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.     

Isolation and characterization of some proteolytic enzyme inhibitors in sweet potato (Ipomoea batatas)

Ten trypsin (EC 3.4.21.4) inhibitors have been isolated and purified by gel filtration and ion-exchange chromatography from the tubers of sweet potato (Ipomoea batatas). The molecular weights of the three most active inhibitors were estimated by molecular sieve chromatography and found to be 12 000, 10 000 and 9300, respectively. They showed maximum activity at pH 7.5–8.5 as well as maximum K_i within this pH range. They displayed different trypsin inhibitory activity, and this activity was completely lost on boiling for 40 min.     

Starch synthases and starch branching enzymes from Pisum sativum

Concentrations of ADPglucose:α-1,4-glucan-4-glucosyltransferase (starch synthase) and α-1,4 glucan: α-1,4-glucan-6-glycosyltransferase (branching enzyme) from developing seeds of Pisum sativum were measured. Primed starch synthase activity increased from 8 to 14 days after anthesis and decreased by 50 % at 26 days. Citrate-stimulated starch synthase activity was highest at 10 days after anthesis decreasing to low levels by 22 days. Branching enzyme activity increased from 8 to 18 days after anthesis and decreased little by 26 days. Two fractions of starch synthase were recovered by gradient elution from DEAE-cellulose of extracts from 12- and 18-day-old seeds. The two fractions differed in primer specificity, K_m for ADPG and relative amounts of citrate-stimulated activity. A major and minor fraction of branching enzyme were observed in extracts from both 12- and 18-day-old seeds. Marked differences in the relative abilities ofthe two branching enzyme fractions to stimulate phosphorylase and to branch amylose as well as pH optima were found. Although the content of the starch synthase and branching enzyme fractions varied with seed age, little difference was seen in the properties of chromatographically similar fractions. Therefore, the changes in starch synthase and branching enzyme activity during pea seed development resulted from changes in the concentrations of a few enzyme forms, but not the appearance of different enzyme forms.     

Involvement of nitrogen in storage root growth and related gene expression in sweet potato (Ipomoea batatas)

• Nitrogen (N) could affect storage root growth and development of sweet potato. To manage external N concentration fluctuations, plants have developed a wide range of strategies, such as growth changes and gene expression.
• Five sweet potato cultivars were used to analyse the functions of N in regulating storage root growth. Growth responses and physiological indicators were measured to determine the physiological changes regulated by different N concentrations. Expression profiles of related genes were analysed via microarray hybridization data and qRT‐PCR analysis to reveal the molecular mechanisms of storage root growth regulated by different N concentrations.
• The growth responses and physiological indicators of the five cultivars were changed by N concentration. The root fresh weight of two of the sweet potato cultivars, SS19 and GS87, was higher under low N concentrations compared with the other cultivars. SS19 and GS87 were found to be having greater tolerance to low N concentration. The expression of N metabolism and storage root growth related genes was regulated by N concentration in sweet potato.
• These results reveal that N significantly regulated storage root growth. SS19 and GS87 were more tolerant to low N concentration and produced greater storage root yield (at 30 days). Furthermore, several N response genes were involved in both N metabolism and storage root growth.

     

Cas9‐mediated mutagenesis of potato starch‐branching enzymes generates a range of tuber starch phenotypes

We investigated whether Cas9‐mediated mutagenesis of starch‐branching enzymes (SBEs) in tetraploid potatoes could generate tuber starches with a range of distinct properties. Constructs containing the Cas9 gene and sgRNAs targeting SBE1, SBE2 or both genes were introduced by Agrobacterium‐mediated transformation or by PEG‐mediated delivery into protoplasts. Outcomes included lines with mutations in all or only some of the homoeoalleles of SBE genes and lines in which homoeoalleles carried several different mutations. DNA delivery into protoplasts resulted in mutants with no detectable Cas9 gene, suggesting the absence of foreign DNA. Selected mutants with starch granule abnormalities had reductions in tuber SBE1 and/or SBE2 protein that were broadly in line with expectations from genotype analysis. Strong reduction in both SBE isoforms created an extreme starch phenotype, as reported previously for low‐SBE potato tubers. HPLC‐SEC and ¹H NMR revealed a decrease in short amylopectin chains, an increase in long chains and a large reduction in branching frequency relative to wild‐type starch. Mutants with strong reductions in SBE2 protein alone had near‐normal amylopectin chain‐length distributions and only small reductions in branching frequency. However, starch granule initiation was enormously increased: cells contained many granules of <4 μm and granules with multiple hila. Thus, large reductions in both SBEs reduce amylopectin branching during granule growth, whereas reduction in SBE2 alone primarily affects numbers of starch granule initiations. Our results demonstrate that Cas9‐mediated mutagenesis of SBE genes has the potential to generate new, potentially valuable starch properties without integration of foreign DNA into the genome.     

The structure of starch can be manipulated by changing the expression levels of starch branching enzyme IIb in rice endosperm

When the starch branching enzyme IIb (BEIIb) gene was introduced into a BEIIb-defective mutant, the resulting transgenic rice plants showed a wide range of BEIIb activity and the fine structure of their amylopectins showed considerable variation despite having the two other BE isoforms, BEI and BEIIa, in their endosperm at the same levels as in the wild-type. The properties of the starch granules, such as their gelatinization behaviour, morphology and X-ray diffraction pattern, also changed dramatically depending on the level of BEIIb activity, even when this was either slightly lower or higher than that of the wild-type. The over-expression of BEIIb resulted in the accumulation of excessive branched, water-soluble polysaccharides instead of amylopectin. These results imply that the manipulation of BEIIb activity is an effective strategy for the generation of novel starches for use in foodstuffs and industrial applications.     

Characterization of major proteins in sweet potato tuberous roots

The tuberous roots, but not other organs, of sweet potato contained large quantities of two proteins which accounted for more than 80% of the total proteins. The two proteins, tentatively named sporamins A and B, were monomeric forms with similar M,s (25 000). They were separated from each other by electrophoresis on polyacrylamide gels in a non-denaturing buffer or a buffer containing sodium dodecyl sulphate without being reduced by dithiothreitol. They were very similar to each other with respect to amino acid composition, peptide map and immunological properties. These proteins decreased in preference to other proteins during sprouting. The amino acid sequencing of the amino terminal part of sporamin A suggested that it consists of at least two molecular species with different combinations of a few amino acids.     

Molecular cloning and nucleotide sequence of cDNA for sporamin,the major soluble protein of sweet potato tuberous roots

Summary Sporamin accounts for more than 80% of the total soluble proteins of tuberous roots of sweet potato, but very little, if any, in other tissues of the same plant. In vitro translation of RNA fractions from the tuberous roots in wheat germ extract and subsequent immunoprecipitation with the antibody to sporamin indicated that this protein is synthesized by membrane-bound polysomes as a precursor 4 000 daltons larger than the mature protein. A cDNA expression library was constructed from the total poly(A)⁺ RNA from the tuberous roots by a vector-primer method, and an essentially full-length cDNA clone for the sporamin mRNA was selected by direct immunological screening of the colonies. Northern blot analysis showed that sporamin mRNA is approximately 950 nucleotides in length and is specifically present in tuberous roots and very little, if any, in leaves, petioles and non-tuberous roots. Nucleotide sequence of the cDNA predicts a 37 amino acid extension in the precursor at the amino-terminus of the mature protein.     

Structural differences in full-length cDNAs for two classes of sporamin,the major soluble protein of sweet potato tuberous roots

Summary Sporamin, which accounts for 80% of the total soluble proteins in sweet potato tuberous roots, consists of two polypeptide classes, A and B. The sporamin cDNA clones can also be classified into sporamin A and B subfamilies based on their sequence homologies, with intra-subfamily homologies being much higher than inter-subfamily homologies. The sequence of an essentially full-length cDNA for sporamin B was compared with that for sporamin A. The coding sequences of two cDNAs share 83% sequence homology. The sequences in the 5- and 3-noncoding regions show many deletions in addition to base substitutions. The endpoints of deletions longer than 4 bp match precisely to the endpoints of short direct repeats present in the other sequence, which suggests that these deletions are generated by slipped mispairing during DNA replication. In the 5- and 3-noncoding region of sporamin B cDNA, there are 5 bp direct repeats with sequences complementary to each other. Since most of these repeats are absent in sporamin A cDNA, these structural features may cause a difference in the secondary structure between A and B mRNAs and affect the translational efficiencies or stabilities of the mRNAs. Precursors for both classes of sporamin carry N-terminal extra-sequences which can be separated into a putative signal peptide segment and a segment enriched with basic amino acids. A two-step processing mechanism for the maturation of sporamin is suggested.     

Introduction of sense and antisense cDNA for branching enzyme in the amylose-free potato mutant leads to physico-chemical changes in the starch

One isoform of the branching enzyme (BE; EC 2.4.1.18) of potato (Solarium tuberosum L.) is known and catalyses the formation of α-1,6 bonds in a glucan chain, resulting in the branched starch component amylopectin. Constructs containing the antisense or sense-orientated distal 1.5-kb part of a cDNA for potato BE were used to transform the amylose-free (amf) mutant of potato, the starch of which stains red with iodine. The expression of the endogenous BE gene was inhibited either largely or fully as judged by the decrease or absence of the BE mRNA and protein. This resulted in a low percentage of starch granules with a small blue core and large red outer layer. There was no effect on the amylose content, degree of branching or λ_max of the iodine-stained starch. However, when the physico-chemical properties of the different starch suspensions were assessed, differences were observed, which although small indicated that starch in the transformants was different from that of theamf mutant.     

Soluble starch synthases and starch branching enzymes from developing seeds of sorghum

Soluble starch synthases and branching enzymes have been partially purified from developing sorghum seeds. Two major fractions and one minor fraction of starch synthase were eluted on DEAE-cellulose chromatography. The minor enzyme eluted first and was similar to the early eluting major synthase in citrate-stimulated activity, faster reaction rates with glycogen primers than amylopectin primers, and in K_m for ADP-glucose (0.05 and 0.08 mM, respectively). The starch synthase peak eluted last had no citrate-stimulated activity, was equally active with glycogen and amylopectin primers, and had the highest K_m for ADP-glucose (0.10 mM). Four fractions of branching enzymes were recovered from DEAE-cellulose chromatography. One fraction eluted in the buffer wash; the other three co-eluted with the three starch synthases. All four fractions could branch amylose or amylopectin, and stimulated α-glucan synthesis catalysed by phosphorylase. Electrophoretic separation and activity staining for starch synthase of crude extracts and DEAE-cellulose fractions demonstrated complex banding patterns. The colour of the bands after iodine staining indicated that branching enzyme and starch synthase co-migrated during electrophoresis.     

Cloning and characterization of a cDNA encoding the cytosolic copper/zinc-superoxide dismutase from sweet potato tuberous root

A full-length cDNA clone encoding a putative copper/zinc-superoxide dismutase (SOD) of sweet potato, Ipomoea batatas (L.) Lam. cv Tainong 57, was isolated from a cDNA library constructed in gt10 from tuber root mRNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprises a complete open reading frame coding for 152 amino acid residues. The deduced amino acid sequence showed higher homology (78–86%) with the sequence of the cytosolic SOD than that of the chloroplast SOD from other plant species. The residues required for coordinating copper and zinc are conserved as they are among all reported Cu/Zn-SOD sequences. In addition, it lacks recognizable plastic or mitochondrial targeting sequences. These data suggest that the isolated sweet potato clone encodes a cytosolic Cu/Zn-SOD.     

Demonstration of in vitro starch branching enzyme activity for a 51/50-kDa polypeptide isolated from developing barley (Hordeum vulgare) caryopses

Starch branching enzyme (SBE, EC 2.4.1.18) activity was followed in developing barley ( Hordeum vulgare L. cv. Golf) caryopses during a period of one month after anthesis. Caryopses with the highest specific activity, and corresponding to a fresh weight of around 60 mg per caryopsis, were homogenized and the soluble extract used for branching enzyme purification by FPLC chromatography. Four branching enzyme activity fractions were resolved. From one of these fractions, which exhibited high activity in both the phosphorylation stimulation and amylose branching assays, a branching enzyme preparation containing two related polypeptides of 51 and 50 kDa was obtained. Native polyacrylamide gel electrophoresis and gel filtration showed that the 51/50-kDa polypeptide is monomeric. A combination of phosphorylation stimulation and amylose branching gel assays, SDS-PAGE, and TLC was used to demonstrate the branching activity of the 51/50-kDa polypeptide. The activity was further confirmed by spectroscopic analyses of iodine-glucan complexes. SBEs from four different plant species were compared using the phosphorylation stimulation gel assay.     

Root and shoot initiation by leaf,stem, and storage root explants of sweet potato

Explants from stem, leaf, and storage root tissue of sweet potato (Ipomoea batatas L.) cv. Jewel, were placed on media conaining 0.1, 1.0, and 10 mg/1NAA with 0.1, 1.0, or 10 mg/1BA in a factorial experiment. Some callus formed in every treatment, but the best callus growth was on media containing 1.0 mg/1NAA and 10 mg/1BA. Roots formed over a range of treatments but were most prolific on the medium containing 1.0 mg/1NAA and 0.1 mg/1BA. Some de novo formed roots subsequently produced shoot buds in culture. Shoot formation increased the longer the original explants remained in culture without subculture. Roots could be subcultured indefinitely on agar solidified medium, but shoot regeneration did not occur after two subcultures. Shoot formation was greatest when the roots were subcultured on medium containing 1.0 mg/NAA and 0.1 mg/1BA. The cultivar Caromex followed the same regeneration pathway, but the number of shoots formed was considerably less. Regeneration in both Jewel and Caromex explants was enhanced by light.Paper No. 8292 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of products named, nor criticism of similar ones not mentioned.This work was done as a partial requirement for the M.S. degree at North Carolina State University.     

葛根淀粉合成关键酶活性动态及其与块根产量和淀粉积累的相关性研究

为探讨葛根发育过程中淀粉合成关键酶活性与块根产量和淀粉积累的关系,以初步揭示其内在的生理机制。该研究以‘桂葛1号’粉葛和‘桂葛8号’野葛为材料,采取生理测定法对农艺性状、直链和支链淀粉的含量、淀粉合成关键酶活性等进行测定,并对葛根发育过程中淀粉合成关键酶活性、农艺性状和淀粉含量动态变化的关系进行相关性分析。结果表明：(1)块根发育过程中,两品种葛根腺苷二磷酸葡萄糖焦磷酸化酶(AGPase)、可溶性淀粉合成酶(SSS)、束缚性结合淀粉合成酶(GBSS)和淀粉分支酶(SBE)的活性呈现先增大后降低的单峰曲线变化,与直链淀粉、支链淀粉和总淀粉含量由缓慢增长到快速增长后趋于稳定的变化趋势基本一致,即在块根形成期至膨大期逐渐增长,至膨大后期达到最大,之后迅速下降,至成熟期缓慢下降,并维持在较高水平。(2)‘桂葛8号’的淀粉含量和产量显著高于‘桂葛1号’,其酶活性也均显著高于‘桂葛1号’。(3)葛块根的根长、根粗、单株重、干物质含量、产量表现为“缓慢-快速-稳定”的变化趋势,淀粉含量表现出类似变化。(4)相关性分析结果显示,4个淀粉合成关键酶活性与块根直链淀粉、支链淀粉及总淀粉含量、根长均呈显著或...     

Calcium chloride enhances the antioxidative system of sweet potato (Ipomoea batatas) under flooding stress

Three sweet potato varieties, Taoyuan 2, Simon 1 and Sushu 18, were pretreated with four levels of CaCl₂ (0, 60, 120 and 180 kg ha^?1) weekly for 50 days from planting before being subjected to non‐flooding (control) and flooding conditions. The experiment used a randomised complete block design with a split‐split plot arrangement of treatments. Young, fully expanded leaves from each plant were clipped for measuring enzyme activities and antioxidant contents. The three genotypes exhibited unique abilities and specificities through the antioxidative systems in response to flooding stress. The level of activity of the antioxidative system in sweet potato leaves was related to CaCl₂ pretreatment during flooding. The ascorbate peroxidase, superoxide dismutase, glutathione reductase, reduced ascorbate, total ascorbate, reduced glutathione and malondialdehyde contents of the three sweet potato varieties under flooding stress significantly increased because of pretreatment with 60 and 120 kg ha^?1 of CaCl₂. Moreover, pretreatment with 60 and 120 kg ha^?1 CaCl₂ enhanced the flooding tolerance of all three sweet potato varieties and mitigated the effects of flooding stress. However, pretreatment with 180 kg ha^?1 CaCl₂ markedly decreased some enzyme activities and antioxidant contents under a flooded condition. Calcium most likely played a role in the antioxidative system in the leaves of these three sweet potato varieties under flooding stress, as an optimum amount strengthened their oxidative systems.     

甘薯块根膨大过程中ATP酶活性、ATP和ABA含量的变化

研究选用鲁薯7号和徐薯18号为材料,对甘薯块根膨大速率变化动态及其块根中可溶性碳水化合物含量、ATP含量、ATP酶活性和脱落酸（ABA）含量的变化进行了研究分析。结果表明：（1）块根膨大速率变化动态呈一双峰曲线,第一个高峰出现在栽秧后50－70d,第二个高峰出现在栽秧后120－165d;（2）块根膨大高峰期,块根中可溶性碳水化合物含量较高,ATP含量则较低;（3）块根中ATP酶活性和ABA含量变化动态与块根膨大速率变化动态相似。讨论了ATP酶和ABA在块根膨大过程中的可能作用。