Mutations in starch BRANCHING ENZYME 2a suppress the traits caused by the loss of ISOAMYLASE1 in barley.

KEY MESSAGE: The hvbe2a mutations restore the starch-deficient phenotype caused by the hvisa1 and hvflo6 mutations in barley endosperm. The genetic interactions among starch biosynthesis genes can be exploited to alter starch properties, but they remain poorly understood due to the various combinations of mutations to be tested. Here, we isolated two novel barley mutants defective in starch BRANCHING ENZYME 2a (hvbe2a-1 and hvbe2a-2) based on the starch granule (SG) morphology. Both hvbe2a mutants showed elongated SGs in the endosperm and increased resistant starch content. hvbe2a-1 had a base change in HvBE2a gene, substituting the amino acid essential for its enzyme activity, while hvbe2a-2 is completely missing HvBE2a due to a chromosomal deletion. Further genetic crosses with barley isoamylase1 mutants (hvisa1) revealed that both hvbe2a mutations could suppress defects in endosperm caused by hvisa1, such as reduction in starch, increase in phytoglycogen, and changes in the glucan chain length distribution. Remarkably, hvbe2a mutations also transformed the endosperm SG morphology from the compound SG caused by hvisa1 to bimodal simple SGs, resembling that of wild-type barley. The suppressive impact was in competition with floury endosperm 6 mutation (hvflo6), which could enhance the phenotype of hvisa1 in the endosperm. In contrast, the compound SG formation induced by the hvflo6 hvisa1 mutation in pollen was not suppressed by hvbe2a mutations. Our findings provide new insights into genetic interactions in the starch biosynthetic pathway, demonstrating how specific genetic alterations can influence starch properties and SG morphology, with potential applications in cereal breeding for desired starch properties.

Mutation in BEIIb mitigates the negative effect of the mutation in ISA1 on grain filling and amyloplast formation in rice.

KEY MESSAGE: Mutation of the BEIIb gene in an isa1 mutant background mitigates the negative effect of the ISA1 mutation on grain filling, and facilitates recovery of amyloplast formation in rice endosperm. In this study, the effect of branching enzyme IIb and isoamylase 1 deficiency on starch properties was demonstrated using high resistant starch rice lines, Chikushi-kona 85 and EM129. Both lines harbored a mutation in the BEIIb and ISA1 genes and showed no BEIIb and ISA1 activity, implying that both lines are beIIb isa1 double mutants. The amylopectin long chain and apparent amylose content of both mutant lines were higher than those of the wild-type. While both mutants contained loosely packed, round starch grains, a trait specific to beIIb mutants, they also showed collapsed starch grains at the center of the endosperm, a property specific to isa1 mutants. Furthermore, beIIb isa1 double mutant F2 lines derived from a cross between Chikushi-kona 85 and Nishihomare (wild-type cultivar) showed significantly heavier seed weight than the beIIb and isa1 single mutant lines. These results suggest that co-occurrence of beIIb and isa1 mutant alleles in a single genetic background mitigates the negative effect of the isa1 allele on grain filling, and contributes to recovery of the amyloplast formation defect in the isa1 single mutant.

Active-type starch synthase (SS) IIa from indica rice partially complements the sugary-1 phenotype in japonica rice endosperm.

KEY MESSAGE: Introduction of higher SSIIa activity to mild-type isa1 mutant by crossing results in restoration of crystallinity, starch granule structure, and production of plump seeds. Isoamylase 1 (ISA1) removes improper α-1, 6 glycosidic branches of amylopectin generated by starch branching enzymes and is essential for the formation of proper amylopectin structure. Rice isa1 (sug-1) mutants in japonica cultivar with less-active starch synthase IIa (SSIIa) and low granule-bound SSI (GBSSI) expression display wrinkled seed phenotype by accumulating water-soluble phytoglycogen instead of insoluble amylopectin. Expression of active SSIIa in transgenic rice produced with a severe-type isa1 mutant accumulated some insoluble glucan with weak B-type crystallinity at the periphery of seeds but their seeds remained wrinkled. To see whether introduction of high levels of SSIIa and/or GBSSI can restore the grain filling of the mild-type sug-1 mutant (EM653), new rice lines (SS2a gbss1L isa1, ss2aL GBSS1 isa1, and SS2a GBSS1 isa1) were generated by crossing japonica isa1 mutant (ss2aL gbss1L isa1) with wild type indica rice (SS2a GBSS1 ISA1). The results showed that SS2a gbss1L isa1 and SS2a GBSS1 isa1 lines generated chalky plump seeds accumulating insoluble amylopectin-like glucans with an increase in DP 13-35, while ss2aL GBSS1 isa1 generated wrinkly seeds and accumulated soluble glucans enriched with DP < 13. Scanning electron microscopic observation of cross-section of the seeds showed that SS2a gbss1L isa1 and SS2a GBSS1 isa1 produced wild type-like polygonal starch granules. These starches showed the A-type crystallinity comparable to the wild type, while the japonica isa1 mutant and the transgenic rice do not show any or little crystallinity, respectively. These results indicate that introduction of higher SSIIa activity can mostly complements the mild-type sug-1 phenotype.

Foliar Application of dsRNA Targeting Endogenous Potato (Solanum tuberosum) Isoamylase Genes ISA1, ISA2, and ISA3 Confers Transgenic Phenotype.

Isoamylase (ISA) is a debranching enzyme found in many plants, which hydrolyzes (1-6)-α-D glucosidic linkages in starch, amylopectin, and ß-dextrins, and is thought to be responsible for starch granule formation (ISA1 and ISA2) and degradation (ISA3). Lipid-modified PEI (lmPEI) was synthesized as a carrier for long double-stranded RNA (dsRNA, 250-bp), which targets the three isoamylase isoforms. The particles were applied to the plant via the foliar spray and were differentially effective in suppressing the expressions of ISA1 and ISA2 in the potato leaves, and ISA3 in the tubers. Plant growth was not significantly impaired, and starch levels in the tubers were not affected as well. Interestingly, the treated plants had significantly smaller starch granule sizes as well as increased sucrose content, which led to an early sprouting phenotype. We confirm the proposal of previous research that an increased number of small starch granules could be responsible for an accelerated turnover of glucan chains and, thus, the rapid synthesis of sucrose, and we propose a new relationship between ISA3 and the starch granule size. The implications of this study are in achieving a transgenic phenotype for endogenous plant genes using a systemic, novel delivery system, and foliar applications of dsRNA for agriculture.

Endosperm sugar accumulation caused by mutation of PHS8/ISA1 leads to pre-harvest sprouting in rice.

Pre-harvest sprouting (PHS) is an unfavorable trait in cereal crops that could seriously decrease grain yield and quality. Although some PHS-associated quantitative trait loci or genes in cereals have been reported, the molecular mechanism underlying PHS remains largely elusive. Here, we characterized a rice mutant, phs8, which exhibits PHS phenotype accompanied by sugary endosperm. Map-based cloning revealed that PHS8 encodes a starch debranching enzyme named isoamylase1. Mutation in PHS8 resulted in the phytoglycogen breakdown and sugar accumulation in the endosperm. Intriguingly, with increase of sugar contents, decreased expression of OsABI3 and OsABI5 as well as reduced sensitivity to abscisic acid (ABA) were found in the phs8 mutant. Using rice suspension cell system, we confirmed that exogenous sugar is sufficient to suppress the expression of both OsABI3 and OsABI5. Furthermore, overexpression of OsABI3 or OsABI5 could partially rescue the PHS phenotype of phs8. Therefore, our study presents important evidence supporting that endosperm sugar not only acts as an essential energy source for seed germination but also determines seed dormancy and germination by affecting ABA signaling.

Heterologous co-expression in E. coli of isoamylase genes from cassava Manihot esculenta Crantz 'KU50' achieves enzyme-active heteromeric complex formation.

KEY MESSAGE: Cloning of two isoamylase genes, MeISA1 and MeISA2, from cassava (Manihot esculenta Crantz) tubers, accompanied by their co-expression in E. coli demonstrates a requirement for heteromeric complex formation to achieve debranching activity. Starch debranching enzyme (DBE) or isoamylase (ISA) (EC.3.2.1.68), an important enzyme in starch metabolism, catalyses the hydrolysis of α-1,6 glycosidic linkages of amylopectin. Isoforms of ISAs have been reported in higher plants and algae (Fujita et al. in Planta 208:283-293, 1999; Hussain et al. in Plant Cell 15:133-149, 2003; Ishizaki et al. in Agric Biol Chem 47:771-779, 1983; Mouille et al. in Plant Cell 8:1353-1366, 1996). In the current work, cassava ISA genes were isolated from cDNA generated from total RNA from tubers of Manihot esculanta Crantz cultivar KU50. MeISA1 and MeISA2 were successfully amplified and cloned into a pETDuet1 vector. The putative MeISA1 and MeISA2 proteins comprised 763 and 882 amino acids, with substantial similarity to StISA1 and StISA2 from potato (84.4% and 68.9%, respectively). Recombinant MeISA1 and MeISA2 were co-expressed in Escherichia coli SoluBL21 (DE3). HistrapTM-Purified rMeISA1 and rMeISA2 showed approximate molecular weights of 87 and 99 kDa, respectively, by SDS-PAGE. Debranching activity was only detectable in the column fractions where both recombinant ISA isoforms were present. The heteromeric DBE from crude extracts of 4-5 h induced cultures analysed by gel filtration chromatography and western blot showed combinations of rMeISA1 and rMeISA2 at ratios of 1:1 to 4:1. Pooled fractions with DBE activity were used for enzyme characterisation, which showed that the enzyme was specific for amylopectin, with optimum activity at 37 °C and pH 7.0. Enzyme activity was enhanced by Co2+, Mg2+ and Ca2+, but was strongly inhibited by Cu2+. Debranched amylopectin products showed chain length distributions typical of plant DBE.

The buffering capacity of stems: genetic architecture of nonstructural carbohydrates in cultivated Asian rice, Oryza sativa.

Harnessing stem carbohydrate dynamics in grasses offers an opportunity to help meet future demands for plant-based food, fiber and fuel production, but requires a greater understanding of the genetic controls that govern the synthesis, interconversion and transport of such energy reserves. We map out a blueprint of the genetic architecture of rice (Oryza sativa) stem nonstructural carbohydrates (NSC) at two critical developmental time-points using a subpopulation-specific genome-wide association approach on two diverse germplasm panels followed by quantitative trait loci (QTL) mapping in a biparental population. Overall, 26 QTL are identified; three are detected in multiple panels and are associated with starch-at-maturity, sucrose-at-maturity and NSC-at-heading. They tag OsHXK6 (rice hexokinase), ISA2 (rice isoamylase) and a tandem array of sugar transporters. This study provides the foundation for more in-depth molecular investigation to validate candidate genes underlying rice stem NSC and informs future comparative studies in other agronomically vital grass species.

Metabolic effectors secreted by bacterial pathogens: essential facilitators of plastid endosymbiosis?

Under the endosymbiont hypothesis, over a billion years ago a heterotrophic eukaryote entered into a symbiotic relationship with a cyanobacterium (the cyanobiont). This partnership culminated in the plastid that has spread to forms as diverse as plants and diatoms. However, why primary plastid acquisition has not been repeated multiple times remains unclear. Here, we report a possible answer to this question by showing that primary plastid endosymbiosis was likely to have been primed by the secretion in the host cytosol of effector proteins from intracellular Chlamydiales pathogens. We provide evidence suggesting that the cyanobiont might have rescued its afflicted host by feeding photosynthetic carbon into a chlamydia-controlled assimilation pathway.

Engineering of isoamylase: improvement of protein stability and catalytic efficiency through semi-rational design.

Isoamylase catalyzes the hydrolysis of α-1,6-glycosidic linkages in glycogen, amylopectin and α/ß-limit dextrins. A semi-rational design strategy was performed to improve catalytic properties of isoamylase from Bacillus lentus. Three residues in vicinity of the essential residues, Arg505, Asn513, and Gly608, were chosen as the mutation sites and were substituted by Ala, Pro, Glu, and Lys, respectively. Thermal stability of the mutant R505P and acidic stability of the mutant R505E were enhanced. The k cat /K m values of the mutant G608V have been promoted by 49%, and the specific activity increased by 33%. This work provides an effective strategy for improving the catalytic activity and stability of isoamylase, and the results obtained here may be useful for the improvement of catalytic properties of other α/ß barrel enzymes.

Distinct functional properties of isoamylase-type starch debranching enzymes in monocot and dicot leaves.

Isoamylase-type starch debranching enzymes (ISA) play important roles in starch biosynthesis in chloroplast-containing organisms, as shown by the strict conservation of both catalytically active ISA1 and the noncatalytic homolog ISA2. Functional distinctions exist between species, although they are not understood yet. Numerous plant tissues require both ISA1 and ISA2 for normal starch biosynthesis, whereas monocot endosperm and leaf exhibit nearly normal starch metabolism without ISA2. This study took in vivo and in vitro approaches to determine whether organism-specific physiology or evolutionary divergence between monocots and dicots is responsible for distinctions in ISA function. Maize (Zea mays) ISA1 was expressed in Arabidopsis (Arabidopsis thaliana) lacking endogenous ISA1 or lacking both native ISA1 and ISA2. The maize protein functioned in Arabidopsis leaves to support nearly normal starch metabolism in the absence of any native ISA1 or ISA2. Analysis of recombinant enzymes showed that Arabidopsis ISA1 requires ISA2 as a partner for enzymatic function, whereas maize ISA1 was active by itself. The electrophoretic mobility of recombinant and native maize ISA differed, suggestive of posttranslational modifications in vivo. Sedimentation equilibrium measurements showed recombinant maize ISA1 to be a dimer, in contrast to previous gel permeation data that estimated the molecular mass as a tetramer. These data demonstrate that evolutionary divergence between monocots and dicots is responsible for the distinctions in ISA1 function.

The simultaneous abolition of three starch hydrolases blocks transient starch breakdown in Arabidopsis.

In this study, we investigated which enzymes are involved in debranching amylopectin during transient starch degradation. Previous studies identified two debranching enzymes, isoamylase 3 (ISA3) and limit dextrinase (LDA), involved in this process. However, plants lacking both enzymes still degrade substantial amounts of starch. Thus, other enzymes/mechanisms must contribute to starch breakdown. We show that the chloroplastic α-amylase 3 (AMY3) also participates in starch degradation and provide evidence that all three enzymes can act directly at the starch granule surface. The isa3 mutant has a starch excess phenotype, reflecting impaired starch breakdown. In contrast, removal of AMY3, LDA, or both enzymes together has no impact on starch degradation. However, removal of AMY3 or LDA in addition to ISA3 enhances the starch excess phenotype. In plants lacking all three enzymes, starch breakdown is effectively blocked, and starch accumulates to the highest levels observed so far. This provides indirect evidence that the heteromultimeric debranching enzyme ISA1-ISA2 is not involved in starch breakdown. However, we illustrate that ISA1-ISA2 can hydrolyze small soluble branched glucans that accumulate when ISA3 and LDA are missing, albeit at a slow rate. Starch accumulation in the mutants correlates inversely with plant growth.

Knockdown of MYB305 disrupts nectary starch metabolism and floral nectar production.

MYB transcription factors have important roles during floral organ development. In this study, we generated myb305 RNAi knockdown tobacco plants and studied the role of MYB305 in the growth of the floral nectary. We have previously shown the MYB305 regulates the expression of flavonoid metabolic genes as well as of nectar proteins (nectarins); however, the myb305 plants showed other floral phenotypes that we investigate in these studies. The nectaries of myb305 plants show juvenile character at late stages of development and secrete reduced levels of nectar. Because starch metabolism is intimately involved in nectar secretion and is strongly regulated during normal nectary development, we examined the accumulation of starch in the nectaries of the myb305 plants. The myb305 plants accumulated lower levels of starch in their nectaries than did wild-type plants. The reduced starch correlated with the reduced expression of the ATP-glucose pyrophosphorylase (small subunit) gene in nectaries of the myb305 plants during the starch biosynthetic phase. Expression of genes encoding several starch-degrading enzymes including ß-amylase, isoamylase 3, and α-amylase was also reduced in the myb305 plants. In addition to regulating nectarin and flavonoid metabolic gene expression, these results suggest that MYB305 may also function in the tobacco nectary maturation program by controlling the expression of starch metabolic genes.

Function of isoamylase-type starch debranching enzymes ISA1 and ISA2 in the Zea mays leaf.

Conserved isoamylase-type starch debranching enzymes (ISAs), including the catalytic ISA1 and noncatalytic ISA2, are major starch biosynthesis determinants. Arabidopsis thaliana leaves require ISA1 and ISA2 for physiological function, whereas endosperm starch is near normal with only ISA1. ISA functions were characterized in maize (Zea mays) leaves to determine whether species-specific distinctions in ISA1 primary structure, or metabolic differences in tissues, are responsible for the differing ISA2 requirement. Genetic methods provided lines lacking ISA1 or ISA2. Biochemical analyses characterized ISA activities in mutant tissues. Starch content, granule morphology, and amylopectin fine structure were determined. Three ISA activity forms were observed in leaves, two ISA1/ISA2 heteromultimers and one ISA1 homomultimer. ISA1 homomultimer activity existed in mutants lacking ISA2. Mutants without ISA2 differed in leaf starch content, granule morphology, and amylopectin structure compared with nonmutants or lines lacking both ISA1 and ISA2. The data imply that both the ISA1 homomultimer and ISA1/ISA2 heteromultimer function in the maize leaf. The ISA1 homomultimer is present and functions in the maize leaf. Evolutionary divergence between monocots and dicots probably explains the ability of ISA1 to function as a homomultimer in maize leaves, in contrast to other species where the ISA1/ISA2 heteromultimer is the only active form.

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.

Enhancing the cyclodextrin production by synchronous utilization of isoamylase and α-CGTase.

Cyclodextrins (CD) are cyclic α-1,4-glucans composed of glucose units, and they have multiple applications in food, pharmaceuticals, cosmetics, agriculture, chemicals, etc. CD are usually produced by cyclodextrin glycosyltransferase (CGTase) from starch. In the present study, a simultaneous conversion approach was developed to improve the yield of CD from starch by conjunction use of isoamylase with α-CGTase. The isoamylase of Thermobifida fusca was cloned and expressed in Escherichia coli BL21(DE3). The biochemical characterization of the enzyme showed that the optimum temperature and pH of the recombinant enzyme was 50 °C and 5.5, respectively, and it maintained 60 %, 85 % and 78 % relative activity at 30 °C, 40 °C and 60 °C, respectively. When the recombinant isoamylase and α-CGTase were used simultaneously to convert potato starch (15 %, w/v) into CD, the optimum conditions were found to be: 10 U of α-CGTase and 48 U of isoamylase per gram of substrate, with reaction temperature of 30 °C and pH 5.6. On the optimum condition, the total yield of CD reached 84.6 % (w/w) after 24 h, which was 31.2 % higher than transformation with α-CGTase alone. This is the first report of synchronous bioconversion of CD by both α-CGTase and isoamylase, and represents the highest efficiency of CD production reported so far.

Purification, characterization and cloning of a thermotolerant isoamylase produced from Bacillus sp. CICIM 304.

A novel thermostable isoamylase, IAM, was purified to homogeneity from the newly isolated thermophilic bacterium Bacillus sp. CICIM 304. The purified monomeric protein with an estimated molecular mass of 100 kDa displayed its optimal temperature and pH at 70 °C and 6.0, respectively, with excellent thermostability between 30 and 70 °C and pH values from 5.5 to 9.0. Under the conditions of temperature 50 °C and pH 6.0, the K m and V max on glycogen were 0.403 ± 0.018 mg/mg and 0.018 ± 0.001 mg/(min mg), respectively. Gene encoding IAM, BsIam was identified from genomic DNA sequence with inverse PCRs. The open reading frame of the BsIam gene was 2,655 base pairs long and encoded a polypeptide of 885 amino acids with a calculated molecular mass of 101,155 Da. The deduced amino acid sequence of IAM shared less than 40 % homology with that of microbial isoamylase ever reported, which indicated it was a novel isoamylase. This enzyme showed its obvious superiority in the industrial starch conversion 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.

[Advances in studying microbial GH13 starch debranching enzyme--a review].

Pullulanase and isoamylase belong to the GH13 family (glycoside hydrolase family 13) with similar sequence, catalytic mechanism and three-dimensional fold ((beta/alpha)8-barrel structure). Starch debranching enzymes can hydrolyze the alpha-1,6-glucosidic bonds at the branch sites of starch, and improve raw material utilization and production efficiency in the starch industry. In this review, the substrate specificity, protein structure, advances and new trends in the study of microbial GH13 starch debranching enzyme were systematically introduced. In addition, some opinions on the research status and prospect for starch debranching enzyme were discussed.

Functional diversity of isoamylase oligomers: the ISA1 homo-oligomer is essential for amylopectin biosynthesis in rice endosperm.

Rice (Oryza sativa) endosperm has two isoamylase (ISA) oligomers, ISA1 homo-oligomer and ISA1-ISA2 hetero-oligomer. To examine their contribution to starch synthesis, expression of the ISA1 or ISA2 gene was differently regulated in various transgenic plants. Although suppression of ISA2 gene expression caused the endosperm to have only the homo-oligomer, no significant effects were detected on the starch phenotypes. In contrast, ISA2 overexpression led to endosperm having only the hetero-oligomer, and starch synthesis in the endosperm was drastically impaired, both quantitatively and qualitatively, because the starch was devoid of typical starch features, such as thermal and x-ray diffraction properties, and water-soluble highly branched maltodextrins were accumulated. In the ISA2 overexpressed line, about 60% to 70% of the ISA1-ISA2 hetero-oligomer was bound to starch, while the ISA homo- and hetero-oligomers from the wild type were mostly present in the soluble form at the early milking stage of the endosperm. Detailed analysis of the relative amounts of homo- and hetero-oligomers in various lines also led us to the conclusion that the ISA1 homo-oligomer is essential, but not the ISA1-ISA2 oligomer, for starch production in rice endosperm. The relative amounts of ISA1 and ISA2 proteins were shown to determine the ratio of both oligomers and the stoichiometry of both ISAs in the hetero-oligomer. It was noted when compared with the homo-oligomer that all the hetero-oligomers from rice endosperm and leaf and potato (Solanum tuberosum) tuber were much more stable at 40°C. This study provides substantial data on the structural and functional diversity of ISA oligomers between plant tissues and species.

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 ß-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.