Starch Synthesis In Plants Research Articles

The effect of trehalose on the contents of starch and oil in microalga Dunaliella parva

Dunaliella parva (D. parva) is a green alga with an extremely salt tolerance, that can synthesize a lot of oil. Its oil composition is similar to that of oil plants. It can accumulate a large amount of oil under stress conditions, making it an ideal raw material for biodiesel production. Previous studies have found that trehalose can inhibit starch synthesis in plants. In this study, D. parva was treated with trehalose at different concentrations (1 mM, 2 mM, 3 mM, 5 mM, 10 mM) to explore the suitable trehalose concentration to increase oil content. The results showed that 3 mM trehalose significantly reduced starch content by inhibiting the expression of ADP-glucose pyrophosphorylase (AGPase) gene and increased oil content. Trehalose also affected the contents of carbohydrate and protein in D. parva. In addition, trehalose treatment increased the proportions of palmitoleic acid, linolenic acid, stearic acid, linoleic acid, and reduced the proportions of myristic acid, palmitic acid, and oleic acid. This study investigated the effect of trehalose on D. parva, and improved its economic benefits.

Comprehensive genomic identification of cotton starch synthase genes reveals that GhSS9 regulates drought tolerance.

Starch metabolism is involved in the stress response. Starch synthase (SS) is the key enzyme in plant starch synthesis, which plays an indispensable role in the conversion of pyrophosphoric acid to starch. However, the SS gene family in cotton has not been comprehensively identified and systematically analyzed. In our study, a total of 76 SS genes were identified from four cotton genomes and divided into five subfamilies through phylogenetic analysis. Genetic structure analysis proved that SS genes from the same subfamily had similar genetic structure and conserved sequences. A cis-element analysis of the SS gene promoter showed that it mainly contains light response elements, plant hormone response elements, and abiotic stress elements, which indicated that the SS gene played key roles not only in starch synthesis but also in abiotic stress response. Furthermore, we also conducted a gene interaction network for SS proteins. Silencing GhSS9 expression decreased the resistance of cotton to drought stress. These findings suggested that SS genes could be related to drought stress in cotton, which provided theoretical support for further research on the regulation mechanism of SS genes on abiotic starch synthesis and sugar levels.

NnABI4-Mediated ABA Regulation of Starch Biosynthesis in Lotus (Nelumbo nucifera Gaertn).

Starch is an important component in lotus. ABA is an important plant hormone, which plays a very crucial role in regulating plant starch synthesis. Using ‘MRH’ as experimental materials, the leaves were sprayed with exogenous ABA before the rhizome expansion. The results showed that stomatal conductance and transpiration rate decreased while net photosynthetic rate increased. The total starch content of the underground rhizome of lotus increased significantly. Meanwhile, qPCR results showed that the relative expression levels of NnSS1, NnSBE1 and NnABI4 were all upregulated after ABA treatment. Then, yeast one-hybrid and dual luciferase assay suggested that NnABI4 protein can promote the expression of NnSS1 by directly binding to its promoter. In addition, subcellular localization results showed that NnABI4 encodes a nuclear protein, and NnSS1 protein was located in the chloroplast. Finally, these results indicate that ABA induced the upregulated expression of NnABI4, and NnABI4 promoted the expression of NnSS1 and thus enhanced starch accumulation in lotus rhizomes. This will provide a theoretical basis for studying the molecular mechanism of ABA regulating starch synthesis in plant.

Melatonin Regulates Chloroplast Protein Quality Control via a Mitogen-Activated Protein Kinase Signaling Pathway.

Serotonin N-acetyltransferase 1 (SNAT1), the penultimate enzyme for melatonin biosynthesis has shown N-acetyltransferase activity toward multiple substrates, including histones, serotonin, and plastid proteins. Under two different light conditions such as 50 or 100 μmol m−2 s−1, a SNAT1-knockout (snat1) mutant of Arabidopsis thaliana ecotype Columbia (Col-0) exhibited small size phenotypes relative over wild-type (WT) Arabidopsis Col-0. Of note, the small phenotype is stronger when growing at the 50 μmol m−2 s−1, exhibiting a dwarfism phenotype and delayed flowering. The snat1 Arabidopsis Col-0 accumulated less starch than the WT Col-0. Moreover, snat1 exhibited lower Lhcb1, Lhcb4, and RBCL protein levels, compared with the WT Col-0, but no changes in the corresponding transcripts, suggesting the involvement of melatonin in chloroplast protein quality control (CPQC). Accordingly, caseinolytic protease (Clp) and chloroplast heat shock proteins (CpHSPs), two key proteins involved in CPQC, as well as ROS defense were suppressed in snat1. In contrast, exogenous melatonin treatment induced expression of Clp, CpHSP, APX1, and GST, but not other growth-related genes such as DWF4, KS, and IAA1. Finally, the induction of ClpR1, APX1, and GST1 in response to melatonin was inhibited in the mitogen-activated protein kinase (MAPK) knockdown Arabidopsis (mpk3/6), suggesting that melatonin-mediated CPQC was mediated, in part, by the MAPK signaling cascade. These results suggest that melatonin is involved in CPQC, which plays a pivotal role in starch synthesis in plants.

Functional characterization of a starch synthesis-related gene AmAGP in Amorphophallus muelleri

ABSTRACT Amorphophallus has attracted tremendous interest because of its high contents of glucomannan and starch. Very few genes regulating glucomannan and starch were reported in Amorphophallus. In this study, an ADP-glucose pyrophosphorylase (AGP) gene that plays a significant role in plant starch synthesis was cloned from Amorphophallus muelleri. It was shown that it encoded a predicted protein containing a conserved plant ADP-Glucose-PP repeat domain and seven potential ligand-binding sites. The real-time quantitative PCR showed that AmAGP was most abundant in tubers, and it was positively correlated with starch content. Additionally, its influencers about temperature and exogenous plant hormone were also discussed, showing that AmAGP expressed highly in tubers under treatments using 25°C and IAA. Furthermore, starch content was closely related to AmAGP expression level, suggesting that AmAGP was involved in the regulation of starch synthesis in A. muelleri. Therefore, identifying the sequence of AmAGP and its expression pattern during tuber enlarging and the changes of its transcript levels in response to temperature and plant hormones would contribute to a better understanding of starch synthesis, and also providing a reference information for future preferable breeding for obtaining more starch or more glucomannan in Amorphophallus.

On the stability of nucleoside diphosphate glucose metabolites: implications for studies of plant carbohydrate metabolism.

Nucleoside diphosphate sugars (NDP-sugars) are the substrates for biosynthesis of oligo- and polysaccharides, such as starch and cellulose, and are also required for biosynthesis of nucleotides, ascorbic acid, several cofactors, glycoproteins and many secondary metabolites. A controversial study that questions the generally accepted pathway of ADP-glucose and starch synthesis in plants is based, in part, on claims that NDP-sugars are unstable at alkaline pH in the presence of Mg2+ and that this instability can lead to unreliable results from in vitro assays of enzyme activities. If substantiated, this claim would have far-reaching implications for many published studies that report on the activities of NDP-sugar metabolizing enzymes. To resolve this controversy, we investigated the stability of UDP- and ADP-glucose using biophysical, namely nuclear magnetic resonance (NMR), and highly specific enzymatic methods. Results obtained with both techniques indicate that NDP-sugars are not as unstable as previously suggested. Moreover, their calculated in vitro half-lives are significantly higher than estimates of their in planta turnover times. This indicates that the physico-chemical stability of NDP-sugars has little impact on their concentrations in vivo and that NDP-sugar levels are determined primarily by the relative rates of enzymatic synthesis and consumption. Our results refute one of the main arguments for the controversial pathway of starch synthesis from imported ADP-glucose produced by sucrose synthase in the cytosol.

Structure/function studies of Diverse Forms of ADPGlucose Pyrophosphorylase

ADPGlucose Pyrophosphorylase (ADPG PPase) is an allosterically regulated enzyme that serves as the rate‐limiting step of starch synthesis in plants and glycogen synthesis in bacteria. Because starch is a source of renewable and biodegradable carbon, ADPG PPase is an attractive target for protein engineering to increase biomass yield. The microbial versions of this enzyme are quite diverse in their regulatory and physical properties in accord with adaptation to different environments; some of these properties would be useful to incorporate into crops to enhance starch production.The enzyme from Deinococcus radiodurans (D. rad), coded for by the glgC gene, is an extremophile resistant to radiation and harsh growth conditions. Using standard assays with magnesium (Mg) as a co‐factor, the purified D. rad enzyme was found to have relatively low apparent affinity for the substrate ATP and Mg with S0.5 values of 8.4 mM and 24 mM, respectively. However, this organism is known to have a high intracellular manganese (Mn) concentration, which correlates with ionizing radiation and desiccation resistance. Because stored glycogen could act as an energy and carbon source during cell recovery, it was hypothesized that this ADPG PPase may be more responsive to Mn. Use of Mn in the assay revealed a ~9.3 fold increase in apparent affinity for ATP, a ~5 fold increase in apparent affinity for Mn (versus Mg), and a two‐fold increase in Vmax which supports the hypothesis. Complete kinetic characterization of the wild‐type (WT) and selected altered enzymes is underway to probe the putative metal binding site(s). Several crystallization conditions were found to yield promising results and further crystallization trials are in progress.In contrast, the enzyme from the thermophile Thermotoga maritima (T.ma) consists of two subunits, coded for by the glgC and glgD genes. Previous studies indicated that the purified WT T.ma glgD alone had no activity but was found to stimulate the relatively low activity of the glgC subunit. The complex could also be activated by FBP while the glgC alone was insensitive to FBP. The apparent affinity for ATP and Mg increased ~2 and ~3 fold in the complex while the Vmax increased 4 fold. Previous results indicated that limited proteolysis of the C‐terminus of glgD resulted in lower activation of the complex. In order to probe this further, a 73 amino acid C‐terminal truncation was generated and the activity of the complexes with the WT and WT glgC/truncated glgD (altered complex) compared. The altered complex exhibited a lower apparent affinity for ATP than the WT complex as well as a 5‐fold reduction in Vmax, which still represented a ~3‐fold stimulation compared to the glgC alone. In the presence of the activator FBP, the altered complex was slightly inhibited by FBP in contrast to the 2‐fold activation of the WT complex. The results appear to support a role for the C‐terminus in allosteric activation by FBP and partial stimulation of the enzyme in the absence of FBP. A 3 Å crystal structure for the glgC subunit was recently solved which was in good agreement with a previously modeled structure based on the A. tumefaciens enzyme structure (PDB 3BRK). Complete kinetic and physical characterization of the altered enzyme complexes in the absence and presence of FBP, as well as crystallization of various complexes are underway.Support or Funding InformationSupported in part by NSF BIO MCB Grant 0448676 and BIO DBI.

Structure‐Function Studies of Deinococcus radiodurans ADP Glucose Pyrophosphorylase: Role of Ser48 in Allosteric Regulation

Adenosine Diphosphate Glucose Pyrophosphorylase (ADPG PPase) is an allosterically regulated enzyme that functions as the rate‐limiting step of starch synthesis in plants and glycogen synthesis in bacteria. Because starch is a source of renewable and biodegradable carbon, ADPG PPase is an attractive target for protein engineering to increase biomass yield in crops. The microbial versions of this enzyme are quite diverse in their regulatory and physical properties; some of these properties would be useful to incorporate into transgenic crops to enhance starch production. While a number of ADPG PPases have been kinetically characterized, there is only one published x ray structure (PDB 3BRK) of an inhibited form. Very little is known about the enzyme from Deinococcus radiodurans (D. rad), an extremophile that is resistant to ionizing radiation and harsh growth conditions. When comparing the amino acid sequence of this enzyme to other characterized ADPG PPases, it was noted that position 48 differed with a serine substituted for alanine in a region known to be important for allostery. To probe the role of Ser‐48, the S48A enzyme was generated by site‐directed mutagenesis and the recombinant altered D. rad ADPG PPases successfully expressed in E. coli and purified via a scheme that includes anion exchange chromatography, size exclusion chromatography, and affinity chromatography. Initial studies on the S48A enzyme in the absence of activators have shown a dramatic 20‐fold increase in the apparent affinity for the substrate ATP, a 3‐fold increase in apparent affinity for the cofactor magnesium, and a 4‐fold increase in Vmax compared to wild‐type. Interestingly, in the presence of the activator FBP there was little change in the apparent binding affinity for substrates or Vmax compared to WT which displays a 14‐fold increase in Vmax and 12‐fold and 3‐fold increase in apparent affinity for ATP and magnesium, respectively. Similarly, in the presence of F6P, there were not a very large increase in Vmax or higher apparent affinity for ATP for the S48A enzyme, but there was a 9‐fold difference in Vmax and a 12‐fold and 2‐fold increase in apparent binding affinity for ATP and magnesium for WT, respectively. The WT enzyme also displayed higher apparent affinity for FBP and F6P compared to the S48A enzyme. The alanine substitution appears to result in an enzyme form that is partially activated but relatively insensitive to activators. Further kinetic characterization of S48A in the presence of sulfate and the inhibitor phosphate is in progress. With respect to physical characterization, two crystallization conditions for S48A, one including imidazole and the other with lithium sulfate and PEG, were found to yield preliminary results. Further crystallization trials are in progress as a first step in elucidating the three‐dimensional structure.Support or Funding InformationSupported in part by NSF and NSF BIO MCB grant #0448676.

Probing the Role of Ser‐48 in the Regulation of Deinococcus radiodurans ADP Glucose Pyrophosphorylase

ADPGlucose Pyrophosphorylase (ADPG PPase) is an allosterically regulated enzyme that serves as the rate-limiting step of starch synthesis in plants and glycogen synthesis in bacteria. Because starch is a source of renewable and biodegradable carbon, ADPG PPase is an attractive target for protein engineering to increase biomass yield. The microbial versions of this enzyme are quite diverse in their regulatory and physical properties; some of these properties would be useful to incorporate into crops to enhance starch production. Little is known about the enzyme from Deinococcus radiodurans (D. rad), an extremophile resistant to radiation and harsh growth conditions. When comparing the sequence of this enzyme to others, position 48 differed with a serine substituted for alanine in a region known to be important for allostery. To probe the role of S48, the S48A enzyme was generated by site-directed mutagenesis and the altered D. rad ADPG PPase successfully expressed and purified. Initial studies on the S48A enzyme in the absence of activators have shown a 20-fold increase in the apparent affinity for the substrate ATP and a 4-fold increase in Vmax compared to wild-type. Interestingly, in the presence of the activators FBP and F6P there were no change in the apparent binding affinity for substrates or Vmax compared to WT which displays a 5-fold increase in Vmax and 12-fold and 3-fold increase in apparent affinity for ATP and magnesium, respectively. The alanine substitution appears to result in an enzyme form that is partially activated but insensitive to activators. Complete kinetic analyses of the S48A enzyme are in progress. Supported in part by NSF Grant 0448676.

Investigating the Electrostatic Role of a Critical Arginine for the Catalysis of E. Coli ADP-Glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase (ADP-Glc PPase) is the regulatory enzyme of the pathway for starch synthesis in plants and glycogen in mammals and enteric bacteria. It exists as a 200 kDa homotetramer (α4) in enteric bacteria, and as a heterotetramer (α2β2) in plants. In both in vivo and in vitro the substrates (Glucose 1-Phosphate; Glc-1P and Adenosine 5'-Triphosphate; ATP) are converted into a glucose donor ADP-Glucose and a pyrophosphate (PPi) via the ADP-Glc PPase enzyme. It has been noted that some residues are conserved in homotetrameric bacterial ADP-Glc PPases, but are not in some plant forms. One of them is Arginine-32 (R32) in the Escherichia coli ADP-Glc PPase. To explore the overall role of this residue and evaluate the structural and electrostatic importance of the Arginine's guanidinium group, we replaced it with Lysine (K, -amino group), Alanine (A, - methyl group), Cysteine (C, -sulfide group), Glutamic (E, - carboxylate group), Glutamine (Q, -amido group) and Leucine (L, -hydrophobic side chain) via site directed mutagenesis. We over-expressed the enzymes, purified them to homogeneity, and measured their kinetic properties. The Specific Activity (U/mg) for the mutants were as follows: WT (90.56), R32A (1.65), R32C (0.57), R32E (0.04), R32K (5.81), R32L (0.65) and R32Q (1.37). Currently, the properties of the R32H (Histidine, -imidizoleum ring) mutant are being investigated. Our results clearly indicate that this guanidinium group of the Arginine-32 residue is critical for catalysis. Modeling of the E. coli enzyme suggests that the two (2) nitrogen atoms of the guanidinium group may interact with the β and γ phosphates of the ATP, helping in the positioning of the substrates, via electrostatic interactions, and making the PPi product a more stable leaving group.

RETRACTED: Molecular strategies in manipulation of the starch synthesis pathway for improving storage starch content in plants (review and prospect for increasing storage starch synthesis)

Starch is the most widespread and abundant storage carbohydrate in plants. We depend upon starch for our nutrition, exploit its unique properties in industry, and use it as a feedstock for bioethanol production. In recent decades, enormous progress has been made in understanding the genetic and biochemical mechanisms of starch synthesis in plants. Yet, despite this remarkable progress and its obvious economic importance, very little has been achieved in terms of adding value to starch or increasing starch content, particularly in cereal crops. In this paper, we first review recent advances in understanding the biochemistry of starch synthesis, particularly in identifying key enzymes involved in starch assembly. We then assess the progress in molecular strategies for improving storage starch content in plants. Finally, we discuss the problems faced in our profession and present ideas to effectively increase storage starch content in the future.

Insights into Glycogen Metabolism in Chemolithoautotrophic Bacteria from Distinctive Kinetic and Regulatory Properties of ADP-Glucose Pyrophosphorylase from Nitrosomonas europaea

Nitrosomonas europaea is a chemolithoautotroph that obtains energy by oxidizing ammonia in the presence of oxygen and fixes CO(2) via the Benson-Calvin cycle. Despite its environmental and evolutionary importance, very little is known about the regulation and metabolism of glycogen, a source of carbon and energy storage. Here, we cloned and heterologously expressed the genes coding for two major putative enzymes of the glycogen synthetic pathway in N. europaea, ADP-glucose pyrophosphorylase and glycogen synthase. In other bacteria, ADP-glucose pyrophosphorylase catalyzes the regulatory step of the synthetic pathway and glycogen synthase elongates the polymer. In starch synthesis in plants, homologous enzymes play similar roles. We purified to homogeneity the recombinant ADP-glucose pyrophosphorylase from N. europaea and characterized its kinetic, regulatory, and oligomeric properties. The enzyme was allosterically activated by pyruvate, oxaloacetate, and phosphoenolpyruvate and inhibited by AMP. It had a broad thermal and pH stability and used different divalent metal ions as cofactors. Depending on the cofactor, the enzyme was able to accept different nucleotides and sugar phosphates as alternative substrates. However, characterization of the recombinant glycogen synthase showed that only ADP-Glc elongates the polysaccharide, indicating that ATP and glucose-1-phosphate are the physiological substrates of the ADP-glucose pyrophosphorylase. The distinctive properties with respect to selectivity for substrates and activators of the ADP-glucose pyrophosphorylase were in good agreement with the metabolic routes operating in N. europaea, indicating an evolutionary adaptation. These unique properties place the enzyme in a category of its own within the family, highlighting the unique regulation in these organisms.

Subtle structural differences crucial for function in similarly engineered ADP-glucose pyrophosphorylase larger subunit in rice and maize

ADP – glucose pyrophosphorylase (AGPase) is a key enzyme for starch synthesis in plants. Heterotetrameric plant AGPase is encoded by two genes, Shrunken-2 (Sh2) and Brittle-2 (Bt2). The Sh2 gene encodes regulatory larger subunit and the Bt2 gene encodes smaller subunit having catalytic properties. A specific mutation in Sh2 gene involving insertion of six nucleotides, without changing the reading frame, resulted in the insertion of two additional amino acid residues serine and tyrosine at specific position at carboxyl end and also in an increase in seed weight up to 11–17%. No increase in seed weight with the same insertion in larger subunit of AGPase enzyme in rice was observed even though the rice and maize subunits have 93% of sequence similarity. In this study, the predicted 3D-structures of larger subunit of normal as well as mutated AGPase in maize and rice, were analyzed and superposed. The segment of six amino acid residues long secondary structure just before the site of insertion of additional amino acids (serine and tyrosine) got reduced in case of mutated maize but not in mutated rice. Therefore, the six residue sequence and corresponding subtle secondary structural difference might be the key factors for functional disparity of engineered larger subunits in the two crops.

Use of TILLING and robotised enzyme assays to generate an allelic series of Arabidopsis thaliana mutants with altered ADP-glucose pyrophosphorylase activity

ADP-glucose pyrophosphorylase (AGPase) catalyses the synthesis of ADP-glucose, and is a highly regulated enzyme in the pathway of starch synthesis. In Arabidopsis thaliana, the enzyme is a heterotetramer, containing two small subunits encoded by the APS1 gene and two large subunits encoded by the APL1-4 genes. TILLING ( Targeting Induced Local Lesions IN Genomes) of a chemically mutagenised population of A. thaliana plants identified 33 novel mutations in the APS1 gene, including 21 missense mutations in the protein coding region. High throughput measurements using a robotised cycling assay showed that maximal AGPase activity in the aps1 mutants varied from <15 to 117% of wild type (WT), and that the kinetic properties of the enzyme were altered in several lines, indicating a role for the substituted amino acid residues in catalysis or substrate binding. These results validate the concept of using such a platform for efficient high-throughput screening of very large populations of mutants, natural accessions or introgression lines. AGPase was estimated to have a flux control coefficient of 0.20, indicating that the enzyme exerted only modest control over the rate of starch synthesis in plants grown under short day conditions (8 h light/16 h dark) with an irradiance of 150 μmol quanta m −2 s −1. Redox activation of the enzyme, via reduction of the intermolecular disulphide bridge between the two small subunits, was increased in several lines. This was sometimes, but not always, associated with a decrease in the abundance of the APS1 protein. In conclusion, the TILLING technique was used to generate an allelic series of aps1 mutants in A. thaliana that revealed new insights into the multi-layered regulation of AGPase. These mutants offer some advantages over the available loss-of-function mutants, e.g. adg1, for investigating the effects of subtle changes in the enzyme's activity on the rate of starch synthesis.

Evolution of the Genes Encoding Starch Synthase in Sorghum and Common Wheat

Starch synthases (SSs) play important roles in plant starch synthesis. Five enzymetic isoforms of starch synthases have been found in plant, some of isoforms have further diverged possibly via whole genome duplication events, which might result in two or three subclasses of the sequences in rice and maize. In this study, we found the retention of GBSS, SSII, and SSIII duplicators except for the SSIV duplicator in genomes of sorghum and wheat. The SSIV gene might have been lost in maize and sorghum genomes based on the synteny relationship among rice maize and sorghum. Expression analyses indicated that the SS duplicators were also diverged from the duplicators of SbGBSSI, SbSSIIIa in expression pattern, and SbSSIIa were expressed mainly in endosperm in sorghum, whereas SbGBSSII, SbSSIIb, and SbSSIIIb of sorghum, TaSSIIb and TaSSIIIb of common wheat, were expressed mainly in leaves. Our findings, in combination with previous studies, indicate that the SSIV duplicator should be not remained in all Gramineae species, while the expression of duplicated SS genes diverged similarly in the studied species.

A novel Ta.AGP.S.1b transcript in Chinese common wheat ( Triticum aestivum L .)

ADP-glucose pyrophosphorylase (AGPase), the key enzyme of starch synthesis in plants, is composed of two small and two large subunits, and has plastidial and cytosolic isoforms. In kernels of wheat ( Triticum aestivum L .) and barley ( Hordeum vulgare L.), transcripts for cytosolic ( Ta.AGP.S1a) and plastidial ( Ta.AGP.S1b) small subunits of AGPase were encoded by the same gene ( Ta.AGP.S.1) by use of the alternative first exons. In this study, a cDNA sequence (1631 bp) [NCBI: EU586278] encoding a novel Ta.AGP.S1b transcript was isolated in kernels of Chinese common wheat cultivars. Compared with another Ta.AGP.S1b transcript [NCBI: FJ643609] isolated in kernels of non-Chinese wheat cultivars, EU586278 lacked a long fragment (117 bp) at its 5′terminal, resulting in a shorten transit peptide. The lacked fragments of Ta.AGP.S1b (EU586278) were universally found in surveyed 22 Chinese common wheat cultivars. Partial genomic DNA sequence [NCBI: FJ907395] of Ta.AGP.S.1 gene, which was corresponded to 5′terminal of EU586278 transcript, was also isolated in Chinese common cultivars and sequencing indicated that FJ907395 contained the corresponding lacked fragment of EU586278 transcript, inferring the lacked fragment in EU586278 transcript was not present in the genome, but possibly occurred at transcription level. Using TargetP software, the predicated transit peptide of putative plastidial SSU encoded by EU586278 contained merely 25 amino acids, considerably shorter than those of other plant AGP. S.1bs (54–70 amino acids). Phylogenetic tree analysis indicated that the amino acid sequence of EU586278 transit peptide was not clustered together with those of other wheat Ta.AGP.S1bs [NCBI: AF536819 and FJ643609] and barley AGP.S1b [NCBI: Z48563]. These implied that EU586278 could be a novel Ta.AGP.S1b transcript. Semi-quantitative PCR analysis indicated that transcripts of EU586278 were abundantly expressed in leaf, moderately in endosperm and stem, and weakly in root.

Ostreococcus tauri ADP-glucose Pyrophosphorylase Reveals Alternative Paths for the Evolution of Subunit Roles

ADP-glucose pyrophosphorylase controls starch synthesis in plants and is an interesting case to study the evolution and differentiation of roles in heteromeric enzymes. It includes two homologous subunits, small (S) and large (L), that originated from a common photosynthetic eukaryotic ancestor. In present day organisms, these subunits became complementary after loss of certain roles in a process described as subfunctionalization. For instance, the potato tuber enzyme has a noncatalytic L subunit that complements an S subunit with suboptimal allosteric properties. To understand the evolution of catalysis and regulation in this family, we artificially synthesized both subunit genes from the unicellular alga Ostreococcus tauri. This is among the most ancient species in the green lineage that diverged from the ancestor of all green plants and algae. After heterologous gene expression, we purified and characterized the proteins. The O. tauri enzyme was not redox-regulated, suggesting that redox regulation of ADP-glucose pyrophosphorylases appeared later in evolution. The S subunit had a typical low apparent affinity for the activator 3-phosphoglycerate, but it was atypically defective in the catalytic efficiency (V(max)/K(m)) for the substrate Glc-1-P. The L subunit needed the S subunit for soluble expression. In the presence of a mutated S subunit (to avoid interference), the L subunit had a high apparent affinity for 3-phosphoglycerate and substrates suggesting a leading role in catalysis. Therefore, the subfunctionalization of the O. tauri enzyme was different from previously described cases. To the best of our knowledge, this is the first biochemical description of a system with alternative subfunctionalization paths.

Role of the N-Terminal Starch-Binding Domains in the Kinetic Properties of Starch Synthase III from Arabidopsis thaliana

Starch synthase III (SSIII), one of the SS isoforms involved in plant starch synthesis, has been reported to play a regulatory role in the synthesis of transient starch. SSIII from Arabidopsis thaliana contains 1025 amino acid residues and has an N-terminal transit peptide for chloroplast localization which is followed by three repeated starch-binding domains (SBDs; SSIII residues 22-591) and a C-terminal catalytic domain (residues 592-1025) similar to bacterial glycogen synthase. In this work, we constructed recombinant full-length and truncated isoforms of SSIII, lacking one, two, or three SBDs, and recombinant proteins, containing three, two, or one SBD, to investigate the role of these domains in enzyme activity. Results revealed that SSIII uses preferentially ADPGlc, although UDPGlc can also be used as a sugar donor substrate. When ADPGlc was used, the presence of the SBDs confers particular properties to each isoform, increasing the apparent affinity and the V max for the oligosaccharide acceptor substrate. However, no substantial changes in the kinetic parameters for glycogen were observed when UDPGlc was the donor substrate. Under glycogen saturating conditions, the presence of SBDs increases progressively the apparent affinity and V max for ADPGlc but not for UDPGlc. Adsorption assays showed that the N-terminal region of SSIII, containing three, two, or one SBD module have increased capacity to bind starch depending on the number of SBD modules, with the D23 protein (containing the second and third SBD module) being the one that makes the greatest contribution to binding. The results presented here suggest that the N-terminal SBDs have a regulatory role, showing a starch binding capacity and modulating the catalytic properties of SSIII.

Subunit interactions specify the allosteric regulatory properties of the potato tuber ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase) catalyzes the first committed step of starch synthesis in plants. The potato tuber enzyme contains a pair of catalytic small subunits (SSs) and a pair of non-catalytic large subunits (LSs). We have previously identified a LS mutant containing a P52L replacement, which rendered the enzyme with down-regulatory properties. To investigate the structure–function relationships between the two subunits with regard to allosteric regulation, putative SS mutants that could reverse the down-regulatory condition of LS P52L were identified by their ability to restore glycogen accumulation in an AGPase-deficient Escherichia coli glgC-strain. Two distinct LS-dependent classes, bona fide SS suppressors dependent on LS P52L but not LS WT and SS up-regulating allosteric mutants, were evident by kinetic analysis. These results indicate that both LS and SS have a regulatory function in controlling allosteric properties through enhancing cooperative subunit interactions.

Three orthologs in rice, Arabidopsis, and Populus encoding starch branching enzymes (SBEs) are different from other SBE gene families in plants

Starch branching enzymes (SBEs) play important roles in plant starch synthesis. Three orthologs encoding SBEs in rice, Arabidopsis thaliana, and Populus trichocarpa are described. Putative amino acid sequences of these three SBE genes show ∼ 30% identity to those of SBEI and SBEII from plants such as maize, barley, and wheat. More interestingly, they share ∼ 31% amino acid sequence identity with those of glycogen-branching enzymes from such animals as mouse, horse, and monkey. The three genes have similar genomic structures, but their structural features are quite different from those of genes of both SBEI and SBEII families in plants. Based on phylogenetic analysis and genomic structure comparison, it is proposed that the three SBE genes represent a new family of SBEs.