Glucan Water Dikinase Research Articles

Glucan, water dikinase (GWD) penetrates the starch granule surface and introduces C6 phosphate in the vicinity of branching points

Starch phosphorylation mediated by α-glucan, water dikinase is an integral part of starch metabolism. So far however, it is not fully understood. For getting deeper insights, several in vitro assays and intensive mass spectrometry analyses were performed. Such analyses allowed us to determine the phosphorylation position within the amylopectin in detail. Thus, unique features of the starch structure and GWD action were correlated. Therefore, recombinant potato GWD (Solanum tuberosum L.; StGWD) was used for detailed analyses of the phosphorylation pattern of various starches. Additionally, oil palm (Elaeis guineensis Jacq.; EgGWD) GWD was cloned and characterized, representing the first characterization of GWD of a monocot species. The distribution patterns of single phosphorylated glucan chains catalyzed by both GWDs were compared. The phosphorylation distribution patterns of both GWDs varied for different starches. It was proven that GWD phosphorylates different positions within the amylopectin of native starch granules. GWD enters the starch granule surface and phosphorylates the glucosyl units in the proximity of branching points to convert the highly ordered glucan chains into a less ordered state and to render them accessible for the downstream acting hydrolases. This enables deciphering the GWD actions and the related structural properties of starch granules.

Suppression of the tonoplast sugar transporter, StTST3.1, affects transitory starch turnover and plant growth in potato.

Transitory starch and vacuolar sugars function as highly dynamic pools of instantly accessible metabolites in plant leaf cells. Their metabolic regulation is critical for plant survival. The tonoplast sugar transporters (TSTs), responsible for sugar uptake into vacuoles, regulate cellular sugar partitioning and vacuolar sugar accumulation. However, whether TSTs are involved in leaf transient starch turnover and plant growth is unclear. Here, we found that suppressing StTST3.1 resulted in growth retardation and pale green leaves in potato plants. StTST3.1-silenced plants displayed abnormal chloroplasts and impaired photosynthetic performance. The subcellular localization assay and the oscillation expression patterns revealed that StTST3.1 encoded a tonoplast-localized protein and responded to photoperiod. Moreover, RNA-seq analyses identified that starch synthase (SS2 and SS6) and glucan water, dikinase (GWD), were downregulated in StTST3.1-silenced lines. Correspondingly, the capacity for starch synthesis and degradation was decreased in StTST3.1-silenced lines. Surprisingly, StTST3.1-silenced leaves accumulated exceptionally high levels of maltose but low levels of sucrose and hexose. Additionally, chlorophyll content was reduced in StTST3.1-silenced leaves. Analysis of chlorophyll metabolic pathways found that Non-Yellow Coloring 1 (NYC1)-like (NOL), encoding a chloroplast-localized key enzyme that catalyzes the initial step of chlorophyll b degradation, was upregulated in StTST3.1-silenced leaves. Transient overexpression of StNOL accelerated chlorophyll b degradation in tobacco leaves. Our results indicated that StTST3.1 is involved in transitory starch turnover and chlorophyll metabolism, thereby playing a critical role in normal potato plant growth.

Carbon pathways during transitory starch degradation in Arabidopsis differentially affect the starch granule number and morphology in the dpe2/phs1 mutant background

The Arabidopsis knockout mutant lacking both the cytosolic disproportionating enzyme 2 (DPE2) and the plastidial phosphorylase (PHS1) had a dwarf-growth phenotype, a reduced and uneven distribution of starch within the plant rosettes, and a lower starch granule number per chloroplast under standard growth conditions. In contrast, a triple mutant impaired in starch degradation by its additional lack of the glucan, water dikinase (GWD) showed improved plant growth, a starch-excess phenotype, and a homogeneous starch distribution. Furthermore, the number of starch granules per chloroplast was increased and was similar to the wild type. We concluded that ongoing starch degradation is mainly responsible for the observed phenotype of dpe2/phs1. Next, we generated two further triple mutants lacking either the phosphoglucan, water dikinase (PWD), or the disproportionating enzyme 1 (DPE1) in the background of the double mutant. Analysis of the starch metabolism revealed that even minor ongoing starch degradation observed in dpe2/phs1/pwd maintained the double mutant phenotype. In contrast, an additional blockage in the glucose pathway of starch breakdown, as in dpe2/phs1/dpe1, resulted in a nearly starch-free phenotype and massive chloroplast degradation. The characterized mutants were discussed in the context of starch granule formation.

Strategies for Efficient Gene Editing in Protoplasts of Solanum tuberosum Theme: Determining gRNA Efficiency Design by Utilizing Protoplast (Research).

Potato (Solanum tuberosum) is a highly diverse tetraploid crop. Elite cultivars are extremely heterozygous with a high prevalence of small length polymorphisms (indels) and single nucleotide polymorphisms (SNPs) within and between cultivars, which must be considered in CRISPR/Cas gene editing strategies and designs to obtain successful gene editing. In the present study, in-depth sequencing of the glucan water dikinase (GWD)1 and the downy mildew resistant 6 (DMR6-1) genes in the potato cultivars Saturna and Wotan, respectively, revealed both indels and a 1.3–2.8 higher SNP prevalence when compared to the heterozygous diploid RH genome sequence as expected for a tetraploid compared to a diploid. This complicates guide RNA (gRNA) and diagnostic PCR designs. High editing efficiencies at the cell pool (protoplast) level are pivotal for achieving full allelic knock-out in tetraploids and for reducing the downstream cumbersome and delicate ex-plant regeneration. Here, CRISPR/Cas ribonucleoprotein particles (RNP) were delivered transiently to protoplasts by polyethylene glycol (PEG) mediated transformation. For each of GWD1 and DMR6-1, 6–10 gRNAs were designed to target regions comprising the 5′ and the 3′ end of the two genes. Similar to other studies including several organisms, editing efficiency of the individual RNPs/gRNAs varied significantly, and some generated specific indel patterns. While RNPs targeting the 5′ end of GWD1 yielded significantly higher editing when compared to targeting the 3′ end, editing efficiencies in the 5′ and 3′ end of DMR6-1 appeared to be somewhat similar. Simultaneous targeting of either the 5′ or the 3′ end with two RNPs (multiplexing) yielded a clear positive synergistic effect on the total editing when targeting the 3′ end of the GWD1 gene only. Multiplexing of the two genes, residing on different chromosomes, yielded no or slightly negative effects on the individual RNP/gRNA editing efficiencies when compared to editing efficiencies obtained in the single RNP/gRNA transformations. These initial findings may instigate larger studies needed for facilitating and optimizing precision breeding in plants.

Genomic regions associated with physiological, biochemical and yield-related responses under water deficit in diploid potato at the tuber initiation stage revealed by GWAS.

Water deficit, which is increasing with climate change, is a serious threat to agricultural sustainability worldwide. Dissection of the genetic architecture of water deficit responses is highly desirable for developing water-deficit tolerant potato cultivars and enhancing the resilience of existing cultivars. This study examined genetic variation in response to water deficit in a panel of diploid potato and identified the QTL governing this trait via a genome-wide association study (GWAS). A panel of 104 diploid potato accessions were evaluated under both well-watered and water deficit treatments at tuber initiation stage. Drought stress index (DTI) was calculated to assess tolerance of the diploid potato genotypes to water deficit. The GWAS was conducted using a matrix of 47K single nucleotide polymorphisms (SNP), recently available for this population. We are reporting 38 QTL, seven for well-watered conditions, twenty-two for water deficit conditions and nine for DTI which explain between 12.6% and 44.1% of the phenotypic variance. A set of 6 QTL were found to be associated with more than one variable. Marker WDP-9.21 was found associated with tuber fresh weigh under WD and gene annotation analysis revealed co-localization with the Glucan/water dikinase (GWD) gene. Of the nine QTL detected from DTI on chromosomes 2,3,5,8,10 and 12, three candidate genes with a feasible role in water deficit response were identified. The findings of this study can be used in marker-assisted selection (MAS) for water- deficit tolerance breeding in potato.

Elucidating the mechanism of MaGWD1-mediated starch degradation cooperatively regulated by MaMADS36 and MaMADS55 in banana

Banana (Musa acuminata) is a common starch-conversion fruit. Starch is rapidly degraded and converted to soluble sugars during postharvest banana fruit ripening. The conversion efficiency directly influences fruit firmness, sweetness, and shelf life. Glucan, water dikinase (GWD) is the first rate-limiting enzyme of starch metabolism. Musa acuminata glucan water dikinase 1 (MaGWD1) plays an important role in banana starch metabolism and its expression is regulated by transcription factors (TFs). However, the regulation of MaGWD1 by MADS-box TFs remains unclear. In this study, the molecular mechanism underlying the cooperative regulation of MaGWD1-mediated starch degradation by MaMADS36 and MaMADS55 was investigated. The results indicated that MaMADS36 interacts with MaMADS55. Both MaMADS36 and MaMADS55 could directly bind to the same position on the CA/T(r)G box within the MaGWD1 promoter. MaMADS36 and MaMADS55 cooperatively regulate banana fruit starch degradation via transcriptional control of MaGWD1.These findings improve our understanding of how TFs regulate starch degradation; this information may be used for banana fruit quality improvement and to support the development of the banana industry.

Mutations in Glucan, Water Dikinase Affect Starch Degradation and Gametophore Development in the Moss Physcomitrella patens

The role of starch degradation in non-vascular plants is poorly understood. To expand our knowledge of this area, we have studied this process in Physcomitrella patens. This has been achieved through examination of the step known to initiate starch degradation in angiosperms, glucan phosphorylation, catalysed by glucan, water dikinase (GWD) enzymes. Phylogenetic analysis indicates that GWD isoforms can be divided into two clades, one of which contains GWD1/GWD2 and the other GWD3 isoforms. These clades split at a very early stage within plant evolution, as distinct sequences that cluster within each were identified in all major plant lineages. Of the five genes we identified within the Physcomitrella genome that encode GWD-like enzymes, two group within the GWD1/GWD2 clade and the others within the GWD3 clade. Proteins encoded by both loci in the GWD1/GWD2 clade, named PpGWDa and PpGWDb, are localised in plastids. Mutations of either PpGWDa or PpGWDb reduce starch phosphate abundance, however, a mutation at the PpGWDa locus had a much greater influence than one at PpGWDb. Only mutations affecting PpGWDa inhibited starch degradation. Mutants lacking this enzyme also failed to develop gametophores, a phenotype that could be chemically complemented using glucose supplementation within the growth medium.

A banana R2R3-MYB transcription factor MaMYB3 is involved in fruit ripening through modulation of starch degradation by repressing starch degradation-related genes and MabHLH6.

Starch degradation is a necessary process determining banana fruit quality during ripening. Many starch degradation-related genes are well studied. However, the transcriptional regulation of starch degradation during banana fruit ripening remains poorly understood. In this study, we identified a MYB transcription factor (TF) termed MaMYB3, as a putative protein binding the promoter of MaGWD1, a member of glucan water dikinase (GWD) family which has been demonstrated as an important enzyme of starch degradation. MaMYB3 was ripening- and ethylene-repressible, and its expression was negatively correlated with starch degradation. Acting as a nucleus-localized transcriptional repressor, MaMYB3 repressed the transcription of 10 starch degradation-related genes, including MaGWD1, MaSEX4, MaBAM7-MaBAM8, MaAMY2B, MaAMY3, MaAMY3A, MaAMY3C, MaMEX1, and MapGlcT2-1, by directly binding to their promoters. Interestingly, a previously identified activator of starch degradation-related genes, MabHLH6, was also suppressed by MaMYB3. The ectopic overexpression of MaMYB3 in tomato down-regulated the expression of starch degradation-related genes, inhibited starch degradation and delayed fruit ripening. Based on these findings, we conclude that MaMYB3 negatively impacts starch degradation by directly repressing starch degradation-related genes and MabHLH6, and thereby delays banana fruit ripening. Collectively, our study expands our understanding of the complex transcriptional regulatory hierarchy modulating starch degradation during fruit ripening.

Modification of Cassava Root Starch Phosphorylation Enhances Starch Functional Properties.

Cassava (Manihot esculenta Crantz) is a root crop used as a foodstuff and as a starch source in industry. Starch functional properties are influenced by many structural features including the relative amounts of the two glucan polymers amylopectin and amylose, the branched structure of amylopectin, starch granule size and the presence of covalent modifications. Starch phosphorylation, where phosphates are linked either to the C3 or C6 carbon atoms of amylopectin glucosyl residues, is a naturally occurring modification known to be important for starch remobilization. The degree of phosphorylation has been altered in several crops using biotechnological approaches to change expression of the starch-phosphorylating enzyme GLUCAN WATER DIKINASE (GWD). Interestingly, this frequently alters other structural features of starch beside its phosphate content. Here, we aimed to alter starch phosphorylation in cassava storage roots either by manipulating the expression of the starch phosphorylating or dephosphorylating enzymes. Therefore, we generated transgenic plants in which either the wild-type potato GWD (StGWD) or a redox-insensitive version of it were overexpressed. Further plants were created in which we used RNAi to silence each of the endogenous phosphoglucan phosphatase genes STARCH EXCESS 4 (MeSEX4) and LIKE SEX4 2 (MeLSF), previously discovered by analyzing leaf starch metabolism in the model species Arabidopsis thaliana. Overexpressing the potato GWD gene (StGWD), which specifically phosphorylates the C6 position, increased the total starch-bound phosphate content at both the C6 and the C3 positions. Silencing endogenous LSF2 gene (MeLSF2), which specifically dephosphorylates the C3 position, increased the ratio of C3:C6 phosphorylation, showing that its function is conserved in storage tissues. In both cases, other structural features of starch (amylopectin structure, amylose content and starch granule size) were unaltered. This allowed us to directly relate the physicochemical properties of the starch to its phosphate content or phosphorylation pattern. Starch swelling power and paste clarity were specifically influenced by total phosphate content. However, phosphate position did not significantly influence starch functional properties. In conclusion, biotechnological manipulation of starch phosphorylation can specifically alter certain cassava storage root starch properties, potentially increasing its value in food and non-food industries.

EARLY STARVATION1 specifically affects the phosphorylation action of starch-related dikinases.

Starch phosphorylation by starch-related dikinases glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) is a key step in starch degradation. Little information is known about the precise structure of the glucan substrate utilized by the dikinases and about the mechanisms by which these structures may be influenced. A 50-kDa starch-binding protein named EARLY STARVATION1 (ESV1) was analyzed regarding its impact on starch phosphorylation. In various invitro assays, the influences of the recombinant protein ESV1 on the actions of GWD and PWD on the surfaces of native starch granules were analyzed. In addition, we included starches from various sources as well as truncated forms of GWD. ESV1 preferentially binds to highly ordered, α-glucans, such as starch and crystalline maltodextrins. Furthermore, ESV1 specifically influences the action of GWD and PWD at the starch granule surface. Starch phosphorylation by GWD is decreased in the presence of ESV1, whereas the action of PWD increases in the presence of ESV1. The unique alterations observed in starch phosphorylation by the two dikinases are discussed in regard to altered glucan structures at the starch granule surface.

Transferring a Biomass Enhancement Biotechnology from Glasshouse to Field: A Case Study on Wheat GWD RNAi

In glasshouse studies we have previously shown that endosperm-specific RNAi suppression of the primary starch phosphorylation enzyme, Glucan, Water Dikinase (GWD) leads to enhanced early vigor, greater leaf biomass, and increases in both head size and yield. To confirm these affects in a field setting, trials were conducted in three Australian environments. Field results were consistent with those in the glasshouse for increased flag leaf area and rachis nodes. However, there was also a decrease in tiller number and consequently a decrease in yield for one event at two sites. These findings provide potentially important information on plant vigor enhancement and highlight the challenges of transferring the modification of complex traits from single plants in controlled environments to the field.

Comparative Analysis of Soybean Root Proteome Reveals Molecular Basis of Differential Carboxylate Efflux under Low Phosphorus Stress.

Carboxylate efflux from roots is a crucial and differential response of soybean genotypes to low phosphorus (P) stress. Exudation of carboxylic acids including oxalate, citrate, succinate and fumarate was induced under low P stress, particularly in P-efficient soybean genotypes. Enhancement of root length, surface area and volume further improved P acquisition under low P stress. To understand the molecular basis of carboxylate efflux under low P stress, the root proteome of contrasting genotypes (P-efficient: EC-232019 and P-inefficient: EC-113396) was compared. Among a total of 325 spots, 105 (32%) were differentially abundant proteins (DAPs) between sufficient (250 µM) and low P (4 µM) levels. Abundance of 44 (14%) proteins decreased by more than two-fold under low P stress, while 61 (19%) proteins increased by more than two-fold. Protein identification and annotation revealed that the DAPs were involved in a myriad of functions including carboxylic acid synthesis, carbohydrate, protein and lipid metabolism. Proteins with significant abundance included malate dehydrogenase, isocitrate dehydrogenase, phosphoglucomutase, phosphoglycerate mutase, fructokinase, enolase, phosphoglycerate kinase, triosephosphate isomerase, alcohol dehydrogenase, glucan water dikinase, glutamine synthetase and argininosuccinate lyase. Inferences from proteomic analysis suggests the crosstalk between various metabolic pathways implicated in conferring superior P acquisition efficiency under stress.

Reduced starch granule number per chloroplast in the dpe2/phs1 mutant is dependent on initiation of starch degradation.

An Arabidopsis double knock-out mutant lacking cytosolic disproportionating enzyme 2 (DPE2) and the plastidial phosphorylase (PHS1) revealed a dwarf-growth phenotype, reduced starch content, an uneven distribution of starch within the plant rosette, and a reduced number of starch granules per chloroplast under standard growth conditions. In contrast, the wild type contained 5–7 starch granules per chloroplast. Mature and old leaves of the double mutant were essentially starch free and showed plastidial disintegration. Several analyses revealed that the number of starch granules per chloroplast was affected by the dark phase. So far, it was unclear if it was the dark phase per se or starch degradation in the dark that was connected to the observed decrease in the number of starch granules per chloroplast. Therefore, in the background of the double mutant dpe2/phs1, a triple mutant was generated lacking the initial starch degrading enzyme glucan, water dikinase (GWD). The triple mutant showed improved plant growth, a starch-excess phenotype, and a homogeneous starch distribution. Furthermore, the number of starch granules per chloroplast was increased and was similar to wild type. However, starch granule morphology was only slightly affected by the lack of GWD as in the triple mutant and, like in dpe2/phs1, more spherical starch granules were observed. The characterized triple mutant was discussed in the context of the generation of starch granules and the formation of starch granule morphology.

Heterologous expression of two Arabidopsis starch dikinases in potato

Starch phosphate esters influence physiochemical properties of starch granules that are essential both for starch metabolism and industrial use of starches. To modify properties of potato starch and understand the effect of starch phosphorylation on starch metabolism in storage starch, the starch dikinases from Arabidopsis thaliana, glucan water dikinase 2 and 3 (AtGWD2 and AtGWD3), were heterologously expressed in potato tubers (Solanum tuberosum) from two genetic backgrounds: the amylose‐containing clone Kardal and the amylose‐free mutant amf. Modified starches showed altered granule morphology, but no significant changes in starch phosphate content were observed. Genes involved in starch metabolism did not show altered expression in the transgenic lines relatively to the control; however, sucrose synthase 4 (SUSY4) was upregulated in transgenic tubers with AtGWD2 expression. Taken together, these results demonstrated that expression of AtGWD2 and AtGWD3 are not suitable for engineering starch with a high phosphate content in potato tubers but can provide new insights in the process of starch phosphorylation in the amyloplast.

Starch phosphorylation: insights and perspectives.

During starch metabolism, the phosphorylation of glucosyl residues of starch, to be more precise of amylopectin, is a repeatedly observed process. This phosphorylation is mediated by dikinases, the glucan, water dikinase (GWD) and the phosphoglucan, water dikinase (PWD). The starch-related dikinases utilize ATP as dual phosphate donor transferring the terminal γ-phosphate group to water and the β-phosphate group selectively to either C6 position or C3 position of a glucosyl residue within amylopectin. By the collaborative action of both enzymes, the initiation of a transition of α-glucans from highly ordered, water-insoluble state to a less order state is realized and thus the initial process of starch degradation. Consequently, mutants lacking either GWD or PWD reveal a starch excess phenotype as well as growth retardation. In this review, we focus on the increased knowledge collected over the last years related to enzymatic properties, the precise definition of the substrates, the physiological implications, and discuss ongoing questions.

Starch Granule Re-Structuring by Starch Branching Enzyme and Glucan Water Dikinase Modulation Affects Caryopsis Physiology and Metabolism.

Starch is of fundamental importance for plant development and reproduction and its optimized molecular assembly is potentially necessary for correct starch metabolism. Re-structuring of starch granules in-planta can therefore potentially affect plant metabolism. Modulation of granule micro-structure was achieved by decreasing starch branching and increasing starch-bound phosphate content in the barley caryopsis starch by RNAi suppression of all three Starch Branching Enzyme (SBE) isoforms or overexpression of potato Glucan Water Dikinase (GWD). The resulting lines displayed Amylose-Only (AO) and Hyper-Phosphorylated (HP) starch chemotypes, respectively. We studied the influence of these alterations on primary metabolism, grain composition, starch structural features and starch granule morphology over caryopsis development at 10, 20 and 30 days after pollination (DAP) and at grain maturity. While HP showed relatively little effect, AO showed significant reduction in starch accumulation with re-direction to protein and β-glucan (BG) accumulation. Metabolite profiling indicated significantly higher sugar accumulation in AO, with re-partitioning of carbon to accumulate amino acids, and interestingly it also had high levels of some important stress-related metabolites and potentially protective metabolites, possibly to elude deleterious effects. Investigations on starch molecular structure revealed significant increase in starch phosphate and amylose content in HP and AO respectively with obvious differences in starch granule morphology at maturity. The results demonstrate that decreasing the storage starch branching resulted in metabolic adjustments and re-directions, tuning to evade deleterious effects on caryopsis physiology and plant performance while only little effect was evident by increasing starch-bound phosphate as a result of overexpressing GWD.

Starch phosphorylation in potato tubers is influenced by allelic variation in the genes encoding glucan water dikinase, starch branching enzymes I and II, and starch synthase III.

Starch phosphorylation is an important aspect of plant metabolism due to its role in starch degradation. Moreover, the degree of phosphorylation of starch determines its physicochemical properties and is therefore relevant for industrial uses of starch. Currently, starch is chemically phosphorylated to increase viscosity and paste stability. Potato cultivars with elevated starch phosphorylation would make this process unnecessary, thereby bestowing economic and environmental benefits. Starch phosphorylation is a complex trait which has been previously shown by antisense gene repression to be influenced by a number of genes including those involved in starch synthesis and degradation. We have used an association mapping approach to discover genetic markers associated with the degree of starch phosphorylation. A diverse collection of 193 potato lines was grown in replicated field trials, and the levels of starch phosphorylation at the C6 and C3 positions of the glucosyl residues were determined by mass spectrometry of hydrolyzed starch from tubers. In addition, the potato lines were genotyped by amplicon sequencing and microsatellite analysis, focusing on candidate genes known to be involved in starch synthesis. As potato is an autotetraploid, genotyping included determination of allele dosage. Significant associations (p < 0.001) were found with SNPs in the glucan water dikinase (GWD), starch branching enzyme I (SBEI) and the starch synthase III (SSIII) genes, and with a SSR allele in the SBEII gene. SNPs in the GWD gene were associated with C6 phosphorylation, whereas polymorphisms in the SBEI and SBEII genes were associated with both C6 and C3 phosphorylation and the SNP in the SSIII gene was associated with C3 phosphorylation. These allelic variants have potential as genetic markers for starch phosphorylation in potato.

Glucan, Water Dikinase Exerts Little Control over Starch Degradation in Arabidopsis Leaves at Night.

The first step on the pathway of starch degradation in Arabidopsis (Arabidopsis thaliana) leaves at night is the phosphorylation of starch polymers, catalyzed by glucan, water dikinase (GWD). It has been suggested that GWD is important for the control of starch degradation, because its transcript levels undergo strong diel fluctuations, its activity is subject to redox regulation in vitro, and starch degradation is strongly decreased in gwd mutant plants. To test this suggestion, we analyzed changes in GWD protein abundance in relation to starch levels in wild-type plants, in transgenic plants in which GWD transcripts were strongly reduced by induction of RNA interference, and in transgenic plants overexpressing GWD. We found that GWD protein levels do not vary over the diel cycle and that the protein has a half-life of 2 d. Overexpression of GWD does not accelerate starch degradation in leaves, and starch degradation is not inhibited until GWD levels are reduced by 70%. Surprisingly, this degree of reduction also inhibits starch synthesis in the light. To discover the importance of redox regulation, we generated transgenic plants expressing constitutively active GWD. These plants retained normal control of degradation. We conclude that GWD exerts only a low level of control over starch degradation in Arabidopsis leaves.

Phosphorylation of transitory starch by α‐glucan, water dikinase during starch turnover affects the surface properties and morphology of starch granules

Glucan, water dikinase (GWD) is a key enzyme of starch metabolism but the physico-chemical properties of starches isolated from GWD-deficient plants and their implications for starch metabolism have so far not been described. Transgenic Arabidopsis thaliana plants with reduced or no GWD activity were used to investigate the properties of starch granules. In addition, using various in vitro assays, the action of recombinant GWD, β-amylase, isoamylase and starch synthase 1 on the surface of native starch granules was analysed. The internal structure of granules isolated from GWD mutant plants is unaffected, as thermal stability, allomorph, chain length distribution and density of starch granules were similar to wild-type. However, short glucan chain residues located at the granule surface dominate in starches of transgenic plants and impede GWD activity. A similarly reduced rate of phosphorylation by GWD was also observed in potato tuber starch fractions that differ in the proportion of accessible glucan chain residues at the granule surface. A model is proposed to explain the characteristic morphology of starch granules observed in GWD transgenic plants. The model postulates that the occupancy rate of single glucan chains at the granule surface limits accessibility to starch-related enzymes.

Increasing Phosphatidylinositol (4,5)-Bisphosphate Biosynthesis Affects Basal Signaling and Chloroplast Metabolism in Arabidopsis thaliana.

One challenge in studying the second messenger inositol(1,4,5)-trisphosphate (InsP3) is that it is present in very low amounts and increases only transiently in response to stimuli. To identify events downstream of InsP3, we generated transgenic plants constitutively expressing the high specific activity, human phosphatidylinositol 4-phosphate 5-kinase Iα (HsPIPKIα). PIP5K is the enzyme that synthesizes phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2); this reaction is flux limiting in InsP3 biosynthesis in plants. Plasma membranes from transgenic Arabidopsis expressing HsPIPKIα had 2–3 fold higher PIP5K specific activity, and basal InsP3 levels in seedlings and leaves were >2-fold higher than wild type. Although there was no significant difference in photosynthetic electron transport, HsPIPKIα plants had significantly higher starch (2–4 fold) and 20% higher anthocyanin compared to controls. Starch content was higher both during the day and at the end of dark period. In addition, transcripts of genes involved in starch metabolism such as SEX1 (glucan water dikinase) and SEX4 (phosphoglucan phosphatase), DBE (debranching enzyme), MEX1 (maltose transporter), APL3 (ADP-glucose pyrophosphorylase) and glucose-6-phosphate transporter (Glc6PT) were up-regulated in the HsPIPKIα plants. Our results reveal that increasing the phosphoinositide (PI) pathway affects chloroplast carbon metabolism and suggest that InsP3 is one component of an inter-organelle signaling network regulating chloroplast metabolism.