Starch Degradation In Plants Research Articles

Natural variation of an autophagy-family gene among rice subspecies affects grain size and weight

Elucidating the genetic basis of natural variation in grain size and weight among rice varieties can help breeders develop high-yielding varieties. We identified a novel gene, GW3a (Grain Weight 3a) (LOC_Os03g27350), that affects rice grain size and weight. gw3a mutants showed higher total starch content and dry matter accumulation than the wild type (WT), Nipponbare, suggesting that GW3a negatively regulates grain size and weight. Moreover, our study found that GW3a interacted with OsATG8 by cleaving it, suggesting that GW3a may be involved in the assembly of autophagosomes and starch degradation in plants. The haplotype analysis of GW3a showed functional differences between indica and japonica rice. Taken together, we conclude that GW3a is expressed in the autophagosome pathway regulating starch metabolism in rice, affecting yield-related traits, such as grain size, grain weight and thousand grain weight (TGW). Our findings also shed new light on autophagy-mediated yield trait regulation, proposing a possible strategy for the genetic improvement of high-yield germplasm in rice.

OsDPE2 Regulates Rice Panicle Morphogenesis by Modulating the Content of Starch

Starch is a carbon sink for most plants, and its biological role changes with response to the environment and during plant development. Disproportionating Enzyme 2 (DPE2) is a 4-α-glycosyltransferase involved in starch degradation in plants at night. LAX1 plays a vital role in axillary meristem initiation in rice. Herein, results showed that Oryza sativa Disproportionating Enzyme 2 (OsDPE2) could rescue the mutant phenotype of lax1-6, LAX1 mutant. OsDPE2 encodes rice DPE2 located in the cytoplasm. In this study, OsDPE2 affected the vegetative plant development of rice via DPE2 enzyme. Additionally, OsDPE2 regulated the reproductive plant development of rice by modulating starch content in young panicles. Furthermore, haplotype OsDPE2(AQ) with higher DPE2 enzyme activity increased the panicle yield of rice. In summary, OsDPE2 can regulate vegetative and reproductive plant development of rice by modulating starch content. Furthermore, DPE2 activities of OsDPE2 haplotypes are associated with the panicle yield of rice. This study provides guidance for rice breeding to improve panicle yield traits.

Amylase Inhibitor SbAI in Potato Species: Structure, Variability and Expression Pattern

Starch degradation in plants is mediated by amylases, the activity of which is post-translationally regulated by inhibitors. It is assumed that the SbAI amylase inhibitor activity is associated with potato cold stress resistance. The study of possible correlations of the SbAI variability with the cold resistance of potato species will improve the understanding of the starch metabolism regulation during plant development and stress response. In the present study, the genome sequences homologous to SbAI were identified in 12 potato species of section Petota. In total, 36 allelic variants of the gene and 31 variants of the protein were revealed. Comparison with data on S. tuberosum cultivars showed that the SbAI cultivar polymorphism is 1.4–1.7 times higher than the interspecific, but the species possess a larger number of allelic variants. Phylogenetic analysis revealed that, for S. demissum, S. acaule, and S. stoloniferum, each species SbAI allelic variants did not get into one clade. The SbAI gene expression pattern was determined in various organs of the species S. tuberosum (cv. Nadezhda), S. rybinii, S. chacoense, and S. kurtzianum. In leaves, S. chacoense SbAI expression level was 13, 50, and 250 times higher than in S. tuberosum, S. kurtzianum, and S. rybinii, respectively. Varied levels of SbAI expression in the species may be due to different initial contents of both synthesized starch and reducing sugars in the leaves. In tubers, S. tuberosum SbAI expression was 5.2, 8.6, and 430 times higher than in S. kurtzianum, S. chacoense, and S. rybinii. In roots, S. rybinii SbAI expression was 2.4 times higher than in S. chacoense and 42.3 times higher than in S. kurtzianum and S. tuberosum. The absence of correlation between the SbAI expression level and the species cold resistance was shown. The obtained expression data suggest that different potato species and cultivars may differently regulate tuber starch content.

Starch granule sizes and degradation in sweet potatoes during storage

Starch granule sizes can greatly influence the quality of both table and processed products of sweet potato (Ipomoea batatas (L.) Lam), an important food, feed and industrial crop. Sweet potatoes require storage under suitable temperatures for year around supply, but there is very little research on starch granule size variation during storage. We characterized dry matter content (DMC), starch content, soluble carbohydrate (SoluCarb), and starch granule length before and after storage at 13 °C for 60 d in the cultivars, SP3388, SP3391, ‘Beauregard’, and ‘Covington’. Tuberous roots with higher DMC tended to have greater DMC, starch content, SoluCarb, and starch granule length after storage. Starch granule sizes ranged nearly continuously from small to large, without frequency peaks of small granules or large granules. Smaller starch granules degraded faster than larger ones during storage. The findings provided insights into starch degradation in plants, can help predict the processing quality of sweet potatoes during storage, and may also assist in their dry matter and starch-related breeding.

Quaternary Structure, Salt Sensitivity, and Allosteric Regulation of β-AMYLASE2 From Arabidopsis thaliana

The β-amylase family in Arabidopsis thaliana has nine members, four of which are both plastid-localized and, based on active-site sequence conservation, potentially capable of hydrolyzing starch to maltose. We recently reported that one of these enzymes, BAM2, is catalytically active in the presence of physiological levels of KCl, exhibits sigmoidal kinetics with a Hill coefficient of over 3, is tetrameric, has a putative secondary binding site (SBS) for starch, and is highly co-expressed with other starch metabolizing enzymes. Here we generated a tetrameric homology model of Arabidopsis BAM2 that is a dimer of dimers in which the putative SBSs of two subunits form a deep groove between the subunits. To validate this model and identify key residues, we generated a series of mutations and characterized the purified proteins. (1) Three point mutations in the putative subunit interfaces disrupted tetramerization; two that interfered with the formation of the starch-binding groove were largely inactive, whereas a third mutation prevented pairs of dimers from forming and was active. (2) The model revealed that a 30-residue N-terminal acidic region, not found in other BAMs, appears to form part of the putative starch-binding groove. A mutant lacking this acidic region was active and did not require KCl for activity. (3) A conserved tryptophan residue in the SBS is necessary for activation and may form π-bonds with sugars in starch. (4) Sequence alignments revealed a conserved serine residue next to one of the catalytic glutamic acid residues, that is a conserved glycine in all other active BAMs. The serine side chain points away from the active site and toward the putative starch-binding groove. Mutating the serine in BAM2 to a glycine resulted in an enzyme with a VMax similar to that of the wild type enzyme but with a 7.5-fold lower KM for soluble starch. Interestingly, the mutant no longer exhibited sigmoidal kinetics, suggesting that allosteric communication between the putative SBS and the active site was disrupted. These results confirm the unusual structure and function of this widespread enzyme, and suggest that our understanding of starch degradation in plants is incomplete.

Arabidopsis β-Amylase2 Is a K+-Requiring, Catalytic Tetramer with Sigmoidal Kinetics.

The Arabidopsis (Arabidopsis thaliana) genome contains nine β-amylase (BAM) genes, some of which play important roles in starch hydrolysis. However, little is known about BAM2, a plastid-localized enzyme reported to have extremely low catalytic activity. Using conservation of intron positions, we determined that the nine Arabidopsis BAM genes fall into two distinct subfamilies. A similar pattern was found in each major lineage of land plants, suggesting that these subfamilies diverged prior to the origin of land plants. Moreover, phylogenetic analysis indicated that BAM2 is the ancestral member of one of these subfamilies. This finding, along with the conservation of amino acids in the active site of BAM2, suggested that it might be catalytically active. We then identified KCl as necessary for BAM2 activity. Unlike BAM1, BAM3, and BAM5, three Arabidopsis BAMs that all exhibited hyperbolic kinetics, BAM2 exhibited sigmoidal kinetics with a Hill coefficient of over 3. Using multi-angle light scattering, we determined that BAM2 was a tetramer, whereas BAM5 was a monomer. Conserved residues from a diverse set of BAM2 orthologs were mapped onto a homology model of the protein, revealing a large, conserved surface away from the active site that we hypothesize is a secondary carbohydrate-binding site. Introduction of bulky methionine for glycine at two points on this surface reduced catalytic activity significantly without disrupting the tetrameric structure. Expression analysis indicated that BAM2 is more closely coexpressed with other starch degradation enzymes than any other BAM, suggesting that BAM2 may play an important role in starch degradation in plants.

Development of fast and simple chromogenic methods for glucan phosphatases in-gel activity assays

Glucan phosphatases are essential for normal starch degradation in plants and glycogen metabolism in mammals. Here we develop two chromogenic methods for the detection of glucan phosphatase activity in situ after non denaturing poliacrylamide gel electrophoresis; one method uses pNPP and the second one applies BCIP/NBT. The assays are sensitive, fast, simple, reliable and cost-effective preventing the use of radioactive or fluorogenic compounds.Taking advantage of an efficient separation method combined with the reported assays it is possible to obtain information about oligomeric state of the active enzymes as well as to simultaneously detect glucan substrate binding and phosphatase activity.

Iminosugar inhibitors of carbohydrate-active enzymes that underpin cereal grain germination and endosperm metabolism.

Starch is a major energy store in plants. It provides most of the calories in the human diet and, as a bulk commodity, it is used across broad industry sectors. Starch synthesis and degradation are not fully understood, owing to challenging biochemistry at the liquid/solid interface and relatively limited knowledge about the nature and control of starch degradation in plants. Increased societal and commercial demand for enhanced yield and quality in starch crops requires a better understanding of starch metabolism as a whole. Here we review recent advances in understanding the roles of carbohydrate-active enzymes in starch degradation in cereal grains through complementary chemical and molecular genetics. These approaches have allowed us to start dissecting aspects of starch degradation and the interplay with cell-wall polysaccharide hydrolysis during germination. With a view to improving and diversifying the properties and uses of cereal grains, it is possible that starch degradation may be amenable to manipulation through genetic or chemical intervention at the level of cell wall metabolism, rather than simply in the starch degradation pathway per se.

Light-Induced Degradation of Starch Granules in Turions of Spirodela polyrhiza Studied by Electron Microscopy

Spirodela polyrhiza forms turions, starch-storing perennial organs. The light-induced process of starch degradation starts with an erosion of the surface of starch grains. The grain size decreases over a period of red irradiation and the surface becomes rougher. The existence of funnel-shaped erosion structures demonstrates that starch degradation is also possible inside the grains. Neither etioplasts nor clues as to their transition into chloroplasts were found in the storage tissue by transmission electron microscopy. Juvenile chloroplasts always contained the starch grains which remained from amyloplasts. No chloroplasts were found which developed independently of starch grains. Amyloplasts are therefore the only source of chloroplasts in the cells of irradiated turions. The intactness of amyloplast envelope membranes could not be directly proved by electron microscopy. However, the light-induced transition of amyloplasts into chloroplasts provides indirect evidence for the integrity of the envelope membranes throughout the whole process. The starch grains are sequestered from the cytosolic enzymes, and only plastid-localized enzymes, which have access to the starch grains, can carry out starch degradation. In this respect the turion system resembles transitory starch degradation as known from Arabidopsis leaves. On the other hand, with α-amylase playing the dominant role, it resembles the mechanism operating in the endosperm of cereals. Thus, turions appear to possess a unique system of starch degradation in plants combining elements from both known starch-storing systems.

Bioinformatics Analysis of Oligosaccharide Phosphorylation Effect on the Stabilization of the β-Amylase Ligand Complex

Starch is the most abundant storage carbohydrate produced in plants. The beginning of transitory starch degradation in plants depends mainly on day cycle, posttranslational regulation of enzyme activity, and starch phosphorylation, but the molecular mechanism of these factors' influence is not yet precisely described. The aim of our analysis was to investigate the effect of phosphorylation on the intermolecular energies for stabilization of the complexes between the set of phosphorylated and nonphosphorylated carbohydrate ligands and Solanum tuberosum (L.) β-amylase model. For performing protein-ligand docking procedures and calculating the binding energies, the DOCK6 and Glide 4.5 program suites were applied. We have observed simultaneously the effect of chain elongation, phosphorylation, and chain branching. Results of flexible ligand docking show that phosphorylation as well as chain elongation increase the stabilization of the ligand-protein complex.

Starch phosphorylation—Maltosidic restrains upon 3′‐ and 6′‐phosphorylation investigated by chemical synthesis, molecular dynamics and NMR spectroscopy

Phosphorylation is the only known in vivo substitution of starch, yet no structural evidence has been provided to explain its implications of the amylosidic backbone and its stimulating effects on starch degradation in plants. In this study, we provide evidence for a major influence on the glucosidic bond in starch specifically induced by the 3-O-phosphate. Two phosphorylated maltose model compounds were synthesized and subjected to combined molecular dynamics (MD) studies and 950 MHz NMR studies. The two phosphorylated disaccharides represent the two possible phosphorylation sites observed in natural starches, namely maltose phosphorylated at the 3'- and 6'-position (maltose-3'-O-phosphate and maltose-6'-O-phosphate). When compared with maltose, both of the maltose-phosphates exhibit a restricted conformational space of the alpha(1-->4) glycosidic linkage. When maltose is phosphorylated in the 3'-position, MD and NMR show that the glucosidic space is seriously restricted to one narrow potential energy well which is strongly offset from the global potential energy well of maltose and almost 50 degrees degrees from the Phi angle of the alpha-maltose crystal structure. The driving force is primarily steric, but the configuration of the structural waters is also significantly altered. Both the favored conformation of the maltose-3'-phosphate and the maltose-6'-phosphate align well into the 6-fold double helical structure of amylopectin when the effects on the glucosidic bond are not taken into account. However, the restrained geometry of the glucosidic linkage of maltose-3'-phosphate cannot be accommodated in the helical structure, suggesting a major local disturbing effect, if present in the starch granule semi-crystalline lattice.

An extra-plastidial -glucan, water dikinase from Arabidopsis phosphorylates amylopectin in vitro and is not necessary for transient starch degradation

Starch phosphorylation catalysed by the alpha-glucan, water dikinases (GWD) has profound effects on starch degradation in plants. The Arabidopsis thaliana genome encodes three isoforms of GWD, two of which are localized in the chloroplast and are involved in the degradation of transient starch. The third isoform, termed AtGWD2 (At4g24450), was heterologously expressed and purified and shown to have a substrate preference similar to potato GWD. Analyses of AtGWD2 null mutants did not reveal any differences in growth or starch and sugar levels, when compared to the wild type. Subcellular localization studies in Arabidopsis leaves and in vitro chloroplast import assays indicated that AtGWD2 was not targeted to the chloroplasts. The AtGWD2 promoter showed a highly restricted pattern of activity, both spatially and temporally. High activity was observed in the companion cells of the phloem, with expression appearing just before the onset of senescence. Taken together, these data indicate that, although AtGWD2 is capable of phosphorylating alpha-glucans in vitro, it is not directly involved in transient starch degradation.

Signal peptide-dependent targeting of a rice alpha-amylase and cargo proteins to plastids and extracellular compartments of plant cells.

alpha-Amylases are important enzymes for starch degradation in plants. However, it has been a long-running debate as to whether alpha-amylases are localized in plastids where starch is stored. To study the subcellular localization of alpha-amylases in plant cells, a rice (Oryza sativa) alpha-amylase, alphaAmy3, with or without its own signal peptide (SP) was expressed in transgenic tobacco (Nicotiana tabacum) and analyzed. Loss-of-function analyses revealed that SP was required for targeting of alphaAmy3 to chloroplasts and/or amyloplasts and cell walls and/or extracellular compartments of leaves and suspension cells. SP was also required for in vitro transcribed and/or translated alphaAmy3 to be cotranslationally imported and processed in canine microsomes. alphaAmy3, present in chloroplasts of transgenic tobacco leaves, was processed to a product with Mr similar to alphaAmy3 minus its SP. Amino acid sequence analysis revealed that the SP of chloroplast localized alphaAmy3 was cleaved at a site only one amino acid preceding the predicted cleavage site. Function of the alphaAmy3 SP was further studied by gain-of-function analyses. beta-Glucuronidase (GUS) and green fluorescence protein fused with or without the alphaAmy3 SP was expressed in transgenic tobacco or rice. The alphaAmy3 SP directed translocation of GUS and green fluorescence protein to chloroplasts and/or amyloplasts and cell walls in tobacco leaves and rice suspension cells. The SP of another rice alpha-amylase, alphaAmy8, similarly directed the dual localizations of GUS in transgenic tobacco leaves. This study is the first evidence of SP-dependent dual translocations of proteins to plastids and extracellular compartments, which provides new insights into the role of SP in protein targeting and the pathways of SP-dependent protein translocation in plants.

The Arabidopsis sex1 Mutant Is Defective in the R1 Protein, a General Regulator of Starch Degradation in Plants, and Not in the Chloroplast Hexose Transporter

The Arabidopsis sex1 Mutant Is Defective in the R1 Protein, a General Regulator of Starch Degradation in Plants, and Not in the Chloroplast Hexose Transporter

The Arabidopsis sex1 Mutant Is Defective in the R1 Protein, a General Regulator of Starch Degradation in Plants, and Not in the Chloroplast Hexose Transporter

The Arabidopsis sex1 Mutant Is Defective in the R1 Protein, a General Regulator of Starch Degradation in Plants, and Not in the Chloroplast Hexose Transporter

The Arabidopsis sex1 mutant is defective in the R1 protein, a general regulator of starch degradation in plants, and not in the chloroplast hexose transporter.

Starch is the major storage carbohydrate in higher plants and of considerable importance for the human diet and for numerous technical applications. In addition, starch can be accumulated transiently in chloroplasts as a temporary deposit of carbohydrates during ongoing photosynthesis. This transitory starch has to be mobilized during the subsequent dark period. Mutants defective in starch mobilization are characterized by high starch contents in leaves after prolonged periods of darkness and therefore are termed starch excess (sex) mutants. Here we describe the molecular characterization of the Arabidopsis sex1 mutant that has been proposed to be defective in the export of glucose resulting from hydrolytic starch breakdown. The mutated gene in sex1 was cloned using a map-based cloning approach. By complementation of the mutant, immunological analysis, and analysis of starch phosphorylation, we show that sex1 is defective in the Arabidopsis homolog of the R1 protein and not in the hexose transporter. We propose that the SEX1 protein (R1) functions as an overall regulator of starch mobilization by controlling the phosphate content of starch.