Starch Branching Enzyme Activity Research Articles

Activities of key enzymes in sucrose-to-starch conversion in wheat grains subjected to water deficit during grain filling.

This study tested the hypothesis that a controlled water deficit during grain filling of wheat (Triticum aestivum) could accelerate grain-filling rate through regulating the key enzymes involved in Suc-to-starch pathway in the grains. Two high lodging-resistant wheat cultivars were field grown. Well-watered and water-deficit (WD) treatments were imposed from 9 DPA until maturity. The WD promoted the reallocation of prefixed 14C from the stems to grains, shortened the grain-filling period, and increased grain-filling rate or starch accumulation rate (SAR) in the grains. Activities of Suc synthase (SuSase), soluble starch synthase (SSS), and starch branching enzyme (SBE) in the grains were substantially enhanced by WD and positively correlated with the SAR. ADP Glc pyrophosphorylase activity was also enhanced in WD grains initially and correlated with SAR with a smaller coefficient. Activities of granule-bound starch synthase and soluble and insoluble acid invertase in the grains were less affected by WD. Abscisic acid (ABA) content in the grains was remarkably enhanced by WD and very significantly correlated with activities of SuSase, SSS, and SBE. Application of ABA on well-watered plants showed similar results as those by WD. Spraying with fluridone, an ABA synthesis inhibitor, had the opposite effect. The results suggest that increased grain-filling rate is mainly attributed to the enhanced sink activity by regulating key enzymes involved in Suc-to-starch conversion, especially SuSase, SSS, and SBE, in wheat grains when subjected to a mild water deficit during grain filling, and ABA plays a vital role in the regulation of this process.

Protein phosphorylation in amyloplasts regulates starch branching enzyme activity and protein-protein interactions.

Protein phosphorylation in amyloplasts and chloroplasts of Triticum aestivum (wheat) was investigated after the incubation of intact plastids with gamma-(32)P-ATP. Among the soluble phosphoproteins detected in plastids, three forms of starch branching enzyme (SBE) were phosphorylated in amyloplasts (SBEI, SBEIIa, and SBEIIb), and both forms of SBE in chloroplasts (SBEI and SBEIIa) were shown to be phosphorylated after sequencing of the immunoprecipitated (32)P-labeled phosphoproteins using quadrupole-orthogonal acceleration time of flight mass spectrometry. Phosphoamino acid analysis of the phosphorylated SBE forms indicated that the proteins are all phosphorylated on Ser residues. Analysis of starch granule-associated phosphoproteins after incubation of intact amyloplasts with gamma-(32)P-ATP indicated that the granule-associated forms of SBEII and two granule-associated forms of starch synthase (SS) are phosphorylated, including SSIIa. Measurement of SBE activity in amyloplasts and chloroplasts showed that phosphorylation activated SBEIIa (and SBEIIb in amyloplasts), whereas dephosphorylation using alkaline phosphatase reduced the catalytic activity of both enzymes. Phosphorylation and dephosphorylation had no effect on the measurable activity of SBEI in amyloplasts and chloroplasts, and the activities of both granule-bound forms of SBEII in amyloplasts were unaffected by dephosphorylation. Immunoprecipitation experiments using peptide-specific anti-SBE antibodies showed that SBEIIb and starch phosphorylase each coimmunoprecipitated with SBEI in a phosphorylation-dependent manner, suggesting that these enzymes may form protein complexes within the amyloplast in vivo. Conversely, dephosphorylation of immunoprecipitated protein complex led to its disassembly. This article reports direct evidence that enzymes of starch metabolism (amylopectin synthesis) are regulated by protein phosphorylation and indicate a wider role for protein phosphorylation and protein-protein interactions in the control of starch anabolism and catabolism.

Effect of high temperature on fine structure of amylopectin in rice endosperm by reducing the activity of the starch branching enzyme

Rice ( Oryza sativa L.) grain quality is affected by the environmental temperature it experiences. To investigate the physiological molecular mechanisms of the effect of high temperatures on rice grain, a non-waxy indica rice was grown under two temperature conditions, (29/35 °C) and (22/28 °C), during the ripening stage in two phytotrons. The activities and gene expression of key enzymes for the biosynthesis of amylose and amylopectin were examined. The activity and expression levels of soluble endosperm starch synthase I were higher at 29/35 °C than that at 22/28 °C. In contrast, the activities and expression levels of the rice branching enzyme1, the branching enzyme3 and the granule bound starch synthase of the endosperm were lower at 29/35 °C than those at 22/28 °C. These results suggest that the decreased activity of starch branching enzyme reduces the branching frequency of the branches of amylopectin, which results in the increased amount of long chains of amylopectin of endosperm in rice grain at high temperature.

Activities of enzymes involved in sucrose-to-starch metabolism in rice grains subjected to water stress during filling

Slow grain filling of rice ( Oryza sativa L.) is a problem due to the heavy use of nitrogen fertilizer or the high lodging resistance of some cultivars. This study investigated if controlled water deficit during grain filling could enhance sink strength by regulating key enzymes involved and lead to faster grain filling. Two rice cultivars with high lodging resistance and slow grain filling were grown in the field and treated with either normal nitrogen (NN) or high nitrogen (HN) at heading. Well-watered (WW) and water-deficit stressed (WS) treatments were imposed from 9 days after anthesis (DAA) until maturity. Leaf water potentials of both cultivars markedly decreased during the day as a result of WS treatments, but completely recovered by early morning. WS promoted the reallocation of prefixed 14 C from stems to grains, facilitated starch accumulation in grains and increased grain-filling rate although it shortened grain-filling period. In contrast, HN behaved in the opposite way. Sucrose synthase (EC 2.4.1.13) activity was substantially enhanced by water stress, and was positively correlated with starch accumulation rate (SAR) in the grains. Both soluble and insoluble invertase (EC 3.2.1.26) activities were less enhanced by WS and showed no significant correlation with SAR. Starch branching enzyme (BE) (EC 2.4.1.18) and soluble starch synthase (EC 2.4.1.18) activities were also enhanced by the WS, with the former enhanced more than the latter, and were significantly correlated with SAR. Adenine diphosphoglucose pyrophosphorylase (EC 2.7.7.27) activity was little affected by WS. The results suggest that WS-increased remobilization and grain-filling rate were attributed to enhanced sink strength by regulating sucrose synthase and starch BE activities in rice grains when subjected to water stress during the grain-filling period.

Characterization of the Difference of Starch Branching Enzyme Activities in Normal and Low-Amylopectin Maize during Kernel Development

In order to determine the reasons for the differences in structure between starch from a normal and a low-amylopectin maize variety the activities of all the enzymes in the committed pathway of starch synthesis were studied throughout kernel development. Levels of ADP glucose pyrophosphorylase and starch synthase activity were found to be broadly similar between the two varieties but the low-amylopectin starch (LAPS) maize variety showed dramatically reduced starch branching enzyme activity, with an almost total absence of the branching enzyme II isoform. Scanning electron microscopy showed a significant alteration in the morphology of the starch granules of the low-amylopectin maize. The results suggest that the increased amylose and the reduction of high molecular weight amylopectin in the LAPS starch results from the absence of the branching enzyme II isoform. This evidence supports the theory that the different branching enzyme isoforms contribute separately to the synthesis and final structure of amylopectin.

Consequences of antisense RNA inhibition of starch branching enzyme activity on properties of potato starch

Antisense constructs containing cDNAs for potato starch branching enzyme (SBE) were introduced into potato ( Solanum tuberosum L.). A population of transgenic plants were generated in which tuber SBE activity was reduced by between 5 and 98% of control values. No significant differences in amylose content or amylopectin branch length profiles of transgenic tuber starches were observed as a function of tuber SBE activity. Starches obtained from low SBE activity plants showed elevated phosphorous content. 31P n.m.r. analysis showed that this was due to proportionate increases in both 3- and 6-linked starch phosphates. A consistent alteration in starch gelatinisation properties was only observed when the level of SBE activity was reduced to below ~5% of that of control values. Starches from these low SBE activity plants showed increases of up to 5 °C in d.s.c. peak temperature and viscosity onset temperature. Studies on melting of crystallites obtained from linear (1 → 4)-α- d-glucan oligomers suggest that an average difference of double helix length of about one glucose residue might be sufficient to account for the observed differences in gelatinisation properties. We speculate that the modification of gelatinisation properties at low SBE activities is due to a subtle alteration in amylopectin branch patterns resulting in small changes in double helix lengths within granules.

Identification of four starch-branching enzymes in barley endosperm: partial purification of forms I, IIa and IIb.

Developing caryopses of barley (Hardeum vulgare L. ev. Golf) were monitored for starch-branching enzyme activity for 1 month after anthesis. Homogenized caryopses from grains with the highest specific activity were used as starting material for purification of branching enzyme by FPLC chromatography. Several branching enzyme activity fractions were resolved. We have previously described the isolation of a 51/50 kDa protein with starch branching activity from one of these fractions. From three other fractions, we have now partially purified starch branching enzymes corresponding to forms I, IIa and IIb.

Relationship between Starch Accumulation and Activities of the Related Enzymes in the Leaf Sheath as a Temporary Sink Organ in Rice (Oryza sativa)

The starch stored temporarily in the leaf sheath of rice plants is translocated to the grain, contributing to the grain yield. In this paper, the relationship between starch accumulation and the activities of enzymes involved in the starch biosynthetic pathway in the leaf sheaths of rice plants was examined to elucidate the regulation mechanism of starch accumulation in the temporary sink organ. When the starch content was compared between different leaf sheath positions on the main stem, the 14th leaf sheath counted from the bottom, which elongated just before anthesis, showed about a four-fold higher value than the 10th leaf sheath. Among the enzymes involved in carbohydrate metabolism, the activity of starch branching enzyme (BE, EC2.4.1.18) was greatly higher in the 14th leaf sheath than in the 10th leaf sheath, while that of ADPglucose pyrophosphorylase (EC2.7.7.27) was similar between the two leaf sheaths. The starch content increased rapidly in the period around anthesis in the 12th and 14th leaf sheaths, but did not in the 10th and 11th leaf sheath. In the higher leaf sheath position, the activity of BE changed with noticeably similar trend to the starch content. Soluble starch synthase (SSS, EC2.4.1.21), granule-bound starch synthase (GBSS, EC2.4.1.21) and plastidial fructose- 1,6-bisphosphatase (FBPase, EC3.1.3.11) were also significantly correlated with the starch content. These results suggest that BE is involved in regulation of starch metabolism, possibly in collaboration with other enzymes such as SSS, GBSS and plastidial FBPase in temporary sink organs like the leaf sheath.

Changes in structure of starch and enzyme activities affected by sugary mutations in developing rice endosperm. Possible role of starch debranching enzyme (R‐enzyme) in amylopectin biosynthesis

In maturing endosperms of a variety of sugary mutants of rice, phytoglycogen‐like polysaccharides with highly branched a‐glucans were accumulated instead of amylopectin. while the amylose content greatly decreased. Measurement of activities per endosperm of the 10 major enzymes involved in starch and sucrose metabolism revealed that the activity of starch debranching enzyme (R‐enzyme) was specifically reduced in the sugary mutants. The activity of starch branching enzyme I (Q‐enzyme I) was also significantly decreased, but less so than the R‐enzyme, in the mutants, suggesting some coordination of the expression of the genes coding for R‐enzyme and Q‐enzyme I. Western blot analysis showed that the sugary mutations of rice resulted in a decrease in the amount of R‐enzyme protein, but not in major modification of the enzyme. These findings strongly suggest that R‐enzyme plays a critical role in determining the amylopectin fine structure, since at the extremely low level of R‐enzyme activity as compared with Q‐enzyme activity, as found in sugary mutants, the rice endosperm produced phytoglycogen. We hypothesize that balance of activities or interaction between Q‐enzyme and R‐enzyme may be responsible for the fine structure of a‐polyglucans in plant tissues.

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.

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

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

The impact of decreased activity of starch-branching enzyme on photosynthetic starch synthesis in leaves of wrinkled-seeded peas

The effect of a reduction of the activity of starch-branching enzyme (1,4-α-(D)-glucan, 1,4-α-(D)-glucan-6-glycosyl transferase; EC 2.4.1.18) on photosynthetic starch synthesis and photosynthate partitioning has been studied in leaves of pea (Pisum sativum L.). Leaves of wrinkled-seeded peas, recessive at the rugosus locus (rr), contained lower activity of branching enzyme than leaves of near-isogenic round-seeded peas, dominant at the rugosus locus (RR). Western blots showed that one isoform of the enzyme is absent from rr leaves, corresponding to the isoform that is absent from rr embryos. RR and rr leaves had identical rates of starch synthesis and photosynthesis at low irradiances. At high irradiances the rate of starch synthesis was decreased by up to 40% in rr relative to RR leaves. There was no corresponding increase of sucrose synthesis in rr leaves; instead, the rate of photosynthesis was decreased. This inhibition of photosynthesis was more marked at low than at high temperatures and was accompanied by increased oscillatory behaviour, rr leaves contained higher levels of ADP glucose and glycerate 3-phosphate than RR leaves in low and high light. The contribution of these results to our understanding of the distribution of control in the pathways of starch and sucrose synthesis is discussed.

Major differences in isoforms of starch-branching enzyme between developing embryos of round- and wrinkled-seeded peas (Pisum sativum L.)

In order to determine whether round-and wrinkled-seeded peas (Pisum sativum L.) differ in the activity and properties of starch-branching enzyme (1,4-α-D-glucan, 1,4-α-D-glucan-6-glycosyl transferase; EC 2.4.1.18) in their developing embryos, essentially isogenic lines of peas, differing only at the r (rugosus) locus that confers the round (RR, Rr) or wrinkled (rr) phenotype, were studied. Activity of the enzyme rises rapidly from an early stage of development in embryos of round peas, but only at later stages in embryos of wrinkled peas. The purified enzyme from mature embryos of round peas can be resolved into two isoforms that differ in molecular weight and in their ability to branch amylose. The purified enzyme from mature embryos of wrinkled peas is a single protein with the same molecular weight and branching properties as one of the isoforms from embryos of round peas. The difference in activity of starch-branching enzyme between embryos of round and wrinkled peas is likely to be due to the absence from embryos of wrinkled peas of one of the isoforms occurring in embryos of round peas.

Some properties of starch branching enzyme from indica rice endosperm ( Oryza sativa L.)

Starch branching enzyme (α-1,4-glucan: α-1,4-glucan-6-glycosyl transferase; EC 2.4.1.18) catalyzes the formation of the α-1,6-bond in branched starch molecules such as amylopectin. Some characteristics of starch branching enzyme in rice endosperm ( Oryza sativa L.) were determined because of the importance of starch structure for rice quality. Two or three peaks of starch branching enzyme activity were resolved by anion-exchange chromatography of extracts from high amylose rice. The properties of rice starch branching enzyme were similar to those found for the enzyme from other plant sources except for a much lower molecular weight. Rice branching enzyme had an apparent molecular weight of 40 000 as estimated by gel permeation chromatography. Multiple forms of starch branching enzyme could also be resolved in milled rice, suggesting that relationships between starch quality and characteristics of starch branching enzyme could be examined in the mature grain after harvest.