most were observed in each peanut upon germination, the latter showing quantitative increases in activity.     

Changes in Seed Constituents During Germination and Seedling Growth of Precociously Matured Soybean Seeds(Glycine max)

During 7 d of precocious maturation of soybean seed (Glycinemax), the starch content declined and soluble sugar levels increasedin patterns similar to natural seed dehydration and maturation.Total seed protein content and total seed dry weight increasedwhereas oil content remained relatively unchanged. Overall,the proportions of the constituents in precociously maturedseeds were comparable to naturally mature seeds. Precociouslymatured soybean seeds showed much the same germination and seedlinggrowth frequency patterns as naturally matured seeds. Duringgermination and seedling growth of precociously matured seeds,starch, soluble sugar, protein and oil levels followed patternssimilar to naturally mature, germinating seeds and seedlings.Therefore, precocious maturation may be used as a model systemto investigate the control of the physiological and biochemicalevents occurring during seed maturation which lead to germinationand subsequently, seedling growth. Glycine max (L.) Merr., soybean, cotyledons, maturation, germination/seedling growth     

Polymorphism in rice amylases at an early stage of seed germination

A polymorphism in rice amylases at an early stage of seed germination is analyzed by zymogram. In non-glutinous cultivars of rice, alpha-amylase isozymes are mainly confirmed in germinating seeds. However, in glutinous cultivars, beta-amylase isozymes, which are not confirmed in nonglutinous cultivars, make up the major part of the total amylase activity and the expression of alpha-amylases are repressed.     

Correlation between seed longevity and RNA integrity in the embryos of rice seeds

The mature embryos of rice seeds contain translatable mRNAs required for the initial phase of germination. To clarify the relationship between seed longevity and RNA integrity in embryos, germinability and stability of embryonic RNAs were analyzed using the seeds of japonica rice cultivars subjected to controlled deterioration treatment (CDT) or long periods of storage. Degradation of RNA from embryos of a japonica rice cultivar “Nipponbare” was induced by CDT before the decline of the germination rate and we observed a positive relationship between seed germinability and integrity of embryonic RNAs. Moreover, this relationship was confirmed in the experiments using aged seeds from the “Nipponbare”, “Sasanishiki” and “Koshihikari” rice cultivars. In addition, the RNA integrity number (RIN) values, calculated using electrophoresis data and Agilent Bioanalyzer software, had a positive correlation with germinability (R²=0.75). Therefore, the stability of embryonic RNAs required for germination is involved in maintaining seed longevity over time and RIN values can serve as a quantitative indicator to evaluate germinability in rice.