The Rice Alpha-Amylase, Conserved Regulator of Seed Maturation and Germination.
ABSTRACT: Alpha-amylase, the major form of amylase with secondary carbohydrate binding sites, is a crucial enzyme throughout the growth period and life cycle of angiosperm. In rice, alpha-amylase isozymes are critical for the formation of the storage starch granule during seed maturation and motivate the stored starch to nourish the developing seedling during seed germination which will directly affect the plant growth and field yield. Alpha-amylase has not yet been studied intensely to understand its classification, structure, expression trait, and expression regulation in rice and other crops. Among the 10-rice alpha-amylases, most were exclusively expressed in the developing seed embryo and induced in the seed germination process. During rice seed germination, the expression of alpha-amylase genes is known to be regulated negatively by sugar in embryos, however positively by gibberellin (GA) in endosperm through competitively binding to the specific promoter domain; besides, it is also controlled by a series of other abiotic or biotic factors, such as salinity. In this review, we overviewed the research progress of alpha-amylase with focus on seed germination and reflected on how in-depth work might elucidate its regulation and facilitate crop breeding as an efficient biomarker.
Project description:Despite being of vital importance for seed establishment and grain quality, starch degradation remains poorly understood in organs such as cereal or legume seeds. In cereals, starch degradation requires the synergetic action of different isoforms of α-amylases. Ubiquitous overexpression of TaAmy2 resulted in a 2.0-437.6-fold increase of total α-amylase activity in developing leaf and harvested grains. These increases led to dramatic alterations of starch visco-properties and augmentation of soluble carbohydrate levels (mainly sucrose and α-gluco-oligosaccharide) in grain. Interestingly, the overexpression of TaAMY2 led to an absence of dormancy in ripened grain due to abscisic acid (ABA) insensitivity. Using an allosteric α-amylase inhibitor (acarbose), we demonstrated that ABA insensitivity was due to the increased soluble carbohydrate generated by the α-amylase excess. Independent from the TaAMY2 overexpression, inhibition of α-amylase during germination led to the accumulation of soluble α-gluco-oligosaccharides without affecting the first stage of germination. These findings support the hypotheses that (i) endosperm sugar may overcome ABA signalling and promote sprouting, and (ii) α-amylase may not be required for the initial stage of grain germination, an observation that questions the function of the amylolytic enzyme in the starch degradation process during germination.
Project description:Previously, <i>AoamyA,</i> the alpha amylase-encoding gene from <i>Aspergillus oryzae,</i> was heterologously expressed in <i>Monascus ruber</i> CICC41233 to promote starch hydrolysis and increase the production of <i>Monascus</i> pigments. The target of this study is to screen the effective alpha-amylases from <i>M. ruber</i> for starch fast degradation and investigated for <i>Monascus</i> pigments production. The 13 types of predicted alpha-amylases in the <i>M. ruber</i> NRRL1597 genome were divided into four classes based on EC number and into five groups based on the glycoside hydrolase sub-family. The predicted alpha-amylases MrAMY1 (protein ID 440333) and MrAMY2 (protein ID 324551) showed the closest match with AOamyA by phylogenetic analysis. The genes encoding alpha-amylase, <i>Mramy1</i>and <i>Mramy2,</i> were cloned from <i>M. ruber</i> CICC41233. However, the gene sequence of <i>Mramy1</i> from <i>M. ruber</i> CICC41233 differed from that of <i>M.ruber</i> NRRL1597 in the length of the intron sequence. Furthermore, the <i>Mramy1-</i>overexpressed strain <i>M.ruber</i> 440333-6A completely degraded the starch of rice grain in 2 d; in contrast, starch (40.32 mg/mL) remained when rice grain was incubated with the <i>Mramy2-</i>overexpressed strain, <i>M. ruber</i> 324551-D even after 2 d, while 45.43 mg/mL and 10.48 mg/mL starch remained after 2 d and 6 d, respectively, in wild type <i>M. ruber</i> CICC41233. Compared to that of <i>M. ruber</i> CICC41233, the total <i>Monascus</i> pigments and ethanol-soluble pigments in <i>M.ruber</i> 440333-6A increased by 71.69% and 119.33% after 6d, respectively; however, it decreased by 21.40%and 26.58% after 6d, respectively, in <i>M. ruber</i> 324551-D. This study demonstrated that alpha-amylase MrAMY1 was superior to MrAMY2, as it effectively degraded the starch of rice grain and enhanced <i>Monascus</i> pigments production.
Project description:An intracellular alpha-amylase from Streptococcus bovis 148 was purified and characterized. The enzyme was induced by maltose and soluble starch and produced about 80% maltotriose from soluble starch. Maltopentaose was hydrolyzed to maltotriose and maltose and maltohexaose was hydrolyzed mainly to maltotriose by the enzyme. Maltotetraose, maltotriose, and maltose were not hydrolyzed. This intracellular enzyme was considered to be a maltotriose-producing enzyme. The enzymatic characteristics and hydrolysis product from soluble starch were different from those of the extracellular raw-starch-hydrolyzing alpha-amylase of strain 148. The deduced amino acid sequence of the intracellular alpha-amylase was similar to the sequences of the mature forms of extracellular liquefying alpha-amylases from Bacillus strains, although the intracellular alpha-amylase did not contain a signal peptide. No homology between the intracellular and extracellular alpha-amylases of S. bovis 148 was observed.
Project description:Wheat starch degradation requires the synergistic action of different amylolytic enzymes. Our spatio-temporal study of wheat ?-amylases throughout grain development shows that AMY3 is the most abundant isoform compared with the other known ?-amylases. Endosperm-specific over-expression of AMY3 resulted in an increase of total ?-amylase activity in harvested grains. Unexpectedly, increased activity did not have a significant impact on starch content or composition but led to an increase of soluble carbohydrate (mainly sucrose) in dry grain. In AMY3 overexpression lines (A3OE), germination was slightly delayed and triacylglycerol (TAG) content was increased in the endosperm of mature grain. Despite increased AMY3 transcript and protein content throughout grain development, alterations of ?-amylase activity and starch granule degradation were not detected until grain maturation, suggesting a post-translational inhibition of ?-amylase activity in the endosperm during the starch filling period. These findings show unexpected effects of a high level of ?-amylase on grain development and composition, notably in carbon partitioning and TAG accumulation, and suggest the presence of a hitherto unknown regulatory pathway during grain filling.
Project description:alpha-Amylase was purified to apparent homogeneity from normal pancreas and a transplantable pancreatic acinar carcinoma of the rat by affinity chromatography on alpha-glucohydrolase inhibitor (alpha-GHI) bound to aminohexyl-Sepharose 4B. Recovery was 95-100% for both pancreas and tumour alpha-amylases. They were monomeric proteins, with Mr approx. 54000 on SDS/polyacrylamide-gel electrophoresis. Isoelectric focusing of both normal and tumour alpha-amylases resolved each into two major isoenzymes, with pI 8.3 and 8.7. Tumour-derived alpha-amylase contained two additional minor isoenzymes, with pI 7.6 and 6.95 respectively. All four tumour isoenzymes demonstrated amylolytic activity when isoelectric-focused gels were treated with starch and stained with iodine. Two-dimensional electrophoresis, on SDS/10-20%-polyacrylamide-gradient gels after isoelectric focusing, separated each major isoenzyme into doublets of similar Mr values. Pancreatic and tumour-derived alpha-amylases had similar Km and Ki (alpha-GHI) values, but the specific activity of the tumour alpha-amylase was approximately two-thirds that of the normal alpha-amylase. Although amino acid analysis and peptide mapping with the use of CNBr, N-chlorosuccinimide or Staphylococcus aureus V8 proteinase gave comparable profiles for the two alpha-amylases, tryptic-digest fingerprint patterns were different. Antibodies raised against the purified pancreatic alpha-amylase and tumour alpha-amylase respectively showed only one positive band on immunoblotting after gel electrophoresis of crude extracts of rat pancreas and carcinoma, at the same position as that of the purified enzyme. More than 95% of the alpha-amylase activity in the pancreas and in the tumour was absorbed by an excess amount of either antibody, indicating that normal and tumour alpha-amylases are immunologically identical. The presence of additional isoenzymes in the carcinoma, and dissimilarity of tryptic-digest patterns, may reflect an alteration in gene expression or in the post-translational modification of this protein in this heterogeneously differentiated transplantable pancreatic acinar carcinoma.
Project description:Global warming impairs grain filling in rice and reduces starch accumulation in the endosperm, leading to chalky-appearing grains, which damages their market value. We found previously that high temperature-induced expression of starch-lytic ?-amylases during ripening is crucial for grain chalkiness. Because the rice genome carries at least eight functional ?-amylase genes, identification of the ?-amylase(s) that contribute most strongly to the production of chalky grains could accelerate efficient breeding. To identify ?-amylase genes responsible for the production of chalky grains, we characterized the histological expression pattern of eight ?-amylase genes and the influences of their overexpression on grain appearance and carbohydrate components through a series of experiments with transgenic rice plants. The promoter activity of most ?-amylase genes was elevated to various extents at high temperature. Among them, the expression of Amy1A and Amy3C was induced in the internal, especially basal to dorsal, region of developing endosperm, whereas that of Amy3D was confined near the ventral aleurone. These regions coincided with the site of occurrence of chalkiness, which was in clear contrast to conventionally known expression patterns of the enzyme in the scutellum and aleurone during seed germination. Furthermore, overexpression of ?-amylase genes, except for Amy3E, in developing endosperm produced various degrees of chalky grains without heat exposure, whereas that of Amy3E yielded normal translucent grains, as was the case in the vector control, even though Amy3E-overexpressing grains contained enhanced ?-amylase activities. The weight of the chalky grains was decreased due to reduced amounts of starch, and microscopic observation of the chalky part of these grains revealed that their endosperm consisted of loosely packed round starch granules that had numerous pits on their surface, confirming the hydrolysis of the starch reserve by ?-amylases. Moreover, the chalky grains contained increased amounts of soluble sugars including maltooligosaccharides at the expense of starch. The integrated analyses proposed that expression of Amy1A, Amy3C, and Amy3D at the specific regions of the developing endosperm could generate the chalkiness. This finding provides the fundamental knowledge to narrow down the targets for the development of high temperature-tolerant premium rice.
Project description:Seed germination plays important roles in the establishment of seedlings and their subsequent growth; however, seed germination is inhibited by salinity, and the inhibitory mechanism remains elusive. Our results indicate that NaCl treatment inhibits rice seed germination by decreasing the contents of bioactive gibberellins (GAs), such as GA1 and GA4, and that this inhibition can be rescued by exogenous bioactive GA application. To explore the mechanism of bioactive GA deficiency, the effect of NaCl on GA metabolic gene expression was investigated, revealing that expression of both GA biosynthetic genes and GA-inactivated genes was up-regulated by NaCl treatment. These results suggest that NaCl-induced bioactive GA deficiency is caused by up-regulated expression of GA-inactivated genes, and the up-regulated expression of GA biosynthetic genes might be a consequence of negative feedback regulation of the bioactive GA deficiency. Moreover, we provide evidence that NaCl-induced bioactive GA deficiency inhibits rice seed germination by decreasing ?-amylase activity via down-regulation of ?-amylase gene expression. Additionally, exogenous bioactive GA rescues NaCl-inhibited seed germination by enhancing ?-amylase activity. Thus, NaCl treatment reduces bioactive GA content through promotion of bioactive GA inactivation, which in turn inhibits rice seed germination by decreasing ?-amylase activity via down-regulation of ?-amylase gene expression.
Project description:A raw-starch-digesting amylase (RSDA) gene from a Cytophaga sp. was cloned and sequenced. The predicted protein product contained 519 amino acids and had high amino acid identity to alpha-amylases from three Bacillus species. Only one of the Bacillus alpha-amylases has raw-starch-digesting capability, however. The RSDA, expressed in Escherichia coli, had properties similar to those of the enzyme purified from the Cytophaga sp.
Project description:In the gap between two closely linked flagellar gene clusters on the Escherichia coli and Salmonella typhimurium chromosomes (at about 42 to 43 min on the E. coli map), we found an open reading frame whose sequence suggested that it encoded an alpha-amylase; the deduced amino acid sequences in the two species were 87% identical. The strongest similarities to other alpha-amylases were to the excreted liquefying alpha-amylases of bacilli, with > 40% amino acid identity; the N-terminal sequence of the mature bacillar protein (after signal peptide cleavage) aligned with the N-terminal sequence of the E. coli or S. typhimurium protein (without assuming signal peptide cleavage). Minicell experiments identified the product of the E. coli gene as a 56-kDa protein, in agreement with the size predicted from the sequence. The protein was retained by spheroplasts rather than being released with the periplasmic fraction; cells transformed with plasmids containing the gene did not digest extracellular starch unless they were lysed; and the protein, when overproduced, was found in the soluble fraction. We conclude that the protein is cytoplasmic, as predicted by its sequence. The purified protein rapidly digested amylose, starch, amylopectin, and maltodextrins of size G6 or larger; it also digested glycogen, but much more slowly. It was specific for the alpha-anomeric linkage, being unable to digest cellulose. The principal products of starch digestion included maltotriose and maltotetraose as well as maltose, verifying that the protein was an alpha-amylase rather than a beta-amylase. The newly discovered gene has been named amyA. The natural physiological role of the AmyA protein is not yet evident.
Project description:Sequencing upstream of the Streptococcus mutans gene for a CcpA gene homolog, regM, revealed an open reading frame, named amy, with homology to genes encoding alpha-amylases. The deduced amino acid sequence showed a strong similarity (60% amino acid identity) to the intracellular alpha-amylase of Streptococcus bovis and, in common with this enzyme, lacked a signal sequence. Amylase activity was found only in S. mutans cell extracts, with no activity detected in culture supernatants. Inactivation of amy by insertion of an antibiotic resistance marker confirmed that S. mutans has a single alpha-amylase activity. The amylase activity was induced by maltose but not by starch, and no acid was produced from starch. S. mutans can, however, transport limit dextrins and maltooligosaccharides generated by salivary amylase, but inactivation of amy did not affect growth on these substrates or acid production. The amylase digested the glycogen-like intracellular polysaccharide (IPS) purified from S. mutans, but the amy mutant was able to digest and produce acid from IPS; thus, amylase does not appear to be essential for IPS breakdown. However, when grown on excess maltose, the amy mutant produced nearly threefold the amount of IPS produced by the parent strain. The role of Amy has not been established, but Amy appears to be important in the accumulation of IPS in S. mutans grown on maltose.