Purification of two forms of starch branching enzyme (Q-enzyme) from developing rice endosperm

Two isoforms of starch branching enzyme (Q-enzyme), QEI and QEII, have been purified to honlogeneity from developing rice endosperm. QEI and QEII, with molecular weights of about 80 and 85 kDa, respectively, could be fully separated by anion-exchange or hydrophobic chromatography. The peptide maps obtained after V₈ proteinase digestion were quite different between the two enzymes. Antibodies prepared against QEI showed no immunological cross-reaction with the QEII protein in Western blot experiments, and anti-QEII serum did not react with the QEI protein. The data indicate that QEI and QEII are distinct proteins encoded by different genes in rice plants.     

Multiple forms of ADPglucose pyrophosphorylase of rice endosperm

ADPglucose pyrophosphorylase from developing rice ( Oryza sativa ) endosperm was purified. The final preparation yielded 6 major protein spots as separated by two-dimensional polyacrylamide electrophoresis. All 6 polypeptides had similar molecular weights of ca 50 kDa and cross-reacted with polyclonal antibodies raised against two main protein bands among them. The results suggest that the rice endosperm ADPglucose pyrophorsphorylase is tetrameric and composed of multiple subunits with similar amino acid structure.     

Absence of amylose in sweet potato [Ipomoea batatas (L.) Lam.] following the introduction of granule-bound starch synthase I cDNA

The full-length sense cDNA for sweet potato granule-bound starch synthase I (GBSSI) driven by the CaMV 35S promoter was introduced into the sweet potato by Agrobacterium tumefaciens-mediated transformation. Out of the 26 transgenic plants obtained, one plant showed the absence of amylose in the tuberous root as determined by the iodine colorimetric method. Electrophoresis analysis failed to detect the GBSSI protein, suggesting that gene silencing of the GBSSI gene occurred in the transgenic sweet potato plant. These results demonstrate that starch composition in the tuberous root of sweet potato can be altered by genetic transformation.     

Induction mechanism of 3-hydroxy-3-methylglutaryl-CoA reductase in potato tuber and sweet potato root tissues

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC1.1.1.34), the key enzyme in isoprenoid biosynthesis, was purified from microsomes of potato tuber tissue, and a polyclonal antibody and two monoclonal antibodies against the purified enzyme were prepared. HMGR protein content was measured by immunotitration and radioimmunoassay using these antibodies. HMGR activity was very low in the fresh tissues of both potato tuber and sweet potato root. The activity in potato tuber was increased by cutting and further by additional fungal infection of the cut tissues. In sweet potato root tissue, the activity was scarcely increased after cutting alone, but was markedly increased by additional fungal infection or chemical treatment. The HMGR protein contents in both fresh potato tuber and sweet potato root tissues were also very low, and increased markedly in response to cutting and fungal infection. From these results, we proposed a hypothesis on the induction mechanism of HMGR after cutting and fungal infection in potato tuber and sweet potato root tissues.     

甘薯叶片超氧化物歧化酶基因克隆及测序

以甘薯（Ｉｐｏｍｏｅａｂａｔａｔａｓ（Ｌ．）Ｐｏｉｒ）叶为材料提取植物总ＲＮＡ,经反转录后,利用多聚酶链式反应技术,扩增并克隆超氧化物歧化酶基因的ｃＤＮＡ,并进行测序分析。该序列全长４８２ｂｐ,其读码框编码１５２个氨基酸,与国外文献报道的甘薯块根ＳＯＤ基因的ｃＤＮＡ序列相比,具有９９％的同源性。     

Organ Specificity of Isoforms of Starch Branching Enzyme (Q-Enzyme) in Rice

The activity and isoenzymes of starch branching enzyme or Q-enzymein the developing endosperm were compared with those in theleaf blade, leaf sheath, culm and root of rice plants. Q-enzymefrom each of these organs could be resolved into two fractions,QE I and QE II, by column chromatography on DEAE cellulose.However, the ratio of the activity of QE I to that of QE IIvaried considerably among the organs. The Q-enzyme from theendosperm was specific for that organ in that the enzyme activity,on the basis of either fresh weight or soluble protein content,was about 100- to 1,000-fold higher than those from the otherorgans. Moreover, in the endosperm, the activity of QE I wasmarkably higher than that of QE II as compared with the relativelevels in other organs. Native polyacrylamide gel electrophoresisfollowed by activity staining revealed that the QE II fractionwas composed of multiple isoforms. The endosperm contained twoisoforms, QE IIa and QE IIb. After electrophoresis on a nativepolyacrylamide gel, QE IIa was detected only in the extractof endosperm, whereas QE IIb was present in extract of all organsexamined. The antiserum raised against QE IIa from the endospermcross-reacted to a considerable extent with QE IIb from thesame organ. However, the antiserum failed to recognize any isoformsof QE II from the other organs. ¹ Present address: National Institute of Sericultural and EntomologicalScience, Tsukuba, Ibaraki, 305 Japan.     

Starch phosphorylase inhibitor from sweet potato

A protein, starch phosphorylase inhibitor, was purified from the root of sweet potato (Ipomoea batatas [L.] Lam. cv Tainon 65). It had a molecular weight of 250,000 and could be composed of five identical subunits. The isoelectric point of the inhibitor was 4.63. It was a noncompetitive inhibitor toward the sweet potato enzyme with a K_i value of 1.3 × 10⁻⁶ molar when glucose-1-P was the variable substrate. Because cross-reacting materials of rabbit antiphosphorylase inhibitor of sweet potato were found in three arbitrarily selected plant materials, viz. potato tuber, spinach leaf, and rice grain, the occurrence of this protein seemed universal in higher plants. By an immunofluorescence technique, the inhibitor was located in the amyloplast and cell wall where phosphorylase was also found. This implies that they may interact in vivo, and the inhibitor may play an unknown regulatory role against the plant enzyme.     

Starch-accumulating Sweet Potato Callus Tissue Devoid of β-Amylase but with Two Starch Phosphorylase Isozymes

By controlling the concentrations of kinetin, auxin, and sucrose in the Murashige–Skoog medium, starch contents in callus culture induced from sweet potato tissues could be manipulated. Activity staining and Western analysis on PAGE plates and activity assays made on starch phosphorylase in the presence and absence of mercuric ions showed that β-amylase is absent in callus cultures regardless of whether their starch content is high or low. This would imply that β-amylase induction in sweet potato calli is not linked to the metabolic control through which the expression of storage function is associated, as proposed by Nakamura et al. [Plant Physiol., 96, 902 (1991)] for sweet potato leaf-petiole cuttings. Analyses of starch phosphorylase in crude extracts suggested the presence of a new starch phosphorylase in tuberous root and callus tissue. This phosphorylase is immunologically different from the tuberous root and leaf enzymes that we studied previously.     

A complex containing both trypsin inhibitor and dehydroascorbate reductase activities isolated from mitochondria of etiolated mung bean (Vigna radiata L. (Wilczek) cv. Tainan no. 5) seedlings

A complex containing trypsin inhibitor (TI) activity was extracted with 0.1 M TRIS buffer (pH 7.9) from trypsin-treated mitochondria of etiolated mung bean seedlings, and further purified with a Superdex 200 FPLC column. This partially purified complex with an M(r) about 820 kDa exhibited additional dehydroascorbate (DHA) reductase activity with specific activities of 0.21, 1.53 and 1.54 mumol ascorbate formed min-1 mg-1 protein at pH 6.0, 6.5 and 7.0, respectively, when glutathione was added. Much lower DHA reductase activity (0.013 and 0.026 mumol ascorbate formed min-1 mg-1 protein at pH 6.5 and 7.0, respectively) was found when glutathione was omitted. The isolated complex gave positive results when it was tested by TI activity staining after SDS-PAGE, and could be recognized by a polyclonal antibody which was raised against 38 kDa sweet potato Kunitz-type TI, one of the root storage proteins of sweet potato. The possible physiological functions of this complex with both TI and DHA reductase activities were discussed.     

Cloning of cDNA for UDP-glucose pyrophosphorylase and the expression of mRNA in rice endosperm

Rice endosperm UDP-glucose pyrophosphorylase (UGPase) cDNA clones were isolated by screening a lambda ZAP II library prepared from poly (A(+)) RNA of japonica rice (cv Sasanishiki) endosperm with a probe of potato UGPase cDNA. One cDNA clone, possessing about 1,700 nucleotides, contained the complete open reading frame of rice UGPase. At the nucleotide-sequence level, the UGPase cDNA of rice endosperm had high homology with the UGPase cDNA of barley endosperm (84%) and potato tuber (71%). The calculated molecular weight (50 kDa) agrees with the value determined by SDS-PAGE (51 kDa). At the amino-acid sequence level, rice UGPase has high homology with the UGPase of barley (92%) and potato (85%). The enzyme contained conserved sequence elements which are thought to be involved in substrate binding and catalytic activity. A Southern-blot analysis indicated that the gene existed as a single copy. Expression of the enzyme in rice endosperm examined by Northern-blot analysis was high at 10-15 days after heading.     

Multiple forms of ADP-glucose pyrophosphorylase from tomato fruit.

ADP-glucose pyrophosphorylase (AGP) was purified from tomato (Lycopersicon esculentum Mill.) fruit to apparent homogeneity. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis the enzyme migrated as two close bands with molecular weights of 50,000 and 51,000. Two-dimensional polyacrylamide gel electrophoresis analysis of the purified enzyme, however, revealed at least five major protein spots that could be distinguished by their slight differences in net charge and molecular weight. Whereas all of the spots were recognized by the antiserum raised against tomato fruit AGP holoenzyme, only three of them reacted strongly with antiserum raised against the potato tuber AGP large subunit, and the other two spots (with lower molecular weights) reacted specifically with antisera raised against spinach leaf AGP holoenzyme and the potato tuber AGP small subunit. The results suggest the existence of at least three isoforms of the AGP large subunit and two isoforms of the small subunit in tomato fruit in vivo. The native molecular mass of the enzyme determined by gel filtration was 220 +/- 10 kD, indicating a tetrameric structure for AGP from tomato fruit. The purified enzyme is very sensitive to 3-phosphoglycerate/inorganic phosphate regulation.     

Lack of fructose-1,6-bisphosphatase in a range of higher plants that store starch.

The aim of this work was to discover whether fructose-1,6-bisphosphatase (FBPase) is present in higher-plant cells that synthesize storage starch. The following were examined: suspension cultures of soybean (Glycine max), tubers of potato (Solanum tuberosum), florets of cauliflower (Brassica oleracea), developing endosperm of maize and of sweet corn (Zea mays), roots of pea (Pisum sativum), and the developing embryos of round and wrinkled varieties of pea. Unfractionated extracts of each tissue readily converted fructose 1,6-bisphosphate to fructose 6-phosphate in assays for both plastidic and cytosolic FBPase. These conversions were not inhibited by 20 microM-fructose 2,6-bisphosphate. Except in extracts of pea embryos and sweet-corn endosperm, treatment with affinity-purified antibodies to pyrophosphate: fructose-6-phosphate 1-phosphotransferase reduced the above fructose 6-phosphate production to the rate found with boiled extracts. The antibody-resistant activity from sweet corn was slight. In immunoblot analyses, antibody to plastidic FBPase did not react positively with any protein in extracts of soybean cells, potato tuber, cauliflower florets, maize endosperm and pea roots. Positive reactions were found for extracts of embryos of both round and wrinkled varieties of peas and endosperm of sweet corn. For pea embryos, but not for sweet-corn endosperm, the Mr of the recognized protein corresponded to that of plastidic FBPase. It is argued that soybean cells, potato tuber, cauliflower florets, maize (var. White Horse Tooth) endosperm and pea roots lack significant activity of plastidic FBPase, but that this enzyme is present in developing embryos of pea. The data for sweet corn (var. Golden Bantam) are not decisive. It is also argued that, where FBPase is absent, carbon for starch synthesis does not enter the amyloplast as triose phosphate.     

Identification of the maize amyloplast stromal 112-kD protein as a plastidic starch phosphorylase

Amyloplast is the site of starch synthesis in the storage tissue of maize (Zea mays). The amyloplast stroma contains an enriched group of proteins when compared with the whole endosperm. Proteins with molecular masses of 76 and 85 kD have been identified as starch synthase I and starch branching enzyme IIb, respectively. A 112-kD protein was isolated from the stromal fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to tryptic digestion and amino acid sequence analysis. Three peptide sequences showed high identity to plastidic forms of starch phosphorylase (SP) from sweet potato, potato, and spinach. SP activity was identified in the amyloplast stromal fraction and was enriched 4-fold when compared with the activity in the whole endosperm fraction. Native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that SP activity was associated with the amyloplast stromal 112-kD protein. In addition, antibodies raised against the potato plastidic SP recognized the amyloplast stromal 112-kD protein. The amyloplast stromal 112-kD SP was expressed in whole endosperm isolated from maize harvested 9 to 24 d after pollination. Results of affinity electrophoresis and enzyme kinetic analyses showed that the amyloplast stromal 112-kD SP preferred amylopectin over glycogen as a substrate in the synthetic reaction. The maize shrunken-4 mutant had reduced SP activity due to a decrease of the amyloplast stromal 112-kD enzyme.     

The Preparation of a Cellulose-like Polymer from 2,3,6-Tri-O (N-pheny1carbamyl)-D-glucopyranose by the Action of Phosphorus Pentoxide in Dimethyl Sulfoxide

Starch phosphorylase was purified from either freshly harvested or stored roots of sweet potato (.Ipomoea batatas (L.) Lam. cv Tain on 65). Both enzyme preparations in their native state showed on polyacrylamide gel electrophoresis a cluster of about six closely located activity bands, which had common antigenic determinants as they were simultaneously probed by monoclonal antibodies. The molecules of enzymes from stored roots were smaller than those from fresh roots. However, the two enzyme preparations had completely fused precipitin lines in double diffusion assays with an antiserum raised against the fresh root preparation. One large subunit and several small ones were found for both enzyme preparations. The small subunits appeared to be the degradation products of the large ones as revealed by peptide mapping and immunoblotting. Immunofluorescence microscopy showed that the enzyme was present in the amyloplasts and cell walls of root storage parenchyma.