Rice debranching enzyme isoamylase3 facilitates starch metabolism and affects plastid morphogenesis

Debranching enzymes, which hydrolyze α-1 and 6-glucosidic linkages in α-polyglucans, play a dual role in the synthesis and degradation of starch in plants. A transposon-inserted rice mutant of isoamylase3 (isa3) contained an increased amount of starch in the leaf blade at the end of the night, indicating that ISA3 plays a role in the degradation of transitory starch during the night. An epitope-tagged ISA3 expressed in Escherichia coli exhibited hydrolytic activity on β-limit dextrin and amylopectin. We investigated whether ISA3 plays a role in amyloplast development and starch metabolism in the developing endosperm. ISA3-green fluorescent protein (GFP) fusion protein expressed under the control of the rice ISA3 promoter was targeted to the amyloplast stroma in the endosperm. Overexpression of ISA3 in the sugary1 mutant, which is deficient in ISA1 activity, did not convert water-soluble phytoglycogen to starch granules, indicating that ISA1 and ISA3 are not functionally redundant. Both overexpression and loss of function of ISA3 in the endosperm generated pleomorphic amyloplasts and starch granules. Furthermore, chloroplasts in the leaf blade of isa3 seedlings were large and pleomorphic. These results suggest that ISA3 facilitates starch metabolism and affects morphological characteristics of plastids in rice.     

Correlation between activities of starch debranching enzyme and α-polyglucan structure in endosperms of sugary-1 mutants of rice

The biochemical lesion of the sugary-1 mutation was examined in five different mutants of rice with varying phenotypes but with mutations at the same locus. The cells in the inner part of the endosperm of all mutants tested contained phytoglycogen instead of starch, while the cells located in the outer part of the endosperm tissue from some mutants were filled with numerous starch granules. The molecular size of phytoglycogen was markedly smaller than that of amylopectin as measured by Sephacryl S-1000 chromatography. Analysis of the distribution of α-1,4 chain lengths revealed that in phytoglycogen the number of A-chains dramatically increased, while long B chains with DP ≥ 37 remarkably decreased or were almost absent, which resulted in the disappearance of the cluster structure. The results suggest that changes in the balance of enzymic activities induced by the mutations brought about a drastic alteration in polyglucan structure and the shape of the polyglucan granule. The greater the extent of phytoglycogen regions in su1 endosperm tissues became, the greater was the phytoglycogen content, and the greater the reduction in the activity of starch debranching enzyme, a type of enzyme referred to as R-enzyme (RE), limit dextrinase or pullulanase. Immunoblot analysis showed that the reduction in RE activity was due to a decrease in the amount of RE protein, and that the reduction in RE was specific since proteins of starch-branching enzymes I and IIa and ADP-glucose pyrophosphorylase were not markedly affected by su1 mutations. The proportion of starch region to the whole endosperm tissue of various su1 mutants was correlated with the RE activity in these endosperms. The results strongly suggest that the reduction in RE activity is involved in the su1 phenotype and that the enzyme plays an essential role in determining the fine structure of the amylopectin molecule     

Brittle-1, an Adenylate Translocator, Facilitates Transfer of Extraplastidial Synthesized ADP-Glucose into Amyloplasts of Maize Endosperms

Amyloplasts of starchy tissues such as those of maize (Zea mays L.) function in the synthesis and accumulation of starch during kernel development. ADP-glucose pyrophosphorylase (AGPase) is known to be located in chloroplasts, and for many years it was generally accepted that AGPase was also localized in amyloplasts of starchy tissues. Recent aqueous fractionation of young maize endosperm led to the conclusion that 95% of the cellular AGPase was extraplastidial, but immunolocalization studies at the electron- and light-microscopic levels supported the conclusion that maize endosperm AGPase was localized in the amyloplasts. We report the results of two nonaqueous procedures that provide evidence that in maize endosperms in the linear phase of starch accumulation, 90% or more of the cellular AGPase is extraplastidial. We also provide evidence that the brittle-1 protein (BT1), an adenylate translocator with a KTGGL motif common to the ADP-glucose-binding site of starch synthases and bacterial glycogen synthases, functions in the transfer of ADP-glucose into the amyloplast stroma. The importance of the BT1 translocator in starch accumulation in maize endosperms is demonstrated by the severely reduced starch content in bt1 mutant kernels.     

Distinct isoforms of ADPglucose pyrophosphorylase occur inside and outside the amyloplasts in barley endosperm

This paper addresses the controversial idea that ADPglucose pyrophosphorylase may be located in the cytosol in some non-photosynthetic plant organs. The intracellular location of the enzyme in developing barley endosperm has been investigated by isolation of intact amyloplasts. Amyloplast preparations contained 13–17% of the total endosperm activity of two plastidial marker enzymes, and less than 0.5% of the total endosperm activity of two cytosolic marker enzymes. Amyloplast preparations contained about 2.5% of the ADPglucose pyrophosphorylase activity, indicating that approximately 15% of the ADPglucose pyrophosphorylase activity in young endosperms is plastidial. Immunoblotting of gels of endosperm and amyloplast extracts also indicated that the enzyme is both inside and outside the amyloplast. Antibodies to the small subunits of the enzyme from barley and maize revealed two bands of protein of different sizes, one of which was located inside and the other outside the amyloplast. The plastidial protein was of the same size as a protein in the chloroplasts of barley leaves which was also recognized by these antibodies. It is suggested that the barley plant contains two distinct isoforms of ADPglucose pyrophosphorylase: one located in plastids (chloroplasts and amyloplasts) and the other in the cytosol of the endosperm. The role of the cytosolic ADPglucose pyrophosphorylase is unknown. Although it may contribute ADPglucose to starch synthesis, the total activity of ADPglucose pyrophosphorylase in the endosperm is far in excess of the rate of starch synthesis and the plastidial isoform is probably capable of catalysing the entire flux of carbon to starch.     

STARCH GRANULE (AMYLOPLAST) DEVELOPMENT IN ENDOSPERM OF SEVERAL ZEA MAYS L. GENOTYPES AFFECTING KERNEL POLYSACCHARIDES

Endosperm cell and starch granule (amyloplast) development of six maize (Zea mays L.) genotypes, normal, amylose-extender (ae), sugary (su), waxy (wx), amylose-extender sugary (ae su), and amylose-extender waxy (ae wx), was compared. Endosperms of all genotypes were indistinguishable at 14 days after pollination. Cells were highly vacuolated and those in the central crown area of the kernel contained small starch granules in close association with the nucleus. Cellular and nuclear enlargement occurred during endosperm development in all genotypes, and major and minor gradients in physiological age of endosperm cells were observed in all kernels. Amyloplast development varied with genotype. Plastid development in normal and wx cells was characterized by an initial starch granule formation followed by granule enlargement to cell maturity. Endosperms homozygous for ae (ae, ae su, and ae wx) developed abnormal plastid-granules. Secondary granule formations preceded development of abnormality in ae and ae su, but not in ae wx endosperms. In contrast to ae and ae su starch granules, ae wx granules were highly birefringent indicating a high degree of crystallinity. In all three ae genotypes, abnormality increased as a function of kernel and physiological cell age. The su mutant had two distinct effects on amyloplast development. First, a mobilization of the initially formed starch, and second a synthesis and accumulation of phytoglycogen and the formation of large rounded plastids. In ae su plastid development, there was a mobilization of the starch initially formed (resulting in irregularly shaped, nonbirefringent granules) but only small amounts of phytoglycogen were produced.     

Interaction of cytochrome b5 with surfactant vesicles.

Lysates of protoplasts from the endosperm of developing grains of wheat (Triticum aestivum) were fractionated on density gradients of Nycodenz to give amyloplasts. Enzyme distribution on the gradients suggested that: (i) starch synthase and ADP-glucose pyrophosphorylase are confined to the amyloplasts; (ii) pyrophosphate: fructose-6-phosphate 1-phosphotransferase and UDP-glucose pyrophosphorylase are confined to the cytosol; (iii) a significant proportion (23-45%) of each glycolytic enzyme, from phosphoglucomutase to pyruvate kinase inclusive, is in the amyloplast. Starch synthase, ADP-glucose pyrophosphorylase and each of the glycolytic enzymes showed appreciable latency when assayed in unfractionated lysates of protoplasts. No activity of fructose-1,6-bisphosphatase was found in amyloplasts or in homogenates of endosperm. Antibody to plastidic fructose-1,6-bisphosphatase did not react positively, in an immunoblot analysis, with any protein in extracts of wheat endosperm. It is argued that wheat endosperm lacks significant plastidic fructose-1,6-bisphosphatase and that carbon for starch synthesis does not enter the amyloplast as a C-3 compound but probably as hexose phosphate.     

Physical association of starch biosynthetic enzymes with starch granules of maize endosperm. Granule-associated forms of starch synthase I and starch branching enzyme II.

Antibodies were used to probe the degree of association of starch biosynthetic enzymes with starch granules isolated from maize (Zea mays) endosperm. Graded washings of the starch granule, followed by release of polypeptides by gelatinization in 2% sodium dodecyl sulfate, enables distinction between strongly and loosely adherent proteins. Mild aqueous washing of granules resulted in near-complete solubilization of ADP-glucose pyrophosphorylase, indicating that little, if any, ADP-glucose pyrophosphorylase is granule associated. In contrast, all of the waxy protein plus significant levels of starch synthase I and starch branching enzyme II (BEII) remained granule associated. Stringent washings using protease and detergent demonstrated that the waxy protein, more than 85% total endosperm starch synthase I protein, and more than 45% of BEII protein were strongly associated with starch granules. Rates of polypeptide accumulation within starch granules remained constant during endosperm development. Soluble and granule-derived forms of BEII yielded identical peptide maps and overlapping tryptic fragments closely aligned with deduced amino acid sequences from BEII cDNA clones. These observations provide direct evidence that BEII exits as both soluble and granule-associated entities. We conclude that each of the known starch biosynthetic enzymes in maize endosperm exhibits a differential propensity to associate with, or to become irreversibly entrapped within, the starch granule.     

Localization of Branching Enzyme in Potato Tuber Cells with the Use of Immunoelectron Microscopy

Potato branching enzyme, a key enzyme in the biosynthesis of starch, was localized in amyloplasts in starch-storage cells of potato (Solanum tuberosum L.) with the use of immunogold electron microscopy. Branching enzyme was found in the amyloplast stroma, concentrated at the interface of the stroma and the surface of the starch granule. ADP-glucose pyrophosphorylase, a key regulatory enzyme in starch synthesis, was localized for comparison to exclude possible artifacts. ADP-glucose pyrophosphorylase, in contrast with branching enzyme, proved to be evenly distributed throughout the stroma. Branching enzyme also appears to be present in a membrane-bounded inclusion body in the stroma, whereas ADP-glucose pyrophosphorylase is not. The presence of branching enzyme predominantly at the surface of the starch granule indicates that branching takes place at that surface and not throughout the amyloplast stroma.     

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.     

Complementation of sugary-1 phenotype in rice endosperm with the wheat isoamylase1 gene supports a direct role for isoamylase1 in amylopectin biosynthesis

To examine the role of isoamylase1 (ISA1) in amylopectin biosynthesis in plants, a genomic DNA fragment from Aegilops tauschii was introduced into the ISA1-deficient rice (Oryza sativa) sugary-1 mutant line EM914, in which endosperm starch is completely replaced by phytoglycogen. A. tauschii is the D genome donor of wheat (Triticum aestivum), and the introduced fragment effectively included the gene for ISA1 for wheat (TaISA1) that was encoded on the D genome. In TaISA1-expressing rice endosperm, phytoglycogen synthesis was substantially replaced by starch synthesis, leaving only residual levels of phytoglycogen. The levels of residual phytoglycogen present were inversely proportional to the expression level of the TaISA1 protein, although the level of pullulanase that had been reduced in EM914 was restored to the same level as that in the wild type. Small but significant differences were found in the amylopectin chain-length distribution, gelatinization temperatures, and A-type x-ray diffraction patterns of the starches from lines expressing TaISA1 when compared with wild-type rice starch, although in the first two parameters, the effect was proportional to the expression level of TaISA. The impact of expression levels of ISA1 on starch structure and properties provides support for the view that ISA1 is directly involved in the synthesis of amylopectin.     

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.     

Enzyme activities associated with developing wheat(Triticum aestivum L.) grain amyloplasts

Intact amyloplasts from endosperm of developing wheat grains have been isolated by first preparing the protoplasts and then fractionating the lysate of the protoplasts on percoll and ficoll gradients, respectively. Amyloplasts isolated as above were functional and not contaminated by cytosol or by organelles likely to be involved in carbohydrate metabolism. The enzyme distribution studies indicated that ADP-glucose pyrophosphorylase and starch synthase were confined to amyloplasts, whereas invertase, sucrose synthase, UDP-glucose pyrophosphorylase, hexokinase, phosphofructokinase-2 and fructose-2,6-P₂ase were absent fro the amyloplast and mainly confined to the cytosol. Triose-P isomerase, glyceraldehyde-3-P dehydrogenase, phosphohexose isomerase, phosphoglucomutase, phosphofructokinase, aldolase, PPi-fructose-6-P-1 phosphotransferase, and fructose-l,6-P₂ase, though predominantly cytosolic, were also present in the amyloplast. Based on distribution of enzymes, a probable pathway for starch biosynthesis in amyloplasts of developing wheat grains has been proposed.     

A low-starch barley mutant,risø 16, lacking the cytosolic small subunit of ADP-glucose pyrophosphorylase,reveals the importance of the cytosolic isoform and the identity of the plastidial small subunit

To provide information on the roles of the different forms of ADP-glucose pyrophosphorylase (AGPase) in barley (Hordeum vulgare) endosperm and the nature of the genes encoding their subunits, a mutant of barley, Ris? 16, lacking cytosolic AGPase activity in the endosperm was identified. The mutation specifically abolishes the small subunit of the cytosolic AGPase and is attributable to a large deletion within the coding region of a previously characterized small subunit gene that we have called Hv.AGP.S.1. The plastidial AGPase activity in the mutant is unaffected. This shows that the cytosolic and plastidial small subunits of AGPase are encoded by separate genes. We purified the plastidial AGPase protein and, using amino acid sequence information, we identified the novel small subunit gene that encodes this protein. Studies of the Ris? 16 mutant revealed the following. First, the reduced starch content of the mutant showed that a cytosolic AGPase is required to achieve the normal rate of starch synthesis. Second, the mutant makes both A- and B-type starch granules, showing that the cytosolic AGPase is not necessary for the synthesis of these two granule types. Third, analysis of the phylogenetic relationships between the various small subunit proteins both within and between species, suggest that the cytosolic AGPase single small subunit gene probably evolved from a leaf single small subunit gene.     

Functional interactions between starch synthase III and isoamylase-type starch-debranching enzyme in maize endosperm

This study characterized genetic interactions between the maize (Zea mays) genes dull1 (du1), encoding starch synthase III (SSIII), and isa2, encoding a noncatalytic subunit of heteromeric isoamylase-type starch-debranching enzyme (ISA1/ISA2 heteromer). Mutants lacking ISA2 still possess the ISA1 homomeric enzyme. Eight du1(-) mutations were characterized, and structural changes in amylopectin resulting from each were measured. In every instance, the same complex pattern of alterations in discontinuous spans of chain lengths was observed, which cannot be explained solely by a discrete range of substrates preferred by SSIII. Homozygous double mutants were constructed containing the null mutation isa2-339 and either du1-Ref, encoding a truncated SSIII protein lacking the catalytic domain, or the null allele du1-R4059. In contrast to the single mutant parents, double mutant endosperms affected in both SSIII and ISA2 were starch deficient and accumulated phytoglycogen. This phenotype was previously observed only in maize sugary1 mutants impaired for the catalytic subunit ISA1. ISA1 homomeric enzyme complexes assembled in both double mutants and were enzymatically active in vitro. Thus, SSIII is required for normal starch crystallization and the prevention of phytoglycogen accumulation when the only isoamylase-type debranching activity present is ISA1 homomer, but not in the wild-type condition, when both ISA1 homomer and ISA1/ISA2 heteromer are present. Previous genetic and biochemical analyses showed that SSIII also is required for normal glucan accumulation when the only isoamylase-type debranching enzyme activity present is ISA1/ISA heteromer. These data indicate that isoamylase-type debranching enzyme and SSIII work in a coordinated fashion to repress phytoglycogen accumulation.     

The failure to express a protein disulphide isomerase-like protein results in a floury endosperm and an endoplasmic reticulum stress response in rice

The rice somaclonal mutant T3612 produces small grains with a floury endosperm, caused by the loose packing of starch granules. The positional cloning of the mutation revealed a deletion in a gene encoding a protein disulphide isomerase-like enzyme (PDIL1-1). In the wild type, PDIL1-1 was expressed throughout the plant, but most intensely in the developing grain. In T3612, its expression was abolished, resulting in a decrease in the activity of plastidial phosphorylase and pullulanase, and an increase in that of soluble starch synthase I and ADP-glucose pyrophosphorylase. The amylopectin in the T3612 endosperm showed an increase in chains with a degree of polymerization 8-13 compared with the wild type. The expression in the mutant's endosperm of certain endoplasmic reticulum stress-responsive genes was noticeably elevated. PDIL1-1 appears to play an important role in starch synthesis. Its absence is associated with endoplasmic reticulum stress in the endosperm, which is likely to underlie the formation of the floury endosperm in the T3612 mutant.     

The encoded primary sequence of a rice seed ADP-glucose pyrophosphorylase subunit and its homology to the bacterial enzyme

Rice seed ADP-glucose pyrophosphorylase cDNA clones were isolated by screening a lambda expression library prepared from rice endosperm poly(A+) RNA with a heterologous antibody raised against the spinach leaf enzyme and subsequently by nucleic acid hybridization. One cDNA plasmid, possessing about 1650 nucleotides, was shown by both DNA and RNA sequence analysis to contain the complete ADP-glucose pyrophosphorylase coding sequence of 483 amino acids. The primary sequence displayed a putative leader peptide presumably required for transport of this nuclear encoded protein into the amyloplasts, a differentiated starch containing plastid. The leader peptide, however, showed little sequence homology with transit peptides displayed by other known nuclear encoded proteins localized in the chloroplasts. A comparison of the primary sequence of the putative mature subunit to the Escherichia coli pyrophosphorylase showed two regions displaying significant homology. These two conserved regions contain residues shown previously to be essential for the allosteric regulation and catalytic activity of the E. coli enzyme. Differences in the primary sequences of the plant and bacterial enzyme may reflect the distinct nature of the allosteric effectors that control these enzymes.     

淀粉分支酶3存在于水稻胚乳的淀粉粒之中

对水稻胚乳淀粉颗粒结合的淀粉分支酶进行了研究.酶活性分析表明水稻胚乳中存在着与淀粉颗粒结合的淀粉分支酶.氨基酸测序分析结果表明结合于水稻胚乳淀粉粒的淀粉分支酶是分子量为84 kD的淀粉分支酶3(rice starch branching enzyme 3; RBE3).从开花后5 d到种子成熟,淀粉颗粒结合的RBE3蛋白都保持较为稳定的含量.Northern 分析表明水稻胚乳发育过程中RBE4最先表达而RBE3和RBE1的表达滞后.综合以上研究结果说明RBE3存在于水稻胚乳的淀粉之中是由于RBE3与淀粉葡聚糖链具有较高亲和性而难以和葡聚糖链解离,进而随着淀粉粒的增长而被其包裹.     

Physicochemical properties and development of wheat large and small starch granules during endosperm development

Wheat mature seeds have large, lenticular A-type starch granules, and small, spherical B-type and irregular C-type starch granules. During endosperm development, large amyloplasts came from proplastid, divided and increased in number through binary fission from 4 to 12 days after flowering (DAF). Large starch granules formed and developed in the large amyloplast. One large amyloplast had only one large starch granule. Small amyloplasts came from the protrusion of large amyloplast envelope, divided and increased in number through envelope protrusion after 12 DAF. B-type starch granules formed and developed in small amyloplast from 12 to 18 DAF, C-type starch granules formed and developed in small amyloplast after 18 DAF. Many B- and C-type starch granules might form and develop in one small amyloplast. The amyloplast envelopes were asynchronously degraded and starch granules released into cell matrix when amyloplasts were full of starch granules. Apparent amylose contents of large starch granules were higher than that of small starch granules, and increased with endosperm development. The swelling powers and crystallinity of large starch granule were lower than that of small starch granules, and decreased with endosperm development. Small starch granules displayed broader gelatinization temperature ranges than did large starch granules.