Rice α-glucosidase isozymes and isoforms showing different starch granules-binding and -degrading ability

Insoluble starch granules stored in plant seeds have generally been considered to be degraded effectively by the combination of amylolytic enzymes following initial attack by de novo synthesized α-amylase at germination. We have shown that rice (Oryza sativa L., var Nipponbare) α-glucosidase isozymes (ONG1, ONG2, and ONG3) are also capable of binding to and degrading starch granules directly, indicating the direct liberation of glucose from starch granules by α-glucosidase at germination. ONG1 and ONG2 are encoded in a distinct locus of the rice genome, while ONG2 and ONG3 are generated by alternative splicing. Interestingly, each of the α-glucosidase isozymes showed different action toward starch granules. In addition, two ONG2 isoforms were found to be produced by post-translational proteolysis. The proteolysis induced changes in binding to and degradation of starch granules.     

Characterization of rice α-amylase isozymes expressed by Saccharomyces cerevisiae

Two rice -amylase isozymes, AmylA and Amy3D, were produced by secretion from genetically engineered strains of Saccharomyces cerevisiae. They have distinct differences in enzymatic characteristics that can be related to the physiology of the germinating rice seed. The rice isozymes were purified with immunoaffinity chromatography. The pH optima for amy3D (pH optimum 5.5) and Amy1A (pH optimum 4.2) correlate with the pH of the endosperm tissue at the times in rice seedling development when these isozymes are produced. Amy3D showed 10–14 times higher reactivity to oligosaccharides than Amy1A. Amy1A, on the other hand, showed higher reactivity to soluble starch and starch granules than Amy3D. These results suggest that the isozyme Amy3D, which is expressed at an early stage of germination, produces sugars from soluble starch during the early stage of seed germination and that the isozyme Amy1A works to initiate hydrolysis of the starch granules.     

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds: 8. Immunohistochemical Localization of beta-Amylase

Rabbit antiserum against β-amylase isolated from germinating seeds of rice was produced, and its specific cross-reactivity with β-amylase was confirmed by means of Ouchterlony double immunodiffusion and immunoelectrophoresis procedures. The cellular localization of β-amylase was studied by indirect fluorescence microscopy of thin sectioned germinating rice seed specimens (1-day stage) which had been fixed and treated with purified rabbit anti-β-amylase immunoglobulin G followed by conjugation with fluorescein isothiocyanate-labeled goat antirabbit immunoglobulin G. It has been demonstrated that β-amylase is uniformly associated with the periphery of starch granules in the starchy endosperm cells. The finding is discussed in relation to the general notion concerning the presence of the latent form of β-amylase bound to protein bodies in cereal seeds.     

刺五加种子结构,后熟作用及其细胞化学研究

刺五加种子为扁肾形,种皮由一层细胞构成。种子脱落时,胚处于心形胚期,胚周围的胚乳细胞解体形成囊腔包囊胚,胚细胞原生质浓厚,胚乳细胞中贮存大量蛋白质和脂类,但两者均未见贮存多糖,有萌发潜能的种子只占全部种子的１２．８０％,种子经变温层积处理６个月即可完成后熟过程,其细胞化学特点是：处理１．５个月时胚细胞中开始积累多糖颗粒,至４个月时达最大量并一直保持至种子萌发。试验地种植条件下饱满种子经１８－２０个     

Asparagine Enhances Starch Accumulation in Developing and Germinating Lupin Seeds

Regulation of starch accumulation in yellow (Lupinus luteus L.), white (L. albus L.), and Andean lupin (L. mutabilis Sweet) developing and germinating seeds was investigated. Research was conducted on cotyledons isolated from developing seeds as well as on organs of germinating seeds, that is, isolated embryo axes, excised cotyledons, and seedling axes and cotyledons. All organs were cultured in vitro for 96 h in different carbon (60 mM sucrose) and nitrogen (35 mM asparagine or 35 mM nitrate) conditions. Ultrastructure observation showed one common pattern of changes in the number and size of starch granules caused by sucrose, asparagine, and nitrate in both developing and germinating seeds. Sucrose increased the number and size of starch granules. Asparagine additionally increased starch accumulation (irrespective of sucrose nutrition) but nitrate had no effect on starch accumulation. Asparagine treatment resulted in a significant decrease in soluble sugar level in all organs of germinating lupin seeds of the three species investigated. The above-mentioned changes were most clearly visible in white lupin organs. In white lupin, starch granules were visible even in cells of sucrose-starved isolated embryo axes where advanced autophagy occurs. The importance of asparagine-increased starch content in the creation of a strong source–sink gradient in developing and germinating lupin seeds is discussed.     

Localization and Activity of a Carboxypeptidase in Germinating Seeds of Scots Pine, Pinus sylvestris

Extracts prepared from the endosperm of germinating seeds of Scots pine, Pinus sylvestris L., hydrolysed two typical carboxypeptidase substrates, Z-Phe-Ala and Z-Phe-Phe, with pH optima at 4.2 and 5.0. The activities were completely destroyed by diisopropylfluorophosphate. Identical heat inactivation curves and elution patterns in gel chromatography on Sephadex G-200 suggest that the two activities are due to a single enzyme. In resting seeds very low carboxypeptidase activity was present in both the endosperm and the embryo. During germination on agar gel at 20°C in the dark the activities, expressed as enzyme units per seed, increased in the seedling and particularly in the endosperm up to the stage when the reserves of the endosperm were completely depleted. The time of rapid increase of activity in the endosperm did not coincide with the onset of storage protein mobilization. On the contrary, the major part of the increase occurred after the bulk of endosperm nitrogen had already been transferred to the seedling. The results suggest that the carboxypeptidase does not play a major role in the mobilization of storage proteins in germinating pine seeds. On the other hand, it probably functions in the proteolytic reactions associated with the senescence of the reserve-depleted endosperm.     

Analysis of Proteome Profile in Germinating Soybean Seed,and Its Comparison with Rice Showing the Styles of Reserves Mobilization in Different Crops

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.     

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds II. Scutellum as the Site of Sucrose Synthesis

In a close parallel to the developmental pattern of α-amylase activity, a rapid increase of maltase activity occurred in the endosperm tissue of germinating rice seeds after about 4 days of the seed imbibition. The overall pattern of the 2 hydrolytic enzyme activities strongly suggest that amylolytic breakdown is the major metabolic route of starch utilization in the germinating rice seeds. Results of the chemical analyses of sugar constituents as well as the measurements of sucrose synthetase activity show that the scutellum is the site of sucrose synthesis in the germinating rice seeds. It is thus supported that glucose derived from the reserve starch in endosperm is transported to scutellum, where it is converted to sucrose. Sucrose is further mobilized to the growing tissues, shoots and roots.     

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds: III. alpha-Amylase Isozymes

The formation of amylase isozymes in germinating rice (Oryza sativa) seeds was studied by isoelectric focusing on polyacrylamide gel disc electrophoresis. Time sequence comparisons of the amylase zymogram were made between extracts from gibberellic acid-treated embryoless and embryo-attached half-endosperm of rice seeds. In both cases, 4 major and 9 to 10 minor isozyme bands were detectable at the maximal stage of the enzyme induction. However, in the embryo-attached half-seeds, bands started to diminish after the 5th day of incubation, in agreement with the results of time sequence analyses of enzyme activities. Nearly identical patterns of amylase isozyme bands on a polyacrylamide gel disc electrophoresis in combination with isoelectric focusing indicate the intrinsic role of gibberellic acid in the starch breakdown in germinating rice seeds. We tentatively assign the newly synthesized enzymes to be α-amylases based on experimental results concerning the lability of the preparation on a prolonged treatment at pH 3.3 and the stability on heat treatment for 15 minutes at 70 C.     

Identification,cDNA cloning and possible roles of seed-specific rice asparaginyl endopeptidase,REP-2

We previously showed that two major cysteine endopeptidases, REP-1 and REP-2, were present in germinated rice ( Oryza sativa L.) seeds, and that REP-1 was the enzyme that digests seed storage proteins. The present study shows that REP-2 is an asparaginyl endopeptidase that acts as an activator of REP-1, and we separated it into two forms, REP-2alpha (39 kDa) and REP-2beta (40 kDa), using ion-exchange chromatography and gel filtration chromatography. Although analysis of the amino terminals revealed that 10 amino acids of both forms were identical, their isoelectric points were different. SDS-PAGE/immunoblot analysis using an antiserum raised against legumain, an asparaginyl endopeptidase from jack bean, indicated that both forms were present in maturing and germinating rice seeds, and that their amounts transiently decreased in dry seeds. Northern blot analysis indicated that REP-2 mRNA was expressed in both maturing and germinating seeds. In germinating seeds, the mRNA was detected in aleurone layers but not in shoot and root tissues. Incubation of the de-embryonated seeds in 10(-6) M gibberellic acid induced the production of large amounts of REP-1, whereas REP-2beta levels declined rapidly. Southern blot analysis showed that there is one gene for REP-2 in the genome, indicating that both REP-2 enzymes are generated from a single gene. The structure of the gene was similar to that of beta-VPE and gamma-VPE isolated from Arabidopsis thaliana.     

Enzymic mechanism of starch breakdown in germinating rice seeds I. An analytical study

Time-sequence analyses of carbohydrate breakdown in germinating rice seeds shows that a rapid breakdown of starch reserve in endosperm starts after about 4 days of germination. Although the major soluble carbohydrate in the dry seed is sucrose, a marked increase in the production of glucose and maltooligosaccharides accompanies the breakdown of starch. Maltotriose was found to constitute the greatest portion of the oligosaccharides throughout the germination stage. α-Amylase activities were found to parallel the pattern of starch breakdown. Assays for phosphorylase activity showed that this enzyme may account for much smaller amounts of starch breakdown per grain, as compared to the amounts hydrolyzed by α-amylase. There was a transient decline in the content of sucrose in the initial 4 days of seed germination, followed by the gradual increase in later germination stages. During the entire germination stage, sucrose synthetase activity was not detected in the endosperm, although appreciable enzyme activity was present in the growing shoot tissues as well as in the frozen rice seeds harvested at the mid-milky stage. We propose the predominant formation of glucose from starch reserves in the endosperm by the action of α-amylase and accompanying hydrolytic enzyme(s) and that this sugar is eventually mobilized to the growing tissues, shoots or roots.     

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.     

The role of two isoenzymes of alpha-amylase of Araucaria araucana (Araucariaceae) on the digestion of starch granules during germination

Starch is the principal reserve of Araucaria araucana seeds, and it is hydrolysed during germination mainly by alpha-amylase. There are several alpha-amylase isoenzymes whose patterns change in the embryo and in the megagametophyte from the one observed in quiescent seeds (T(0)) to a different one observed 90 h after imbibition (T(90)). The objective of this research was to study the roles of two purified alpha-amylase isoenzymes by in vitro digestion of starch granules extracted from the tissues at two times of imbibition: one is abundant in quiescent seeds and the other is abundant after 90 h of imbibition. The isoenzymes digested the starch granules of their own stage of germination better, since the isoenzyme T(0) digested starch granules mainly from quiescent seeds, while the isoenzyme T(90) digested starch mainly at 90 h of imbibition. The sizes of the starch granule and the tissue from which these granules originated make a difference to digestion by the isoenzymes. Embryonic isoenzyme T(0) digested large embryonic starch granules better than small and medium-sized granules, and better than those isolated from megagametophytes. Similarly isoenzyme T(90) digested small embryonic starch granules better than medium-sized and large granules, and better than those isolated from megagametophytes. However, a mixture of partially purified megagametophytic isoenzymes T(0) and T(90) digested the megagametophytic granules better than those isolated from embryos. Studies of in vitro sequential digestion of starch granules with these isoenzymes corroborated their specificity. The isoenzyme T(90) digested starch granules previously digested by the isoenzyme T(0). This suggests that in vivo these two isoenzymes may act sequentially in starch granule digestion.     

Pullulanase from rice endosperm

Pullulanase (EC 3.2.1.41) in non-germinating seeds was compared with that in germinating seeds. Moreover, pullulanase from the endosperm of rice (Oryza sativa L., cv. Hinohikari) seeds was isolated and its properties investigated. The pI value of pullulanase from seeds after 8 days of germination was almost equal to that from non-germinating seeds, which shows that these two enzymes are the same protein. Therefore, the same pullulanase may play roles in both starch synthesis during ripening and starch degradation during germination in rice seeds. The enzyme was isolated by a procedure that included ammonium sulfate fractionation, DEAE-cellulofine column chromatography, preparative isoelectric focusing, and preparative disc gel electrophoresis. The enzyme was homogeneous by SDS/PAGE. The molecular weight of the enzyme was estimated to be 100 000 based on its mobility on SDS/PAGE and 105 000 based on gel filtration with TSKgel super SW 3000, which showed that it was composed of a single unit. The isoelectric point of the enzyme was 4.7. The enzyme was strongly inhibited by beta-cyclodextrin. The enzyme was not activated by thiol reagents such as dithiothreitol, 2-mercaptoethanol or glutathione. The enzyme most preferably hydrolyzed pullulan and liberated only maltotriose. The pullulan hydrolysis was strongly inhibited by the substrate at a concentration higher than 0.1%. The degree of inhibition increased with an increase in the concentration of pullulan. However, the enzyme hydrolyzed amylopectin, soluble starch and beta-limit dextrin more rapidly as their concentrations increased. The enzyme exhibited alpha-glucosyltransfer activity and produced an alpha-1,6-linked compound of two maltotriose molecules from pullulan.     

Molecular cloning and gibberellin-induced expression of multiple cysteine proteinases of rice seeds (oryzains)

We screened a cDNA library of germinating rice seeds with a cDNA for aleurain (cysteine proteinase from barley) and obtained three distinct types of cDNA clones encoding three species of cysteine proteinases (oryzains alpha, beta, and gamma). The deduced amino acid sequences are distinct in part, but, on the whole, are similar to one another. The three sequences all contain the catalytic triad Cys25-His159-Asn175 (papain numbering). The three oryzains are similar to one another and also to other known cysteine proteinases such as papain and cathepsin H with respect to the sequences around the active site residues and the COOH-terminal Trp-rich region. Amino acid sequence comparison revealed that oryzains alpha and beta are similar not only to each other (70% similarity) but also to actinidin and papain (about 50%), whereas oryzain gamma was rather similar to aleurain (85%) and cathepsin H (60%). Northern blot analysis revealed that the mRNAs for the three oryzains are expressed only in seeds, not in shoots or roots, and show different expression profiles during germination and when the seeds are treated with gibberellic acid. Oryzains alpha and gamma are expressed continuously during germination with a maximum expression 5 days from the start of germination, but are present in neither ripening nor ripened seeds. On the other hand, oryzain beta is expressed not only during germination, but also in ripened seeds before germination. It was noted that the expression of the three oryzain mRNAs is enhanced in different manners by gibberellic acid but is not enhanced by other plant hormones such as auxin. The induction of oryzain beta mRNA is transient, reaching a maximum in 4 h from the addition of giberellic acid and diminishing rapidly thereafter, while the induction of oryzain alpha and gamma mRNAs continues over 5 days. Thus, multiple systems involving cysteine proteinases must be differentially involved in the germination process, probably under hormonal control.     

Increases in 1,5-anhydroglucitol levels in germinating amaranth seeds and in ripening banana

To examine whether 1,5-anhydroglucitol (AG) is derived from starch degradation in plant tissues, we colorimetrically measured AG contents of germinating amaranth seeds and ripening banana pulp. In both cases, as starch degradation proceeded, AG levels were significantly increased, but were 1,700-5,000 times lower than those of total soluble carbohydrates. alpha-1,4-Glucan lyase activity, which is measured by the 1,5-anhydrofructose (AF) liberated from non-reducing glucose residues of starch or glycogen, was too low to be detected in amaranth or banana by the 3,5-dinitrosalicylic acid method. On the other hand, AF reductase, which reduces AF to AG, was detected in germinating amaranth seeds and banana pulp. Thus, the increases in AG levels are conceived to be derived from starch breakdown, although further investigation is needed to answer whether the starch degradation pathway via alpha-1,4-glucan lyase/AF reductase exists in plant tissues.     

Increases in 1,5-Anhydroglucitol Levels in Germinating Amaranth Seeds and in Ripening Banana

To examine whether 1,5-anhydroglucitol (AG) is derived from starch degradation in plant tissues, we colorimetrically measured AG contents of germinating amaranth seeds and ripening banana pulp. In both cases, as starch degradation proceeded, AG levels were significantly increased, but were 1,700-5,000 times lower than those of total soluble carbohydrates. α-1,4-Glucan lyase activity, which is measured by the 1,5- anhydrofructose (AF) liberated from non-reducing glucose residues of starch or glycogen, was too low to be detected in amaranth or banana by the 3,5-dinitrosalicylic acid method. On the other hand, AF reductase, which reduces AF to AG, was detected in germinating amaranth seeds and banana pulp. Thus, the increases in AG levels are conceived to be derived from starch breakdown, although further investigation is needed to answer whether the starch degradation pathway via α-1,4-glucan lyase/AF reductase exists in plant tissues.