Isoamylase Activity Research Articles

Biochemical analysis of a new sugary-type rice mutant, Hemisugary1, carrying a novel allele of the sugary-1 gene.

A novel allele of the sugary-1 rice mutant was isolated. The single amino acid change led to isoamylase activity reduction and accumulation of high-molecular-weight phytoglycogen in seeds. A new sugary rice variety with an improved seed appearance has been isolated and designated Hemisugary1. This mutant, which was derived from Japonica-type cultivar Tsugaruroman treated with sodium azide, has about half the isoamylase activity of seeds in the original Tsugaruroman. The mutant also accumulates significant phytoglycogen, albeit approximately 40% of the total phytoglycogen in the existing sugary cultivar Ayunohikari which is defective in its most isoamylase activity. The site of mutation was identified using a re-sequence of the whole genome and a cleaved amplified polymorphic sequence (CAPS) marker. The hemisugary phenotypes of the F2 progeny were entirely consistent with the results of genotyping using the CAPS marker. Segregation analysis of the F2 population showed that the hemisugary phenotype was controlled by a single recessive gene, which was produced by a G → A single nucleotide polymorphism in the sugary-1 gene, resulting in a missense mutation from glycine to aspartic acid at amino acid position 333. Zymogram showed that this amino acid replacement resulted in a decrease in isoamylase activity with a concomitant reduction in the formation of isoamylase complexes. Phytoglycogen molecules from Hemisugary1 seeds were 3.5 times larger and contained more short glucan chains than did Ayunohikari seeds. Our data provide new insights into the relationship between isoamylase structure and phytoglycogen formation.

Implications of Stisa2 catalytic residue restoration through site directed mutagenesis

Abstract Objective Restoration of catalytic activity of Isa2 from Solanum tuberosum (Stisa2) through restoration of conserved catalytic residues by site directed mutagenesis. Methods The six conserved amino acid residues absent in the Stisa2 gene were restored by mutation using the overlap extension PCR and the asymmetrical overlap extension PCR methods. Next, mutant Stisa2 with restored catalytic residues was expressed in E. coli Rosetta 2 under optimized conditions. Evaluation of debranching activity on starch, amylopectin and β-limit dextrin was carried out by measuring the amount of glucose equivalents released using the bicinchoninic acid assay. Results Both qualitative and quantitative analysis showed that the restoration of the conserved residues in the catalytic site did not restore starch debranching activity. Molecular modeling showed greater than expected distances between the catalytic triad in mutant Stisa2. These additional distances are likely to prevent hydrogen bonding which stabilizes the reaction intermediate, and are critical for catalytic activity. Conclusions These results suggest that during evolution, mutations in other highly conserved regions have caused significant changes to the structure and function of the catalytic network. Catalytically inactive Isa2, which is conserved in starch-producing plants, has evolved important non-catalytic roles such as in substrate binding and in regulating isoamylase activity.

Can Serum Pancreatic Amylase and Lipase Levels Be Used as Diagnostic Markers to Distinguish Between Patients With Mucinous Cystic Lesions of the Pancreas, Chronic Pancreatitis, and Pancreatic Ductal Adenocarcinoma?

This study aimed to assess the presence of pancreatic hyperenzymemia in patients with pancreatic cystic lesions as compared to other chronic diseases of the pancreas. Ninety-one patients were studied: 32 had mucinous cystic lesions, 35 had chronic pancreatitis (CP), and 24 had pancreatic ductal adenocarcinoma (PDAC). Surgery was carried out in 10 of the 32 patients with mucinous cystic lesion (7 of them had severe dysplasia), in 5 patients with CP, and in 9 patients with PDAC. Abnormally high serum pancreatic isoamylase activity was present in 11 (34.4%) patients with mucinous cystic lesions, in 14 (40.0%) patients with CP, and none in patients with PDAC (P = 0.002); whereas serum lipase activity was abnormally high in 8 (25.0%) patients with mucinous cystic lesion, in 17 (48.6%) patients with CP, and in 3 (12.5%) patients with PDAC (P = 0.009). In 7 patients with mucinous cystic lesions and histologically confirmed severe dysplasia, abnormally high levels of both serum pancreatic amylase and lipase were present in 3 (42.9%) patients. High serum concentrations of pancreatic amylase and lipase were found in no more than half of the patients with mucinous cystic lesions. High levels of pancreatic enzymes were not associated with a greater risk of malignancy.

Enhanced Production of Recombinant Thermobifida fusca Isoamylase in Escherichia coli MDS42.

Isoamylase, an industrially significant enzyme used primarily in the saccharification of starch, is commonly obtained through recombinant expression in Escherichia coli. To improve the yield of this important enzyme, the isoamylase from Thermobifida fusca was expressed in the reduced-genome E. coli strain MDS42. Expression conditions were initially optimized in shake flasks. The optimal induction temperature was 37°C and IPTG was superior to lactose as an inducer due to the low intracellular β-galactosidase activity in E. coli MDS42 expression system. Then, expression conditions were optimized in a 3.6-L fermenter. In the fermenter, optimal isoamylase expression was obtained when cells were grown at 37°C and expression was induced at mid-log phase using 0.25mM IPTG. The greatest isoamylase activity (22,983.0U/mL of culture) and production (18.8mg/mL) were obtained 24h after induction of expression. These values are the highest ever reported, suggesting that E. coli MDS42 is a suitable host for the production of enzymes for industrial use.

Comparison of amylopectin structure and activities of key starch synthesis enzymes in the grains of rice single-segment substitution lines with different Wx alleles

The Wx gene is the major gene controlling amylose synthesis in rice endosperm. So far, the Wx gene can be mainly classified into five common alleles, wx, Wx t, Wx g1, Wx g2, and Wx g3, according to their amylose phenotypes. Besides, the Wx allelic variation was also found to play an important role in regulating other physicochemical properties of rice starch. However, the specific physiological and starch structural mechanisms are not clear yet. With a set of single-segment substitution lines harboring five different Wx alleles, the genotype differences in amylopectin structure and dynamic changes of activities of key starch synthesis enzymes were investigated. The distinct Wx genotype difference in chain length distribution of amylopectin was confirmed by size exclusion chromatography. There were clear allelic differences in activities of starch synthesis enzymes during grain filling. The Wx allelic variation had the highest impact on the activities of starch branching enzyme, followed by pullulanase, and soluble starch synthase, while no influence on isoamylase activity. Differences in structural features of amylopectin and mean enzyme activities during grain filling period correlated well with most of the variations in starch property traits. These findings indicate that the Wx allelic variation may affect starch structure and physicochemical properties through selectively influencing activities of starch synthesis enzymes in developing rice grains. The present results will contribute to our understanding of the regulation of starch biosynthesis in rice endosperm.

Development of a new selection method and quality improvement of sugary-1 rice mutants.

Brown rice of sugary-1 mutants has a wrinkled character because of the presence of phytoglycogen instead of starch in the inner part of the endosperm. Because the wrinkled phenotype was used as a sole selection marker for progeny of the sugary-1 strain, identification of mutant seeds with improved appearance is very difficult. We found that sugary-1 varieties contained not only phytoglycogen but also free glucose in the endosperm, and these were positively correlated. In the segregated F2 seeds that resulted from crossing Hokurikutou237 (sugary-1) and Koshihikari strains, glucose and phytoglycogen were also significantly correlated. Thus, we identified new sugary types with improved appearance from these progeny using glucose measurements. The F4 seeds of the improved strain had moderate phytoglycogen contents and seed germination characteristics. Native-PAGE showed that pullulanase activity in the improved strain increased in developing seeds compared with Hokurikutou237, although isoamylase activity was extremely low and similar to that in sugary-1 types. The new selection method in this study efficiently aids the development of improved sugary rice types that lack the wrinkled phenotype.

Starch degradation by alpha-amylase in tobacco leaves during the curing process

Tobacco (Nicotiana tabacum L.) leaf starch is degraded to sugars through curing (42°C/82.3% relative humidity/72 h). Total carbohydrate content remained almost constant, starch content decreased markedly, and soluble sugar content (mostly glucose) increased. α-Amylase and starch phosphorylase activities increased sixfold and threefold, respectively, whereas β-amylase activity was unaltered and isoamylase activity decreased. Increased α-amylase activity was accompanied by increased α-amylase protein levels. Although tobacco has four α-amylase gene members, only NtAMY1 mRNA levels increased. For other starch degradation genes, such as NtBAM1 and NtBMY2 (β-amylase), NtISO1 and NtISO2 (isoamylase) and NtGWD1 and NtGWD3 (glucan water dikinase), the mRNA levels remained unaltered during the first 48 h of curing. NtAMY1 expression was induced by osmotic stress but was unaffected by high temperature and/or injury stresses. Similarly, soluble sugar contents were largely increased by osmotic stress. This suggests that starch is degraded by α-amylase during curing and that α-amylase is coded by NtAMY1, induced by osmotic stress.

Constitutive expression of a novel isoamylase from Bacillus lentus in Pichia pastoris for starch processing

Isoamylase is essential to saccharifying starch by cleavage of 1,6-glucoside linkages in starch molecules. In this study, a novel isoamylase gene from Bacillus lentus JNU3 was cloned. The open reading frame of the gene was 2412 base pairs long and encoded a polypeptide of 804 amino acids with a calculated molecular mass of 90kDa. The deduced amino acid sequence shared less than 40% homology with that of microbial isoamylase ever reported, which indicated it was a novel isoamylase. A constitutive GAP promoter was used to express the recombinant isoamylase in the yeast Pichia pastoris by continuous high cell-density fermentation to avoid the use of methanol, which resulted in 318U/mL extracellular isoamylase activity after 72h in a 10L fermenter. The recombinant enzyme was purified and characterized. It had an estimated molecular mass of 90kDa, with its optimal activity at 70°C, pH 6.5 and was quite stable between 30°C and 70°C. The recombinant isoamylase proves to be superior to pullulanase as an auxiliary enzyme in maltose production from starch. Therefore it will contribute significantly to the starch debranching process.

Identification of the isoamylase 3 gene in common wheat and its expression profile during the grain-filling period

In higher plants, isoamylase-type starch debranching enzyme catalyzes the α-1,6-glucosidic linkages of glycogen and phytoglycogen. We cloned an isoamylase-type starch debranching enzyme ISA3 cDNA sequence (2883 bp), designated as TaISA3, from common wheat (Triticum aestivum), using the rapid amplification of cDNA ends method. The open reading frame of TaISA3 was found to have 2331 bp, and its deduced amino acid sequence was found to share high similarity with those of other gramineous plant ISA3 proteins. It contains a putative transit peptide (68 amino acids), N-terminus domain (107 amino acids), and a catalytic domain (173 amino acids). We extracted the expressed TaISA3 protein from Escherichia coli (BL21), and measured starch isoamylase activity. During the wheat grain-filling period, transcripts of the TaISA3 gene reached a maximum level at the early developmental stage, then declined, and increased again near the final maturation stage of the grain. We confirm that the ISA3 gene is present in common wheat; it appears to play a role in starch synthesis during early and late stages of the grain-filling period.

Analysis of Physiological and Biochemical Characteristics of Six Mutants with Stable High Percentage of Chalkiness in Rice Grains

For explaining the mechanism of chalkiness formation in rice ( Oryza sativa L.), the physiological and biochemical factors for chalkiness formation were analyzed using 6 mutants with chalky percentage higher than 80%, which were obtained from a T-DNA insertion mutant pool of cultivar Nipponbare. The appearance of high chalky percentages of the 6 mutants was proven to result from genetic factors because high temperature stress did not occur during grain-filling period. Based on the activities of key enzymes associated with starch synthesis, sucrose content, starch content, and ratio of amylose to starch in mature grains, the 6 mutants were classified into 3 groups. Compared with the wild type, mutants mu1 and mu2 had lower activities of sucrose synthase (SS) during the whole grain-filling period, higher activity of adenosine diphosphoglucose pyrophosphorylase (AGPase) at early grain-filling stage, and higher activities of SS I and total soluble starch synthase (SSS) at late grain-filling stage. Mutants mu3 and mu6 had lower activities of SS I, total SSS, pullulanase, and isoamylase at early filling stage and higher isoamylase activity at late filling stage than the wide type. Compared with the wild type, mutants mu4 and mu5 exhibited higher SS activity at early and middle stages of grain filling and lower activity of total SSS during the whole grain-filling period; the isoamylase activity increased from a lower level than the wild type at early filling stage to a higher level at late filling stage. All the 6 mutants had white belly grains with no visible differences to the wild type after iodine staining. The contents of fructose, sucrose, and total soluble sugar were higher in the 6 mutants than in the wild type, especially in mu2 and mu5. These results primarily revealed the physiological basis of chalkiness formation during rice grain development.

α-Amylase Activity of <I>Rhyzopertha dominica</I> (Coleoptera: Bostrichidae) Reared on Several Wheat Varieties and Its Inhibition with Kernel Extracts

Total progeny of Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) reared on 10 wheat, Triticum aestivum L., varieties was evaluated. Higher amylase activities were detected in populations with few individuals, whereas the opposite was observed in higher populations. As protein ingested increased, reproductive success increased. However, consumption of wheat protein was inversely correlated with amylase activity levels (r = -0.66). Amylase activity in homogenates of R. dominica populations showed variable inhibition by wheat extracts prepared from wheat varieties on which they were reared. Insect populations with lowest amylase activities were inhibited more by wheat extracts than those with higher amylase activity (r = -0.77). An electrophoretic analysis revealed four phenotypes showing combinations of three isoamylases (Rm 0.70, 0.79, and 0.90) in different populations of R. dominica. Some of the insect progeny that emerged from resistant wheat varieties contained the three isoamylases, whereas progeny that emerged from the most susceptible varieties showed reduced activity of isoamylases 0.70 or 0.90. These results suggest that the alpha-amylase activity levels and the composition of isoamylases in R. dominica populations are modulated by diet and that the alpha-amylase inhibitory activity of the wheat kernels influences these variations.

Recovery of Isoamylase Using an Aqueous Two-Phase System

This study attempted to recover starch debranching enzyme, isoamylase, from the fermentation broths by using an aqueous two-phase system. A series of poly (ethylene glycol) (PEG) combined with different salt systems were used to prepare the partition systems. In these experiments, the partition coefficient of isoamylase for the PEG1500(6%)/ (NH4)2SO4 (15%) system could be up to 40 when a crude enzyme solution with an isoamylase activity of 3386U/mL was used, and the enzyme activity was 5.0-fold concentrated in the top phase as compared to the crude enzyme solution. Based on the recovery and concentration fold of isoamylase, a pH around 4~5 was found to be the optimal condition for this system. The polymer design and combination of salts could be manipulated to achieve different volume ratios between the top and the bottom phases, and could also achieve the purpose of enzyme concentration. For the PEG1500(6%)/(NH4)2SO4 (15%) system, experimental results indicated a 6.0-fold volume reduction in the top phase as compared to that of the crude enzyme solution, and the isoamylase activity retention in the top phase was higher than 80%. With PEG systems of various molecular weights and concentrations, the partition coefficients of isoamylase tended to decrease with the increases of PEG molecular weight and concentration. These behaviors could be interpreted by the influence of hydrophobic interaction as well as free volume change.

Mutants of Arabidopsis Lacking a Chloroplastic Isoamylase Accumulate Phytoglycogen and an Abnormal Form of Amylopectin

Mutant lines defective for each of the four starch debranching enzyme (DBE) genes (AtISA1, AtISA2, AtISA3, and AtPU1) detected in the nuclear genome of Arabidopsis (Arabidopsis thaliana) were produced and analyzed. Our results indicate that both AtISA1 and AtISA2 are required for the production of a functional isoamylase-type of DBE named Iso1, the major isoamylase activity found in leaves. The absence of Iso1 leads to an 80% decrease in the starch content in both lines and to the accumulation of water-soluble polysaccharides whose structure is similar to glycogen. In addition, the residual amylopectin structure in the corresponding mutant lines displays a strong modification when compared to the wild type, suggesting a direct, rather than an indirect, function of Iso1 during the synthesis of amylopectin. Mutant lines carrying a defect in AtISA3 display a strong starch-excess phenotype at the end of both the light and the dark phases accompanied by a small modification of the amylopectin structure. This result suggests that this isoamylase-type of DBE plays a major role during starch mobilization. The analysis of the Atpu1 single-mutant lines did not lead to a distinctive phenotype. However, Atisa2/Atpu1 double-mutant lines display a 92% decrease in starch content. This suggests that the function of pullulanase partly overlaps that of Iso1, although its implication remains negligible when Iso1 is present within the cell.

Arabidopsis mutants Atisa1 and Atisa2 have identical phenotypes and lack the same multimeric isoamylase, which influences the branch point distribution of amylopectin during starch synthesis

The aim of this work was to evaluate the function of isoamylase in starch granule biosynthesis in Arabidopsis leaves. A reverse-genetic approach was used to knockout AtISA1, one of three genes in Arabidopsis encoding isoamylase-type debranching enzymes. The mutant (Atisa1-1) lacks functional AtISA1 transcript and the major isoamylase activity (detected by native gels) in crude extracts of leaves. The same activity is abolished by mutation at the DBE1 locus, which encodes a second isoamylase-type protein, AtISA2. This is consistent with the idea that ISA1 and ISA2 proteins are subunits of the same enzyme in vivo. Atisa1-1, Atisa2-1 (dbe1), and the Atisa1-1/Atisa2-1 double mutant all have identical phenotypes. Starch content is reduced compared with the wild type but substantial quantities of the soluble glucan phytoglycogen are produced. The amylopectin of the remaining starch and the phytoglycogen in the mutants are structurally related to each other and differ from wild-type amylopectin. Electron micrographs reveal that the phytoglycogen-accumulating phenotype is highly tissue-specific. Phytoglycogen accumulates primarily in the plastids of the palisade and spongy mesophyll cells. Remarkably, other cell types appear to accumulate only starch, which is normal in appearance but is altered in structure. As phytoglycogen accumulates during the day, its rate of accumulation decreases, its structure changes and intermediates of glucan breakdown accumulate, suggesting that degradation occurs simultaneously with synthesis. We conclude that the AtISA1/AtISA2 isoamylase influences glucan branching pattern, but that this may not be the primary determinant of partitioning between crystalline starch and soluble phytoglycogen.

Isolation and characterisation of novel starch mutants of oats

Abstract Mutagenised grains of the diploid oat, Avena strigosa Schreb., were screened for altered starch granules in the endosperm. Three mutant lines were identified: mutants lam-1 and lam-2 had granules that stained red with iodine solution and sga-1 had endosperm containing soluble material that stained red with iodine solution, as well as blue-staining starch granules. The lam-1 and lam-2 lines lack or have severely reduced amounts of granule-bound starch synthase activity, the granule-bound starch synthase I protein and the amylose component of starch in the endosperm. They therefore represent mutations of the waxy type. The red-staining, soluble material in the sga-1 mutant is very similar in appearance and chain-length profile to the phytoglycogen that accumulates in the endosperms of the sugary-1 mutants of cereals. All sugary-1 mutants lack or have reduced amounts of isoamylase activity. However, native gel assays reveal a normal isoamylase activity in the developing endosperms of sga-1 suggesting that it is not of the sugary-1 type. The nature of the mutation responsible for the sga-1 phenotype has yet to be discovered.

Starch granule initiation is controlled by a heteromultimeric isoamylase in potato tubers.

Starch granule initiation is not understood, but recent evidence implicates a starch debranching enzyme, isoamylase, in the control of this process. Potato tubers contain isoamylase activity attributable to a heteromultimeric protein containing Stisa1 and Stisa2, the products of two of the three isoamylase genes of potato. To discover whether this enzyme is involved in starch granule initiation, activity was reduced by expression of antisense RNA for Stisa1 or Stisa2. Transgenic tubers accumulated a small amount of a soluble glucan, similar in structure to the phytoglycogen of cereal, Arabidopsis, and Chlamydomonas mutants lacking isoamylase. The major effect, however, was on the number of starch granules. Transgenic tubers accumulated large numbers of tiny granules not seen in normal tubers. These data indicate that the heteromultimeric isoamylase functions during starch synthesis to suppress the initiation of glucan molecules in the plastid stroma that would otherwise crystallize to nucleate new starch granules.

Antisense inhibition of isoamylase alters the structure of amylopectin and the physicochemical properties of starch in rice endosperm.

This is the first report on regulation of the isoamylase1 gene to modify the structure of amylopectin and properties of starch by using antisense technology in plants. The reduction of isoamylase1 protein by about 94% in rice endosperm changed amylopectin into a water-insoluble modified amylopectin and a water-soluble polyglucan (WSP). As compared with wild-type amylopectin, the modified amylopectin had more short chains with a degree of polymerization of 5-12, while their molecular sizes were similar. The WSP, which structurally resembled the phytoglycogen in isoamylase-deficient sugary-1 mutants, accounted for about 16% of the total alpha-polyglucans in antisense endosperm, and it was distributed throughout the whole endosperm unlike in sugary-1 mutant. The reduction of isoamylase activity markedly lowered the gelatinization temperature from 54 to 43 degrees C and the viscosity, and modified X-ray diffraction pattern and the granule morphology of the starch. The activity of pullulanase, the other type of starch debranching enzyme, in the antisense endosperm was similar to that in wild-type, whereas it is deficient in sugary-1 mutants. These results indicate that the isoamylase1 is essential for amylopectin biosynthesis in rice endosperm, and that alteration of the isoamylase activity is an effective means to modify the physicochemical properties and granular structure of starch.

Three isoforms of isoamylase contribute different catalytic properties for the debranching of potato glucans.

Isoamylases are debranching enzymes that hydrolyze alpha-1,6 linkages in alpha-1,4/alpha-1,6-linked glucan polymers. In plants, they have been shown to be required for the normal synthesis of amylopectin, although the precise manner in which they influence starch synthesis is still debated. cDNA clones encoding three distinct isoamylase isoforms (Stisa1, Stisa2, and Stisa3) have been identified from potato. The expression patterns of the genes are consistent with the possibility that they all play roles in starch synthesis. Analysis of the predicted sequences of the proteins suggested that only Stisa1 and Stisa3 are likely to have hydrolytic activity and that there probably are differences in substrate specificity between these two isoforms. This was confirmed by the expression of each isoamylase in Escherichia coli and characterization of its activity. Partial purification of isoamylase activity from potato tubers showed that Stisa1 and Stisa2 are associated as a multimeric enzyme but that Stisa3 is not associated with this enzyme complex. Our data suggest that Stisa1 and Stisa2 act together to debranch soluble glucan during starch synthesis. The catalytic specificity of Stisa3 is distinct from that of the multimeric enzyme, indicating that it may play a different role in starch metabolism.

Starch granule initiation and growth are altered in barley mutants that lack isoamylase activity.

Two mutant lines of barley, Risø 17 and Notch-2, were found to accumulate phytoglycogen in the grain. Like the sugary mutants of maize and rice, these phytoglycogen-accumulating mutants of barley lack isoamylase activity in the developing endosperm. The mutants were shown to be allelic, and to have lesions in the isoamylase gene, isa1 that account for the absence of this enzyme. As well as causing a reduction in endosperm starch content, the mutations have a profound effect on the structure, number and timing of initiation of starch granules. There are no normal A-type or B-type granules in the mutants. The mutants have a greater number of starch granules per plastid than the wild-type and, particularly in Risø 17, this leads to the appearance of compound starch granules. These results suggest that, as well as suppressing phytoglycogen synthesis, isoamylase in the wild-type endosperm plays a role in determining the number, and hence the form, of starch granules.

Two loci control phytoglycogen production in the monocellular green alga Chlamydomonas reinhardtii.

The STA8 locus of Chlamydomonas reinhardtii was identified in a genetic screen as a factor that controls starch biosynthesis. Mutations of STA8 cause a significant reduction in the amount of granular starch produced during nutrient limitation and accumulate phytoglycogen. The granules remaining in sta8 mutants are misshapen, and the abundance of amylose and long chains in amylopectin is altered. Mutations of the STA7 locus, which completely lack isoamylase activity, also cause accumulation of phytoglycogen, although sta8 and sta7 mutants differ in that there is a complete loss of granular starch in the latter. This is the first instance in which mutations of two different genetic elements in one plant species have been shown to cause phytoglycogen accumulation. An analytical procedure that allows assay of isoamylase in total extracts was developed and used to show that sta8 mutations cause a 65% reduction in the level of this activity. All other enzymes known to be involved in starch biosynthesis were shown to be unaffected in sta8 mutants. The same amount of total isoamylase activity (approximately) as that present in sta8 mutants was observed in heterozygous triploids containing two sta7 mutant alleles and one wild-type allele. This strain, however, accumulates normal levels of starch granules and lacks phytoglycogen. The total level of isoamylase activity, therefore, is not the major determinant of whether granule production is reduced and phytoglycogen accumulates. Instead, a qualitative property of the isoamylase that is affected by the sta8 mutation is likely to be the critical factor in phytoglycogen production.