Water Dikinase Research Articles

Starch phosphorylation—A needle in a haystack

Phosphoesterification is the only naturally occurring covalent starch modification identified to date, and it has a major impact on overall starch metabolism. The incorporation of phosphate groups mediated by dikinases [α-glucan, water dikinase (GWD), EC 2.7.9.4; phosphoglucan, water dikinase (PWD), EC 2.7.9.5] massively alters the starch granule properties; however, previous studies did not determine whether the starch-related dikinases bind the phosphate to the glucosyl units within the amylopectin molecules in a specific pattern or randomly. In order to answer this challenging question, a number of approaches were initially pursued until a protocol could be established that enabled a massive step forward in the in vitro analysis of phosphorylated glucan chains obtained from starch. For this purpose, phosphorylation by GWD was investigated, including the final state of phosphorylation i.e., the state of substrate saturation when GWD lacks further free hydroxyl groups at OH-C6 for the catalysis of monophosphate esters. Since the separated phosphorylated glucan chains were required for the analysis, isoamylase digestion was performed to cleave the α-1,6-glycosidic bonds and to allow for the removal of the huge number of existing neutral chains by means of anion exchange chromatography. Via Matrix-Assisted Laser Desorption/Ionization–Time of Flight (MALDI-TOF) MS and MALDI-MS/MS, the phosphorylated α-glucan chains were analysed, and the position of the phosphate group within the chain in relation to the reducing end was determined. Here, we demonstrate a protocol that enables the analysis of phosphorylated oligosaccharides, even in small quantities.

Exploring the function of MaPHO1 in starch degradation and its protein interactions in postharvest banana fruits

Banana is a quintessential fruit known for its starch conversion process. During development, substantial starch accumulates in banana fruit and subsequently undergoes rapid degradation, contributing to flavor development during postharvest ripening. However, the genes orchestrating this crucial starch degradation process have not been comprehensively studied thus far. Through temporal transcriptomic analysis, we screened 63 active genes that were actively expressed in association with starch degradation during the ripening of banana fruits. The results found that phosphorolytic enzymes primarily regulate starch degradation at the early to middle stages of fruit ripening and that hydrolytic enzymes significantly contribute to starch degradation at the later stage. The expression of Musa acuminata starch phosphorylase 1 (MaPHO1) was the highest at the early to middle stages of banana ripening, characterized by the insertion of an L80 domain. While MaPHO1 was predominately found in the chloroplast, MaPHO2 was localized to the cell membrane. Starch accumulation was significantly increased by transient attenuation of MaPHO1 expression, whereas a drastic reduction in starch content was triggered by transient overexpression of MaPHO1, affirming its functional role in banana fruit ripening. By constructing a co-expression network, 29 categories, including 74 proteins interacting with MaPHO1 were identified. Tripartite interactions between MaPHO1 and both glucan, water dikinase 1 (GWD1) and enolase 1 (ENO1) were revealed by further validation through yeast two-hybrid and glutathione-S-transferase pull-down analyses.

Peculiar properties of tuber starch in a potato mutant lacking the α-glucan water dikinase 1 gene GWD1 created by targeted mutagenesis using the CRISPR/dMac3-Cas9 system.

Glucose chains in starch are phosphorylated and contribute to structural stabilization. Phosphate groups contained in starch also play a role in retaining moisture. α-Glucan water dikinase 1 (GWD1) is involved in the phosphorylation of glucose chains in starch. In this study, we generated potato mutants of the GWD1 gene using the CRISPR/dMac3-Cas9 system. Observation of the phenotypes of the GWD1-deficient mutants revealed their physiological roles in tuber starch formation. The 4-allele mutants showed growth retardation and a delay in tuber formation. A significant decrease in phosphorus content was detected in the tuber starch of the gwd1 mutant. This mutant starch showed a higher amylose content than the wild-type starch, whereas its gelatinization temperature was slightly lower than that of the WT starch. The peak viscosity of the mutant starch was lower than that of the WT starch. These observations revealed that the starch of the gwd1 mutants had peculiar and unique properties compared to those of WT, sbe3 and gbss1 mutant starches. The amount of tissue-released water due to freeze-thawing treatment was determined on tubers of the gwd1 mutant and compared with those of WT and the other mutants. Significantly less water loss was found in the gwd1, sbe3 and gbss1 mutant tubers than in the WT tubers. Our results indicate that the GWD1 gene is not only important for potato growth, but also largely effective for the traits of tuber starch.

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.

Key roles of amylopectin synthesis and degradation enzymes in the establishment and reactivation of chronic toxoplasmosis

Toxoplasma gondii (T. gondii) is an obligate intracellular parasite with a wide range of hosts, including humans and many warm-blooded animals. The parasite exists in two interconvertible forms, namely tachyzoites and bradyzoites in intermediate hosts that are responsible for acute and chronic infections respectively. Mature bradyzoites accumulate large amounts of amylopectin granules but their roles have not been fully characterized. In this study, the predicted key enzymes involved in amylopectin synthesis (UDP-sugar pyrophospharylase, USP) and degradation (alpha-glucan water dikinase, GWD) of ME49 strain were individually knocked out, and then bradyzoite-related phenotyping experiments in vitro and in vivo were performed to dissect their roles during parasite growth and development. Deletion of the usp or gwd gene in the type II strain ME49 reduced the replication rates of tachyzoites in vitro and parasite virulence in vivo, suggesting that amylopectin metabolism is important for optimal tachyzoite growth. Interestingly, the Δusp mutant grew slightly faster than the parental strain under stress conditions that induced bradyzoite transition, which was likely due to the decreased efficiency of bradyzoite formation of the Δusp mutant. Although the Δgwd mutant could convert to bradyzoite robustly in vitro, it was significantly impaired in establishing chronic infection in vivo. Both the Δusp and Δgwd mutants showed a dramatic reduction in the reactivation of chronic infection in an in vitro model. Together, these results suggest that USP and GWD, which are involved in amylopectin synthesis and degradation have important roles in tachyzoite growth, as well as in the formation and reactivation of bradyzoites in T. gondii.

Improved Hydrolysis of Granular Starches by a Psychrophilic α-Amylase Starch Binding Domain-Fusion.

Degradation of starch granules by a psychrophilic α-amylase, AHA, from the Antarctic bacterium Pseudoalteromonas haloplanktis TAB23 was facilitated by C-terminal fusion to a starch-binding domain (SBD) from either Aspergillus niger glucoamylase (SBDGA) or Arabidopsis thaliana glucan, water dikinase 3 (SBDGWD3) via a decapeptide linker. Depending on the waxy, normal or high-amylose starch type and the botanical source, the AHA-SBD fusion enzymes showed up to 3 times higher activity than AHA wild-type. The SBD-fusion thus increased the density of enzyme attack-sites and binding-sites on the starch granules by up to 5- and 7-fold, respectively, as measured using an interfacial catalysis approach that combined conventional Michaelis-Menten kinetics, with the substrate in excess, and inverse kinetics, having enzyme in excess, with enzyme-starch granule adsorption isotherms. Higher substrate affinity of the SBDGA compared to SBDGWD3 was accompanied by the superior activity of AHA-SBDGA in agreement with the Sabatier principle of adsorption limited heterogenous catalysis.

Genetic loci and candidate genes with pleiotropic effects for tofu and soymilk weights across multiple environments in soybean (Glycine max)

AbstractSoybean provides large amounts of protein for human, and the most popular consumption pattern is tofu and soymilk. Recently, the demand of tofu and soymilk is increased, and the breeding of special variety becomes very urgent. To illustrate the genetic basis of these traits, 269 accessions were evaluated under six environments, and 32 associated SNPs were identified on nine chromosomes across multiple environments or conferring diverse traits. Of these SNPs, 16 were demonstrated by BLUP values. Seven adjacent‐SNPs were first identified to associate with tofu and soymilk weights in an 1.02‐Mb region on chromosome 8, which were verified by BLUP‐values. Meanwhile, four neighbouring SNPs in a 0.32‐Mb region on chromosome 12 were first identified to associate with these traits across multiple environments, of which three were detected in BLUP values. Furthermore, some candidate genes, that is, α‐glucan water dikinase Glyma.08G283700 and E3 SUMO‐protein ligase Glyma.12G071300, were screened out with differential expressions between the high‐ and low‐tofu/soymilk‐output accessions. Thus, these associated SNPs and candidate genes provide new insight into the genetic basis of tofu and soymilk processing quality in soybean.

LIKE EARLY STARVATION 1 alters the glucan structures at the starch granule surface and thereby influences the action of both starch-synthesizing and starch-degrading enzymes.

For starch metabolism to take place correctly, various enzymes and proteins acting on the starch granule surface are crucial. Recently, two non-catalytic starch-binding proteins, pivotal for normal starch turnover in Arabidopsis leaves, namely, EARLY STARVATION 1 (ESV1) and its homolog LIKE EARLY STARVATION 1 (LESV), have been identified. Both share nearly 38% sequence homology. As ESV1 has been found to influence glucan phosphorylation via two starch-related dikinases, α-glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD), through modulating the surface glucan structures of the starch granules and thus affecting starch degradation, we assess the impact of its homolog LESV on starch metabolism. Thus, the 65-kDa recombinant protein LESV and the 50-kDa ESV1 were analyzed regarding their influence on the action of GWD and PWD on the surface of the starch granules. We included starches from various sources and additionally assessed the effect of these non-enzymatic proteins on other starch-related enzymes, such as starch synthases (SSI and SSIII), starch phosphorylases (PHS1), isoamylase and β-amylase. The data obtained indicate that starch phosphorylation, hydrolyses and synthesis were affected by LESV and ESV1. Furthermore, incubation with LESV and ESV1 together exerted an additive effect on starch phosphorylation. In addition, a stable alteration of the glucan structures at the starch granule surface following treatment with LESV and ESV1 was observed. Here, we discuss all the observed changes that point to modifications in the glucan structures at the surface of the native starch granules and present a model to explain the existing processes.

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.

Melatonin delays dark-induced leaf senescence by inducing miR171b expression in tomato.

Melatonin functions in multiple aspects of plant growth, development, and stress response. Nonetheless, the mechanism of melatonin in plant carbon metabolism remains largely unknown. In this study, we investigated the influence of melatonin on the degradation of starch in tomato leaves. Results showed that exogenous melatonin attenuated carbon starvation-induced chlorophyll degradation and leaf senescence. In addition, melatonin delayed leaf starch degradation and inhibited the transcription of starch-degrading enzymes after sunset. Interestingly, melatonin-alleviated symptoms of leaf senescence and starch degradation were compromised when the first key gene for starch degradation, α-glucan water dikinase (GWD), was overexpressed. Furthermore, exogenous melatonin significantly upregulated the transcript levels of several microRNAs, including miR171b. Crucially, the GWD gene was identified as a target of miR171b, and the overexpression of miR171b ameliorated the carbon starvation-induced degradation of chlorophyll and starch, and inhibited the expression of the GWD gene. Taken together, these results demonstrate that melatonin promotes plant tolerance against carbon starvation by upregulating the expression of miR171b, which can directly inhibit GWD expression in tomato leaves.

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.

Allelic variation for alpha-Glucan Water Dikinase is associated with starch phosphate content in tetraploid potato

Key messageAssociation analysis resulted in the identification of specific StGWD alleles causing either an increase or decrease in starch phosphate content which was verified in diploid and tetraploid potato mapping populations.Potatoes are grown for various purposes like French fries, table potatoes, crisps and for their starch. One of the most important aspects of potato starch is that it contains a high amount of phosphate ester groups which are considered to be important for providing improved functionalization after derivatization processes. Little is known about the variation in phosphate content as such in different potato varieties and thus we studied the genetic diversity for this trait. From other studies it was clear that the phosphate content is controlled by a quantitative trait locus (QTL) underlying the candidate gene α-Glucan Water Dikinase (StGWD) on chromosome 5. We performed direct amplicon sequencing of this gene by Sanger sequencing. Sequences of two StGWD amplicons from a global collection of 398 commercial cultivars and progenitor lines were used to identify 16 different haplotypes. By assigning tag SNPs to these haplotypes, each of the four alleles present in a cultivar could be deduced and linked to a phosphate content. A high value for intra-individual heterozygosity was observed (Ho = 0.765). The average number of different haplotypes per individual (Ai) was 3.1. Pedigree analysis confirmed that the haplotypes are identical-by-descent (IBD) and offered insight in the breeding history of elite potato germplasm. Haplotypes originating from introgression of wild potato accessions carrying resistance genes could be traced. Furthermore, association analysis resulted in the identification of specific StGWD alleles causing either an increase or decrease in starch phosphate content varying from 12 nmol PO4/mg starch to 38 nmol PO4/mg starch. These allele effects were verified in diploid and tetraploid mapping populations and offer possibilities to breed and select for this trait.

Effect of Short-Term Cold Treatment on Carbohydrate Metabolism in Potato Leaves.

Plants are often challenged by an array of unfavorable environmental conditions. During cold exposure, many changes occur that include, for example, the stabilization of cell membranes, alterations in gene expression and enzyme activities, as well as the accumulation of metabolites. In the presented study, the carbohydrate metabolism was analyzed in the very early response of plants to a low temperature (2 °C) in the leaves of 5-week-old potato plants of the Russet Burbank cultivar during the first 12 h of cold treatment (2 h dark and 10 h light). First, some plant stress indicators were examined and it was shown that short-term cold exposure did not significantly affect the relative water content and chlorophyll content (only after 12 h), but caused an increase in malondialdehyde concentration and a decrease in the expression of NDA1, a homolog of the NADH dehydrogenase gene. In addition, it was shown that the content of transitory starch increased transiently in the very early phase of the plant response (3–6 h) to cold treatment, and then its decrease was observed after 12 h. In contrast, soluble sugars such as glucose and fructose were significantly increased only at the end of the light period, where a decrease in sucrose content was observed. The availability of the monosaccharides at constitutively high levels, regardless of the temperature, may delay the response to cold, involving amylolytic starch degradation in chloroplasts. The decrease in starch content, observed in leaves after 12 h of cold exposure, was preceded by a dramatic increase in the transcript levels of the key enzymes of starch degradation initiation, the α-glucan, water dikinase (GWD-EC 2.7.9.4) and the phosphoglucan, water dikinase (PWD-EC 2.7.9.5). The gene expression of both dikinases peaked at 9 h of cold exposure, as analyzed by real-time PCR. Moreover, enhanced activities of the acid invertase as well as of both glucan phosphorylases during exposure to a chilling temperature were observed. However, it was also noticed that during the light phase, there was a general increase in glucan phosphorylase activities for both control and cold-stressed plants irrespective of the temperature. In conclusion, a short-term cold treatment alters the carbohydrate metabolism in the leaves of potato, which leads to an increase in the content of soluble sugars.

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.

Impact of Drought on Soluble Sugars and Free Proline Content in Selected Arabidopsis Mutants.

Simple SummaryDrought has severe effects on plants, negatively impacting economic, agricultural and environmental processes. Depending on the duration and the strength of water stress conditions, plants adjust a series of physiological, cellular, and molecular mechanisms aimed at providing a correct stress response and, if possible, at establishing stress tolerance. The model plant Arabidopsis thaliana is one of the best tools for analyzing the involvement of specific genes in the response to drought. Thanks to this tool, the role of two genes encoding enzymes involved in sugars metabolism, and one gene encoding an enzyme involved in proline synthesis, have been investigated. In addition to suggesting an interaction between the metabolism of proline and that of soluble sugars, results broaden our understanding of the predominant role played by the accumulation of soluble sugars in counteracting mild osmotic stress.Water shortage is an increasing problem affecting crop yield. Accumulation of compatible osmolytes is a typical plant response to overcome water stress. Sucrose synthase 1 (SUS1), and glucan, water dikinase 2 (GWD2) and δ1-pyrroline-5-carboxylate synthetase 1 (P5CS1) are members of small protein families whose role in the response of Arabidopsis thaliana plants to mild osmotic stress has been studied in this work. Comparative analysis between wild-type and single loss-of-function T-DNA plants at increasing times following exposure to drought showed no differences in the content of water-insoluble carbohydrate (i.e., transitory starch and cell wall carbohydrates) and in the total amount of amino acids. On the contrary, water-soluble sugars and proline contents were significantly reduced compared to wild-type plants regardless of the metabolic pathway affected by the mutation. The present results contribute to assigning a physiological role to GWD2, the least studied member of the GWD family; strengthening the involvement of SUS1 in the response to osmotic stress; showing a greater contribution of soluble sugars than proline in osmotic adjustment of Arabidopsis in response to drought. Finally, an interaction between proline and soluble sugars emerged, albeit its nature remains speculative and further investigations will be required for complete comprehension.

Differential Evolution of α-Glucan Water Dikinase (GWD) in Plants.

The alpha-glucan water dikinase (GWD) enzyme catalyzes starch phosphorylation, an integral step in transitory starch degradation. The high phosphate content in stored starch has great industrial value, due to its physio–chemical properties making it more versatile, although the phosphate content of stored starch varies depending on the botanical source. In this study, we used various computational approaches to gain insights into the evolution of the GWD protein in 48 plant species with possible roles in enzyme function and alteration of phosphate content in their stored starch. Our analyses identified deleterious mutations, particularly in the highly conserved 5 aromatic amino acid residues in the dual tandem carbohydrate binding modules (CBM-45) of GWD protein in C. zofingiensis, G. hirsutum, A. protothecoides, P. miliaceum, and C. reinhardtii. These findings will inform experimental designs for simultaneous repression of genes coding for GWD and the predicted interacting proteins to elucidate the role this enzyme plays in starch degradation. Our results reveal significant diversity in the evolution of GWD enzyme across plant species, which may be evolutionarily advantageous according to the varying needs for phosphorylated stored starch between plants and environments.

Quantitative phosphoproteomics analysis reveals that protein modification and sugar metabolism contribute to sprouting in potato after BR treatment

Brassinosteroids (BRs), a new class of steroid hormones, are involved in the regulation of plant cell elongation and seed germination. Nevertheless, the molecular mechanism of the effect of BRs on tuber sprouting remains largely unknown. In this study, quantitative phosphoproteomics was employed to investigate the protein phosphorylation changes in sprouting induced by BRs. Our results showed that BRs accelerated the conversion of starch into soluble sugar in tubers. A functional enrichment cluster analysis suggested that the “amino acid metabolism pathway” was upregulated and that “plant hormone signal transduction and protein export” were downregulated. BR treatment also changed the phosphorylation of proteins involved in the BR, ABA, starch and sugar signal transduction pathways, such as serine/threonine-protein kinase (BSK), 14-3-3, alpha-glucan water dikinase (GWD), sucrose-phosphate synthase (SPS), sucrose synthase (SS) and alkaline/neutral invertase (A/N-INV). These results shed more light on the pattern of protein phosphorylation in BR promoting potato sprouting.

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.

Functional Roles of Starch Binding Domains and Surface Binding Sites in Enzymes Involved in Starch Biosynthesis.

Biosynthesis of starch is catalyzed by a cascade of enzymes. The activity of a large number of these enzymes depends on interaction with polymeric substrates via carbohydrate binding sites, which are situated outside of the catalytic site and its immediate surroundings including the substrate-binding crevice. Such secondary binding sites can belong to distinct starch binding domains (SBDs), classified as carbohydrate binding modules (CBMs), or be surface binding sites (SBSs) exposed on the surface of catalytic domains. Currently in the Carbohydrate-Active enZYmes (CAZy) database SBDs are found in 13 CBM families. Four of these families; CBM20, CBM45, CBM48, and CBM53 are represented in enzymes involved in starch biosynthesis, namely starch synthases, branching enzymes, isoamylases, glucan, water dikinases, and α-glucan phosphatases. A critical role of the SBD in activity has not been demonstrated for any of these enzymes. Among the well-characterized SBDs important for starch biosynthesis are three CBM53s of Arabidopsis thaliana starch synthase III, which have modest affinity. SBSs, which are overall less widespread than SBDs, have been reported in some branching enzymes, isoamylases, synthases, phosphatases, and phosphorylases active in starch biosynthesis. SBSs appear to exert roles similar to CBMs. SBSs, however, have also been shown to modulate specificity for example by discriminating the length of chains transferred by branching enzymes. Notably, the difference in rate of occurrence between SBDs and SBSs may be due to lack of awareness of SBSs. Thus, SBSs as opposed to CBMs are not recognized at the protein sequence level, which hampers their identification. Moreover, only a few SBSs in enzymes involved in starch biosynthesis have been functionally characterized, typically by structure-guided site-directed mutagenesis. The glucan phosphatase Like SEX4 2 from A. thaliana has two SBSs with weak affinity for β-cyclodextrin, amylose and amylopectin, which were indicated by mutational analysis to be more important than the active site for initial substrate recognition. The present review provides an update on occurrence of functional SBDs and SBSs in enzymes involved in starch biosynthesis.

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.