The Unique Branching Patterns of Deinococcus Glycogen Branching Enzymes Are Determined by Their N-Terminal Domains

Glycogen branching enzymes (GBE) or 1,4-α-glucan branching enzymes (EC 2.4.1.18) introduce α-1,6 branching points in α-glucans, e.g., glycogen. To identify structural features in GBEs that determine their branching pattern specificity, the Deinococcus geothermalis and Deinococcus radiodurans GBE (GBE_Dg and GBE_Dr, respectively) were characterized. Compared to other GBEs described to date, these Deinococcus GBEs display unique branching patterns, both transferring relatively short side chains. In spite of their high amino acid sequence similarity (88%) the D. geothermalis enzyme had highest activity on amylose while the D. radiodurans enzyme preferred amylopectin. The side chain distributions of the products were clearly different: GBE_Dg transferred a larger number of smaller side chains; specifically, DP5 chains corresponded to 10% of the total amount of transferred chains, versus 6.5% for GBE_Dr. GH13-type GBEs are composed of a central (β/α) barrel catalytic domain and an N-terminal and a C-terminal domain. Characterization of hybrid Deinococcus GBEs revealed that the N2 modules of the N domains largely determined substrate specificity and the product branching pattern. The N2 module has recently been annotated as a carbohydrate binding module (CBM48). It appears likely that the distance between the sugar binding subsites in the active site and the CBM48 subdomain determines the average lengths of side chains transferred.Glycogen is an energy reserve polymer of many animals and microorganisms. It is composed of a backbone of glucose residues linked by α-1,4 glycosidic bonds with α-1,6-linked side chains (7, 31). In bacteria, the linear α-1,4-glucan is synthesized from ADP-glucose by the enzyme glycogen synthase, which is thought to be involved in both initiation and elongation of the chain (40). Side chains are introduced by glycogen branching enzyme (GBE) or 1,4-α-glucan branching enzyme (EC 2.4.1.18). This enzyme catalyzes formation of α-1,6 branch points by cleaving an α-1,4 glycosidic linkage in the donor substrate and transferring the nonreducing end-terminal fragment of the chain to the C-6 hydroxyl position of an internal glucose residue that acts as the acceptor substrate (4). Depending on its source, GBEs have a preference for transferring different lengths of glucan chains (1, 23). Most GBEs are members of subfamily 8 (Eukaryota) or 9 (Bacteria) of glycoside hydrolase family 13 (GH13) (34). Recently, the first GBE from family GH57 was described (28) (http://www.cazy.org).GH13-type GBEs are composed of three major domains of secondary structure, a central (β/α) barrel catalytic domain or A domain, an N-terminal domain, and a C-terminal domain (1). Domain A is present in all members of family GH13 and consists of a highly symmetrical fold of eight parallel β-strands encircled by eight α-helices. However, some variations occur in GBEs (a missing α-helix 5 and insertion of extra α-helices) (1). Domain A contains the four conserved amino acid regions (I to IV) typical for enzymes of family GH13 (35). In most GH13 enzymes, an extra domain is present, inserted between β-strand 3 and α-helix 3 (domain B), which affects their catalysis and product specificity (16). In GBEs, the length of this loop is only 40 residues, not long enough to be considered a separate domain (1). Domain C is found in most GH13 enzymes and is believed to shield the hydrophobic residues of the catalytic domain from contacts with the solvent. Domain C has also been suggested to be involved in substrate binding (25).Domain N is typical for GH13 enzymes cleaving or forming endo-α-1,6 linkages (17), namely, isoamylase (EC 3.2.1.68; subfamily 11) (18), pullulanase (EC 3.2.1.41; subfamilies 12 to 14) (27), and both starch (subfamily 8) and glycogen branching enzymes (subfamilies 8 and 9). An exception is the 4-α-d-{(1→4)-α-d-glucano}trehalose trehalohydrolase (EC 3.2.1.141; subfamily 10), which hydrolyzes linear maltooligosaccharide-like substrates (6) (39). The crystal structures of most of these enzymes with their N domains (all or part) have been published previously (1, 6, 18, 27, 39). The exact function of this N domain has remained unclear, and the similarity between the N domains in these different enzymes is low. They vary in length, and some of them consist of two or three modules. However, they all possess one common module that was recently classified as a family 48 carbohydrate-binding-module (CBM48) (19) (http://www.cazy.org/).In GBEs domain N comprises a module of 150 amino acids, termed the N2 module, that contains the putative CBM48. In some branching enzymes, it is preceded by a module of 100 to 150 amino acids, termed the N1 module. It has been proposed that the N1 module has originated from a DNA duplication of the N2 module (24). Based on the architecture and length of the N domain, GBEs can be divided into group 1, containing both the N1 and the N2 modules, and group 2, containing only the N2 module (12). A 112-amino-acid truncation of the N1 module in E. coli GBE (group 1) resulted in a 40% reduction of enzyme activity (2) and an altered branching pattern (3). Further investigations of this N1 module, by sequential N-terminal deletions, showed that enzymes with the shorter N1 module transferred longer glucan chains (5). No studies have been reported thus far investigating the role of the N2 module (containing the putative CBM48 domain) in GBEs as well as in other GH13 members.Here, we report a detailed biochemical characterization of two GH13 GBEs from the extremophilic bacteria Deinococcus geothermalis and Deinococcus radiodurans. These two GBEs (GBE_Dg and GBE_Dr, respectively) generate unique branching patterns by transferring glucosidic chains that are shorter than those of other GBEs reported to date (9, 36, 38, 41). To investigate the role of the different domains in these enzymes, chimeras of GBE_Dg and GBE_Dr were constructed. Their characterization revealed that substrate and chain length specificity in these Deinococcus GH13 GBEs are largely determined by the putative CBM48 part of the N domain.     

Biochemical and Crystallographic Characterization of the Starch Branching Enzyme I (BEI) from Oryza sativa L

Starch branching enzyme (SBE) catalyzes the cleavage of α-1.4-linkages and the subsequent transfer of α-1.4 glucan to form an α-1.6 branch point in amylopectin. We overproduced rice branching enzyme I (BEI) in Escherichia coli cells, and the resulting enzyme (rBEI) was characterized with respect to biochemical and crystallographic properties. Specific activities were calculated to be 20.8 units/mg and 2.5 units/mg respectively when amylose and amylopectin were used as substrates. Site-directed mutations of Tyr235, Asp270, His275, Arg342, Asp344, Glu399, and His467 conserved in the α-amylase family enzymes drastically reduced catalytic activity of rBEI. This result suggests that the structures of BEI and the other α-amylase family enzymes are similar and that they share common catalytic mechanisms. Crystals of rBEI were grown under appropriate conditions and the crystals diffracted to a resolution of 3.0 Å on a synchrotron X-ray source.     

Differential Chain-Length Specificities of Two Isoamylase-Type Starch-Debranching Enzymes from Developing Seeds of Kidney Bean

Plant isoamylase-type starch-debranching enzymes (ISAs) hydrolyze α-1,6-linkages in α-1,4/α-1,6-linked polyglucans. Two ISAs, designated PvISA1/2 and PvISA3, were purified from developing seeds of kidney bean by ammonium sulfate fractionation and several column chromatographic procedures. The enzymes displayed different substrate specificities for polyglucans: PvISA1/2 showed broad chain-length specificities, whereas PvISA3 liberated specific chains with a DP of 2 to 4.     

Preparation and Characterization of Octenyl Succinic Anhydride Modified Taro Starch Nanoparticles

The polar surface and hydrophilicity of starch nanoparticles (SNPs) result in their poor dispersibility in nonpolar solvent and poor compatibility with hydrophobic polymers, which limited the application in hydrophobic system. To improve their hydrophobicity, SNPs prepared through self-assembly of short chain amylose debranched from cooked taro starch, were modified by octenyl succinic anhydride (OSA). Size via dynamic light scattering of OSA-SNPs increased compared with SNPs. Fourier transform infrared spectroscopy data indicated the OSA-SNPs had a new absorption peak at 1727 cm^-1, which was the characteristic peak of carbonyl, indicating the formation of the ester bond. The dispersibility of the modified SNPs in the mixture of water with nonpolar solvent increased with increasing of degree of substitution (DS). OSA-SNPs appear to be a potential agent to stabilize the oil-water systems.     

Production and Biochemical Characterization of Insecticidal Enzymes from Aspergillus fumigatus Toward Callosobruchus maculatus

In the present work, Aspergillus fumigatus is described as a higher producer of hydrolytic enzymes secreted in response to the presence of the Callosobruchus maculatus bruchid pest. This fungus was able to grow over cowpea weevil shells as a unique carbon source, secreting alkaline proteolytic and chitinolytic enzymes. Enzyme secretion in A. fumigatus was induced by both C. maculatus exoskeleton as well as commercial chitin, and alkaline proteolytic and chitinolytic activities were detected after 48 hours of growth. Furthermore, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the production of specific proteins. Among them, two extracellular alkaline proteinases from culture enriched with C. maculatus exoskeleton were purified after chromatographic procedures using ion exchange and affinity columns. These proteins, named AP15 and AP30, had apparent molecular masses of 15,500 and 30,000 Da, respectively, as estimated by SDS-PAGE electrophoresis and mass spectrometry. AP30 was classified as a serine proteinase because it was inhibited by 5 mM phenylmethylsulfonyl fluoride (100%) and 50 μM leupeptin (67.94%).     

On the Modified and Inflated Discrete Distributions

Firstly, a modified bivariate discrete distribution is considered where a set of counts are misreported as another set of counts with different modification rates. Variances and covariances are put in the closed form and for the case when all modification rates are the same, these variances and covariances are expressed as parabolic functions and they are actually evaluated for the bivariate negative binomial. Regarding the asymptotic distributions of the estimates, elements of variance-covariance matrix are obtained. Next, a multivariate inflated discrete distribution is taken up. For the case of inflated multivariate negative binomial, Bayesian estimates of inflation as well as those of parameters are given.     

水稻籽粒淀粉分支酶活性的遗传分析

用由247个株系组成的珍汕97B/密阳46重组自交系群体及其含207个分子标记的连锁图谱,在2002年和2003年分别测定亲本和重组自交系群体开花后10 d和20 d籽粒的淀粉分支酶的活性,检测到3个控制开花后10 d Q酶活性的主效应QTL(qnantitative trait loci),联合贡献率为10%,其中qQ10-6与环境发生显著的互作;分别检测到5对和2对染色体区间对开花后10 d、20 d Q酶活性的影响具有加性×加性上位性作用,其中开花后10 d的3对染色体区间具有显著的上位性×环境互作效应.由此可见,水稻籽粒Q酶活性相关基因的表达,受到环境因子的极大影响.     

玉米淀粉分支酶基因启动子的克隆与功能分析

以玉米品种“吉糯1号”的基因组DNA为模板,通过PCR扩增得到玉米淀粉分支酶基因的启动子序列,克隆到pMD18-TVector上,经测序,该启动子大小为934bp。与已报道的序列比较仅有14个核苷酸发生改变,同源性为98.5%。用该启动子取代植物表达载体pBI121的35S启动子,与GUS基因编码区连接,构建成融合质粒pSBE-GUS。经农杆菌介导法转化烟草,获得了转基因植株。GUS活性检测结果表明,由该启动子序列引导的GUS基因能在种子中表达,而在其他组织中表达微弱或未表达,证实该启动子具有种子特异性表达的功能。     

Characterization of the Negative Binomial and Gamma Distributions

Characterization of the negative binomial and gamma distributions by a conditional distribution and a linear regression, and the gamma distribution by the negative binomial distribution are given. An application to a random shock model is discussed.     

淀粉含量不同的玉米自交系籽粒淀粉积累及其关键酶活性

以2个高淀粉和2个低淀粉玉米自交系为材料,分析了玉米籽粒淀粉的动态积累规律,同时对高低淀粉玉米籽粒灌浆过程中淀粉生物合成关键酶活性的动态变化及其与淀粉积累动态的相关性进行讨论分析。研究结果表明:灌浆过程中4个自交系淀粉含量变化趋势均呈sigmoid型曲线。灌浆过程中ADPG-PPase(腺苷二磷酸葡萄糖焦磷酸化酶)、SSS(可溶性淀粉合成酶)、GBSS(颗粒结合淀粉合成酶)活性均呈单峰曲线变化,峰值都出现在20～30DAP(授粉后天数)。2个高淀粉自交系的Q酶(淀粉分支酶)活性也呈单峰曲线变化,峰值也出现在20DAP,而2个低淀粉自交系的Q酶活性则呈双峰曲线变化,2个峰值分别出现在15～20DAP和30～35DAP。4个自交系籽粒淀粉的积累速率与各自交系ADPG-PPase、SSS和GBSS的活性变化呈极显著正相关。各自交系关键酶活性之间,ADPG-PPase、SSS和GBSS三者间活性变化呈极显著正相关,这3种酶活性变化与Q酶活性变化也呈不同程度的正相关。     

The Control of Vascular Branching in Coleus 2. The Corner Traces

Corner trace connections are less well defined than those ofthe side bundle in Coleus, the locations of branch points, branchpartners, and number of connections made by a corner trace beingmore variable. The auxin balance between corner traces was alteredby leaf removal and by application of exogenous auxin. Branchingof new strands was shifted toward the pre-existing strand withthe lower auxin flux, but only within a narrow range of developmentalstages and with the imposition of a large auxin imbalance. Branchingoccurred only in nodal regions, as in control plants. Thus,auxin balance can be made to control xylem strand branching,but it does not account fully for the control of vascular branchingin intact plants. In the intact pattern, corner trace branchesappear to be directed toward the pre-existing strand with thehigher auxin flux. It is proposed that, in the vicinity of astrand with high flux, auxin is transported laterally withinthe nodal vascular cambium, facilitating vessel differentiationbetween strands in the derivatives of the vascular cambium.These vessels comprise the connections between traces. Coleus, vascular differentiation, vascular anatomy, vascular branching, vascular patterns, auxin, auxin balance, node     

The Control of Vascular Branching in Coleus 1. The Side Bundle

Xylary branching at the proximal end of a differentiating sidebundle was modified by surgical alteration of the surroundingleaf traces and manipulation of their auxin fluxes. Incisionsthrough one corner trace, with the other pre-existing traceintact, resulting in xylem differentiation in the branch ofthe newly formed side bundle toward only the severed trace.Application of IAA to the cut trace allowed xylem branchingof the new strand in both directions. With sufficient auxinimbalance created by increasing the concentration of the appliedIAA, the new xylem strand branched away from the higher auxinsource. Auxin relations were thus able to regulate the courseof differentiation of vascular strands, but their role in regulatingbranching patterns in intact plants may be questioned. Xylembranched exclusively toward an incised trace only when the auxinflux of the incised trace was virtually eliminated. Phloem andprocambium of the differentiating strand were unaffected bythis treatment. Coleus, vascular differentiation, vascular anatomy, vascular branching, vascular patterns, auxin, auxin balance, node     

On the Plasticization Process of Potato Starch: Preparation and Characterization

This study aimed to gain a deep understanding of the preparation mechanism of the thermoplastic potato starch (TPPS) by using melt-mixing as a production method, to pursue the changes occurred on the microstructure, morphology and thermal properties of potato starch, TPPS was prepared using a mixture of potato starch with glycerol and water as plasticizer in an internal mixer. The steps of the phase transition, happening by applying harsh conditions (60 rpm, 160 °C, and 7 min), were followed by monitoring the evolution of torque during the mixing time. It was shown that the granules structure was destroyed and a new phase was formed. This was proved by SEM which gave the evidence that the morphology of the TPPS was homogeneous with the smooth surface means that the mixing conditions used in this work were good enough to obtain the thermoplastic starch with a high level of homogeneity in all dimensions. FTIR analysis allowed deducing the formation of new H-bonds between the starch and plasticizers molecules instead of intra and intermolecular H-bonds in the native starch that was destructed through the melt-mixing process., These caused starch chains gain mobility and as the results decreasing in crystallinity, where the XRD analysis exhibited that the crystallinity decreased from 14.5% resulting from B-type in native potato starch to 9% resulting from B-type and VH-type in TPPS. TGA and DSC analysis proved a decreasing in the thermal stability in the TPPS as compared to the starch granules.     

Organ Specificity of Isoforms of Starch Branching Enzyme (Q-Enzyme) in Rice

The activity and isoenzymes of starch branching enzyme or Q-enzymein the developing endosperm were compared with those in theleaf blade, leaf sheath, culm and root of rice plants. Q-enzymefrom each of these organs could be resolved into two fractions,QE I and QE II, by column chromatography on DEAE cellulose.However, the ratio of the activity of QE I to that of QE IIvaried considerably among the organs. The Q-enzyme from theendosperm was specific for that organ in that the enzyme activity,on the basis of either fresh weight or soluble protein content,was about 100- to 1,000-fold higher than those from the otherorgans. Moreover, in the endosperm, the activity of QE I wasmarkably higher than that of QE II as compared with the relativelevels in other organs. Native polyacrylamide gel electrophoresisfollowed by activity staining revealed that the QE II fractionwas composed of multiple isoforms. The endosperm contained twoisoforms, QE IIa and QE IIb. After electrophoresis on a nativepolyacrylamide gel, QE IIa was detected only in the extractof endosperm, whereas QE IIb was present in extract of all organsexamined. The antiserum raised against QE IIa from the endospermcross-reacted to a considerable extent with QE IIb from thesame organ. However, the antiserum failed to recognize any isoformsof QE II from the other organs. ¹ Present address: National Institute of Sericultural and EntomologicalScience, Tsukuba, Ibaraki, 305 Japan.     

淀粉分支酶sbeIIb基因RNA干涉片段转化玉米自交系的研究

玉米高直链淀粉育种是玉米分子育种的一个重要研究方向.本实验中,首先研究了不同诱导愈伤培养基对再生体系的影响,确定了LS+2,4-D 2.0 mg/L+L-pro 700 mg/L+CH 500 mg/L+3 %蔗糖为诱导培养基.同时,构建并验证了含有淀粉分支酶sbeIIb基因双干涉片段载体和胚乳特异性启动子的表达载体pCAMBIA 1301+Glu+1620,并转入根癌农杆菌EHA105,以农杆菌转化法转化玉米自交系178.通过PCR检测,5株转化株表现阳性,初步证明了干涉片段已整合入玉米基因组中.     

Focus on Metabolism: Molecular Genetic Analysis of Glucan Branching Enzymes from Plants and Bacteria in Arabidopsis Reveals Marked Differences in Their Functions and Capacity to Mediate Starch Granule Formation

The major component of starch is the branched glucan amylopectin, the branching pattern of which is one of the key factors determining its ability to form semicrystalline starch granules. Here, we investigated the functions of different branching enzyme (BE) types by expressing proteins from maize (Zea mays BE2a), potato (Solanum tuberosum BE1), and Escherichia coli (glycogen BE [EcGLGB]) in Arabidopsis (Arabidopsis thaliana) mutant plants that are deficient in their endogenous BEs and therefore, cannot make starch. The expression of each of these three BE types restored starch biosynthesis to differing degrees. Full complementation was achieved using the class II BE ZmBE2a, which is most similar to the two endogenous Arabidopsis isoforms. Expression of the class I BE from potato, StBE1, resulted in partial complementation and high amylose starch. Expression of the glycogen BE EcGLGB restored only minimal amounts of starch production, which had unusual chain length distribution, branch point distribution, and granule morphology. Nevertheless, each type of BE together with the starch synthases and debranching enyzmes were able to create crystallization-competent amylopectin polymers. These data add to the knowledge of how the properties of the BE influence the final composition of starch and fine structure of amylopectin.Starch is composed of two glucan polymers: amylopectin and amylose. Amylopectin constitutes around 80% of the mass of most starches and is a large, branched polymer with a tree-like architecture. The positioning and frequency of branch points together with the distribution of chain lengths are thought to be critical factors allowing amylopectin to adopt a semicrystalline state. Within amylopectin molecules, clusters of unbranched chain segments align, and adjacent chains form double helices. These pack into crystalline lamellae that alternate with amorphous regions containing the branch points. Longer chain segments span from one cluster to the next (Zeeman et al., 2010).Amylopectin is synthesized by three enzyme activities. First, starch synthases (SSs) transfer the glucosyl part of ADP-Glc to the nonreducing end of existing glucan chains, forming new α-1,4 glucosidic bonds. Second, branching enzymes (BEs) cleave part of an α-1,4-linked chain and through an inter- or intramolecular transfer reaction, reattach it, creating α-1,6-branch points. This reaction creates additional nonreducing ends on which SSs can act. Third, debranching enzymes (DBEs) hydrolyze some of these branches, tailoring the structure of the polymer to promote its crystallization.Several SS and BE isoforms are involved in starch synthesis in plants. There are five conserved classes of SSs (granule-bound starch synthase [GBSS] and SS1–SS4) and two conserved classes of BEs (classes I and II; also referred to as classes B and A, respectively; Nougué et al., 2014). In addition, plants contain two classes of DBEs: isoamylases (ISAs) and limit dextrinases (LDAs; also called pullulanases). One ISA, a multimeric enzyme composed of either a mixture of ISA1 and ISA2 subunits or just ISA1 subunits, is primarily involved in amylopectin synthesis (James et al., 1995; Mouille et al., 1996; Nakamura et al., 1996; Delatte et al., 2005). The other DBEs (i.e. ISA3 and LDA) are primarily involved in starch degradation (Wattebled et al., 2005; Delatte et al., 2006).Based on the in vitro analysis of purified or recombinant proteins and the phenotypes of mutant plants, the different SS isoforms are proposed to have distinct, albeit overlapping, functions. SS1 is thought to preferentially elongate short chains produced by the branching reactions to between 8 and 12 Glc units (Delvallé et al., 2005; Fujita et al., 2006). SS2 is proposed to elongate such chains farther to about 20 Glc units, optimal for cluster formation (Edwards et al., 1999; Umemoto et al., 2002; Zhang et al., 2008). The precise role of SS3 is less clear, although it has been proposed to generate long, cluster-spanning chains (Fujita et al., 2007). SS4 has a distinct role in initiating and/or coordinating granule formation (Roldán et al., 2007; Crumpton-Taylor et al., 2013).The two different BE classes are also proposed to have distinct functions in amylopectin synthesis. In vitro analyses of maize (Zea mays), rice (Oryza sativa), and potato (Solanum tuberosum) enzymes suggest that the class I enzymes preferentially act on amylose and transfer longer chains, whereas class II enzymes preferentially act on branched substrates, such as amylopectin, and transfer shorter chains (Guan and Preiss, 1993; Rydberg et al., 2001; Nakamura et al., 2010). This knowledge derives largely from experiments where linear or branched substrates were provided to recombinant or purified enzymes and the increased degree of branching was monitored. Similar conclusions were gained by recombinant protein expression in Escherichia coli and yeast (Saccharomyces cerevisiae) strains deficient in their endogenous glycogen BEs (Guan et al., 1995; Seo et al., 2002), where chain elongation by glycogen synthases occurred concurrently with branching.Models have been proposed in which both BE classes help create the final cluster structure of amylopectin: class I BEs initiate branching by transferring long or branched chains, which are subsequently acted on by class II BEs to create more numerous shorter chains. These shorter chains are then elaborated by the SSs to create the clusters (Nakamura et al., 2010). After the branching reactions, a degree of debranching occurs, which is thought to control branch number and positioning and thereby, facilitate amylopectin crystallization (Myers et al., 2000; Zeeman et al., 2010). Several studies have shown that isa1-deficient mutants produce starch with an altered amylopectin, accumulate a related soluble polymer (phytoglycogen), or both (James et al., 1995; Mouille et al., 1996; Nakamura et al., 1996; Delatte et al., 2005).Despite the wide conservation of the two BE classes, major alterations in starch properties are only observed when genes encoding class II enzymes are mutated or repressed. Loss of class I BE activity in maize endosperm, rice endosperm, or potato tuber did not alter starch content and caused only minor differences in amylopectin structure (e.g. the distribution of chain lengths and branch points) and/or starch properties (e.g. gelatinization or digestibility; Safford et al., 1998; Blauth et al., 2002; Satoh et al., 2003; Xia et al., 2011). In contrast, loss of class II BE results in significant changes, such as decreased starch content and a high apparent amylose content. This has been observed in several species, including maize (Stinard et al., 1993), potato (Jobling et al., 1999), pea (Pisum sativum; Bhattacharyya et al., 1990), rice (Mizuno et al., 1993), barley (Hordeum vulgare; Regina et al., 2010), and wheat (Triticum aestivum; Regina et al., 2006). The high apparent amylose content was caused at least in part by the accumulation of less-frequently branched amylopectin that stains with a higher wavelength of maximal absorption (λ_max) than that of the wild type (Boyer et al., 1976). In potato, this phenotype was enhanced by the simultaneous suppression of BE1 (Schwall et al., 2000), a result also shown recently in barley (Carciofi et al., 2012).Arabidopsis (Arabidopsis thaliana) has three genes annotated as BEs, At3g20440 (BE1), At5g03650 (BE2), and At2g36390 (BE3), but it seems that only BE2 and BE3 are active. Both BE2 and BE3 are class II BEs, making Arabidopsis somewhat unusual in not possessing a class I BE. The gene annotated as BE1 encodes a related protein that falls into a separate clade to either class I or II BEs (Dumez et al., 2006; Han et al., 2007; Wang et al., 2010). It was initially suggested that plants with mutations in this gene had a wild-type phenotype (Dumez et al., 2006), but subsequent work indicated that homozygous be1 mutation causes embryo lethality (hence, its alternative name EMBRYO DEFECTIVE2729; Wang et al., 2010). Thus, the function of the protein encoded at At3g20440 is currently unknown but unlikely to be a functional BE.The be2 and be3 single mutants have phenotypes that closely resemble the wild type, indicating that there is a high degree of redundancy between the enzymes. However, be2be3 double mutants lack starch (Dumez et al., 2006). Instead, the plants accumulate large amounts of maltose and other linear malto-oligosaccharides (MOSs). This is presumably because linear chains produced by the SSs are cleaved by starch-degrading enzymes (α- and β-amylases; Dumez et al., 2006). The altered metabolism of these double-mutant plants impedes growth, and they are smaller and paler than the wild type. The precise reason for this is unclear.In addition to mutagenesis, there have been several studies where BEs were overexpressed in transgenic plants. Overexpression of the E. coli glycogen BE (EcGLGB) in potato tubers or rice endosperm resulted in an increased degree of branching of amylopectin (Shewmaker et al., 1994; Kortstee et al., 1996; Kim et al., 2005). Overexpression of endogenous plant BE2 genes has also been performed in both rice and potato, increasing the proportion of shorter amylopectin chains (Tanaka et al., 2004; Brummell et al., 2015), and rice, leading to the accumulation of highly branched, water-soluble polysaccharides (Tanaka et al., 2004). Transgenic expression of genes from different photosynthetic organisms has also shown the degree of functional conservation within the plant BE classes. Sawada et al. (2009) showed that class II BE from Chlorella kessleri could rescue the BE2b-deficient phenotype in rice endosperm.The aim of this work was to investigate the capacity of different types of BEs to mediate starch granule formation by assessing their ability to function in the context of an otherwise intact starch biosynthesis pathway. To do this, we used the Arabidopsis be2be3 double mutants as a line in which to express three types of BEs. We chose BE2a from maize (required for leaf starch synthesis and similar to the endogenous Arabidopsis proteins; Yandeau-Nelson et al., 2011), BE1 from potato (represents the plant class I BEs that Arabidopsis lacks; Safford et al., 1998), and GLGB (the BE from E. coli involved in glycogen biosynthesis). This approach differs from previous investigations, because the activity of each BE type (working in planta with the same set of SSs and DBEs) can be assessed, and the results can be directly compared. In addition, we sought to address whether a glycogen BE was sufficient for starch production—in other words, whether the remaining starch biosynthetic enzymes are capable of generating a crystallization competent polymer, even when partnered with a BE with a different specificity. In previously described transgenic plants expressing E. coli GLGB, the endogenous plant BEs were still present (Shewmaker et al., 1994; Kortstee et al., 1996; Kim et al., 2005).In the transgenic lines generated here, we analyzed glucan synthesis, starch structure, and composition. Our results show that all three BE types can mediate starch granule production but to differing degrees. In each case, the structure of amylopectin and the amylose content depend on the type of BE present, as does starch granule morphology. We discuss the reasons for these differences in relation to previously reported BE properties.     

稻米淀粉品质形成的关键酶及其分子生物学研究进展

稻米淀粉的形成是影响水稻产量和品质的决定性因素之一。因此,开展稻米淀粉形成过程中所涉及关键酶的研究是非常必要的。随着分子生物学技术的快速发展,有关稻米淀粉品质的研究也越来越深入,并取得了较大进展。该文对水稻淀粉品质形成过程中的关键酶及其分子生物学研究进展进行了较为详尽的综述,主要包括ADP葡萄糖焦磷酸化酶、淀粉合成酶、淀粉分支酶和淀粉去分支酶等,并对该领域的发展趋势进行了展望。     

Visualization of Gluten and Starch Distributions in Dough by Fluorescence Fingerprint Imaging

A novel method combining imaging techniques and fluorescence fingerprint (FF) data measurement was developed to visualize the distributions of gluten and starch in dough without any preprocessing. Fluorescence images of thin sections of gluten, starch, and dough were acquired under 63 different combinations of excitation and emission wavelengths, resulting in a set of data consisting of the FF data for each pixel. Cosine similarity values between the FF of each pixel in the dough and those of gluten and starch were calculated. Each pixel was colored according to the cosine similarity value to obtain a pseudo-color image showing the distributions of gluten and starch. The dough sample was then fluorescently stained for gluten and starch. The stained image showed patterns similar to the pseudo-color FF image, validating the effectiveness of the FF imaging method. The method proved to be a powerful visualization tool, applicable in fields other than food technology.