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Starch metabolism is regulated by a complex catalytic network, one of the key enzymes of which is the plastid starch phosphorylase PHO1. In this study, using the CRISPR-Cas9 system, we obtained tobacco (Nicotiana tabacum L.) plants with a partial knockout of the NtPHO1-L1 gene due to deletion variants of the catalytic domain of the NtPHO1-L1 protein, leading to the formation of nonfunctional forms of the enzyme. The edited lines differed from wild-type plants by increased starch accumulation and decreased content of sugars, chlorophylls, and carotenoids in the leaves. It was shown that, compared to the control, the edited plants were characterized by differential expression of starch (NtPHO1-L1, NtGWD, NtBAM1, NtBAM9, NtAI) and carotenoid (NtPSY2, NtPDS, NtZDS, NtCRTISO, NtVDE) metabolism genes, as well as genes encoding MADS-domain transcription factors (NtFUL1, NtSEP1, NtSEP2, NtSEP3), which are presumably involved in the regulation of transcription of the studied metabolic genes. These data suggest that partial knockout of NtPHO1-L1 alters the functional activity of tobacco starch phosphorylase. This, in turn, may influence the coordinated activity of starch catabolism enzymes, as well as chlorophyll and carotenoid synthesis enzymes, possibly through differential expression of MADS-box genes. The results highlight the critical regulatory role of plastid starch phosphorylase in transient starch metabolism and in stimulating plant photosynthesis. [ABSTRACT FROM AUTHOR]

Chinese rose (Rosa chinensis) is an important ornamental plant, with economic, cultural, and symbolic significance. During the application of outdoor greening, adverse environments such as high temperature and drought are often encountered, which affect its application scope and ornamental quality. The starch phosphorylase (Pho) gene family participate in the synthesis and decomposition of starch, not only related to plant energy metabolism, but also plays an important role in plant stress resistance. The role of Pho in combating salinity and high temperature stress in R. chinensis remains unknown. In this work, 4 Phos from R. chinensis were detected with Pfam number of Pho (PF00343.23) and predicted by homolog-based prediction (HBP). The Phos are characterized by sequence lengths of 821 to 997 bp, and the proteins are predicted to subcellularly located in the plastid and cytoplasm. The regulatory regions of the Phos contain abundant stress and phytohormone-responsive cis-acting elements. Based on transcriptome analysis, the Phos were found to respond to abiotic stress factors such as drought, salinity, high temperature, and plant phytohormone of jasmonic acid and salicylic acid. The response of Phos to abiotic stress factors such as salinity and high temperature was confirmed by qRT-PCR analysis. To evaluate the genetic characteristics of Phos, a total of 69 Phos from 17 species were analyzed and then classified into 3 groups in phylogenetic tree. The collinearity analysis of Phos in R. chinensis and other species was conducted for the first time. This work provides a view of evolution for the Pho gene family and indicates that Phos play an important role in abiotic stress response of R. chinensis. [ABSTRACT FROM AUTHOR]

Worldwide, potato (Solanum tuberosum L.) is one of the most essential agro-economically food crops. To ensure its presence throughout the year for food processors and to extend its shelf life, it should be stored in cold temperatures. Despite the benefits of storage at low temperatures, it causes undesirable phenomena; one of them is cold-induced sweetening, which reduces the quality and the commercial value of the potato tubers. In this study, the CRISPR technology ″Clustered Regularly Interspaced Short Palindromic Repeats″ has been used in potato cultivar Desiree via Agrobacterium-mediatied transformation to edit starch phosphorylase gene that is responsible for starch degradation in cold temperature storage, after regeneration steps. The regenerated plants were used in the screening of genome editing by Sanger sequencing and the ICE program. Screening results showed that Desiree 2 (DE2), Desiree 7 (DE7) and Desiree 27 (DE27) lines have mutations in sgRNA sequence that indicated a significant reduction in reduced sugar contents of tubers after cold storage for 90 and 120 days. The reduction mean values were 29.224, 35.078 and 35.862 in DE2, DE7 and DE27 respectively after 90 days and 83.212, 56.674 and 34.109 after 120 days without visible differences in plant morphology and tubers. [ABSTRACT FROM AUTHOR]

Reserve starch from seeds and tubers is a crucial plant product for human survival. Much research has been devoted to quantitative and qualitative aspects of starch synthesis and its relation to abiotic factors of importance in agriculture. Certain aspects of genetic factors and enzymes influencing carbon assimilation into starch granules remain elusive after many decades of research. Starch phosphorylase (Pho) can operate, depending on metabolic conditions, in a synthetic and degradative pathway. The plastidial form of the enzyme is one of the most highly expressed genes in potato tubers, and the encoded product is imported into starch-synthesizing amyloplasts. We identified that the genomic locus of a Pho1a-type starch phosphorylase is duplicated in potato. Our study further shows that the enzyme is of importance for a normal starch granule phenotype in tubers. Null mutants created by genome editing display rounded starch granules in an increased number that contained a reduced ratio of apparent amylose in the starch. [ABSTRACT FROM AUTHOR]

Starch phosphorylase (PHO) is a multimeric enzyme with two distinct isoforms: plastidial starch phosphorylase (PHO1) and cytosolic starch phosphorylase (PHO2). PHO1 specifically resides in the plastid, while PHO2 is found in the cytosol. Both play a critical role in the synthesis and degradation of starch. This study aimed to report the detailed structure, function, and evolution of genes encoding PHO1 and PHO2 and their protein ligand-binding sites in eight monocots and four dicots. "True" orthologs of PHO1 and PHO2 of Oryza sativa were identified, and the structure of the enzyme at the protein level was studied. The genes controlling PHO2 were found to be more conserved than those controlling PHO1; the variations were mainly due to the variable sequence and length of introns. Cis-regulatory elements in the promoter region of both genes were identified, and the expression pattern was analyzed. The real-time quantitative polymerase chain reaction indicated that PHO2 was expressed in all tissues with a uniform pattern of transcripts, and the expression pattern of PHO1 indicates that it probably contributes to the starch biosynthesis during seed development in Zea mays. Under abscisic acid (ABA) treatment, PHO1 was found to be downregulated in Arabidopsis and Hordeum vulgare. However, we found that ABA could up-regulate the expression of both PHO1 and PHO2 within 12 h in Zea mays. In all monocots and dicots, the 3D structures were highly similar, and the ligand-binding sites were common yet fluctuating in the position of aa residues. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Agricultural Science & Technology (1008-0864) is the property of Journal of Agricultural Science & Technology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of Agricultural Science & Technology (1008-0864) is the property of Journal of Agricultural Science & Technology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

To examine the roles of starch phosphatases in potatoes, transgenic lines were produced where orthologs of SEX4 and LIKE SEX FOUR2 (LSF2) were repressed using RNAi constructs. Although repression of either SEX4 or LSF2 inhibited leaf starch degradation, it had no effect on cold-induced sweetening in tubers. Starch amounts were unchanged in the tubers, but the amount of phosphate bound to the starch was significantly increased in all the lines, with phosphate bound at the C6 position of the glucosyl units increased in lines repressed in StSEX4 and in the C3 position in lines repressed in StLSF2 expression. This was accompanied by a reduction in starch granule size and an alteration in the constituent glucan chain lengths within the starch molecule, although no obvious alteration in granule morphology was observed. Starch from the transgenic lines contained fewer chains with a degree of polymerization (DP) of less than 17 and more with a DP between 17 and 38. There were also changes in the physical properties of the starches. Rapid viscoanalysis demonstrated that both the holding strength and the final viscosity of the high phosphate starches were increased indicating that the starches have increased swelling power due to an enhanced capacity for hydration. [ABSTRACT FROM AUTHOR]

Starch phosphorylase (Pho), especially the plastidial type, plays an important role in starch biosynthesis. The availability of a complete cassava (Manihot esculenta Crantz) genome database allowed the identification of five Pho family members, which could be grouped into the plastidial type (MePho1a, MePho1b, and MePho1c) and cytosolic type (MePho2 and MePho3) on the basis of phylogenetic analysis. Spatial gene expression analysis of various cassava tissues revealed that plastidial MePho1b had the highest transcriptional activity in root stele, whereas the cytosolic MePho2 was expressed with higher levels in leaves and petiole. Spatial expression analysis showed that MePho1b was strongly expressed in the tuber during root development process, especially at 70 and 180 d after planting, whereas MePho1a had highest expression in leaves at 180 d after planting. Expression of MePho1b and MePho2 could be induced by salt, drought, exogenous abscisic acid, methyl jasmonate, and salicylic acid signal in the leaves of abiotic stress-treated plantlets. In addition, the expression profiles of MePho1b and starch branching enzyme gene member MeSBE2.2 exhibited significant correlation under diverse stress environments, indicating that they may have coordinated functions. [ABSTRACT FROM AUTHOR]

Gel-forming starch phosphates with a content of acidic phosphate groups ranging from 2.1 to 3.8 mmol/g were obtained in a phosphoric acid-urea system. The rates of starch phosphate degradation in buffer solution in the absence of amylase and in the presence of this enzyme were investigated in vitro. Starch phosphate gels were shown to be prone to enzymatic degradation. However, the rate of biodegradation decreased gradually as the content of phosphate groups increased. The use of microgels with average particle sizes in the range of 7.8-60.1 μm for the production of controlled-release preparations of interferon-alpha 2b has been demonstrated to be possible in principle. [ABSTRACT FROM AUTHOR]

To decrease the accumulation of reducing and non-reducing sugars in potato tubers stored at low temperature, a multiple gene-silencing vector pBIPhLSAR1-IR possessing a part of starch phosphorylase L and starch-associated R1 genes was constructed and transformed into potato (Solanum tuberosumL.) cultivars Agria and Marfona. Polymerase chain reaction (PCR) and southern blotting indicated that the RNAi construct was transformed successfully into the genome. Real-time reverse transcription PCR (RT-PCR) analysis showed that the expression level of starch phosphorylase L and starch-associated R1 genes was reduced in transgenic microtubers in relation to the non-transgenic plants. The accumulation of total and reducing sugars in transgenic microtubers was significantly decreased (up to 58% in line AM6) compared to the control. Repression of these genes decreased the phosphate content of starch and increased starch content in transgenic microtubers, implying that silencing of starch phosphorylase L and starch-associated R1 genes reduced starch breakdown during cold storage conditions. [ABSTRACT FROM PUBLISHER]

Two isoforms of starch phosphorylase (PHO; EC 2.4.1.1), plastidic PHO1 and cytosolic PHO2, have been found in all plants studied to date. Another starch phosphorylase-like gene, PHO3, which is an ortholog of Chlamydomonas PHOB, has been detected in some plant lineages. In this study, we identified three PHO isoform (LjPHO) genes in the Lotus japonicus genome. Expression of the LjPHO3 gene was observed in all tissues tested in L. japonicus, and the LjPHO3 protein was located in the chloroplast. Overexpression of LjPHO3 in L. japonicus resulted in a drastic decline in starch granule sizes and starch content in leaves. The LjPHO3 overexpression transgenic seedlings were smaller, and showed decreased pollen fertility and seed set rate. Our results suggest that LjPHO3 may participate in transitory starch metabolism in L. japonicus leaves, but its catalytic properties remain to be studied. [ABSTRACT FROM AUTHOR]

Main conclusion: Consistent with its essential role in starch biosynthesis at low temperatures, the plastidial starch phosphorylase from rice endosperm is highly active at low temperature. Moreover, contrary to results on other higher plant phosphorylases, the L80 peptide, a domain unique to plant phosphorylases and not present in orthologous phosphorylases from other organisms, is not involved in enzyme catalysis. Starch phosphorylase (Pho) is an essential enzyme in starch synthesis in developing rice endosperm as the enzyme plays a critical role in both the early and maturation phases of starch granule formation especially at low temperature. In this study, we demonstrated that the rice Pho1 maintains substantial enzyme activity at low temperature (<20 °C) and its substrate affinities for branched α-glucans and glucose-1-phosphate were significantly increased at the lower reaction temperatures. Under sub-saturating substrate conditions, OsPho1 displayed higher catalytic activities at 18 °C than at optimal 36 °C, supporting the prominent role of the enzyme in starch synthesis at low temperature. Removal of the highly charged 80-amino acid sequence L80 peptide, a region found exclusively in the plastidial Pho1 of higher plants, did not significantly alter the catalytic and regulatory properties of OsPho1 but did affect heat stability. Our kinetic results support the low temperature biosynthetic role of OsPho1 in rice endosperm and indicate that its L80 region is unlikely to have a direct enzymatic role but provides stability of the enzyme under heat stress. [ABSTRACT FROM AUTHOR]

Under stressful conditions, the non-model marine microalga Tetraselmis subcordiformis can accumulate a substantial amount of starch, making it a potential feedstock for the production of fuel ethanol. Investigating the interactions of the enzymes and the regulatory factors involved in starch metabolism will provide potential genetic manipulation targets for optimising the starch productivity of T. subcordiformis. For this reason, the proteome of T. subcordiformis was utilised to predict the first protein-protein interaction (PPI) network for this marine alga based on orthologous interactions, mainly from the general PPI repositories. Different methods were introduced to evaluate the credibility of the predicted interactome, including the confidence value of each PPI pair and Pfam-based and subcellular location-based enrichment analysis. Functional subnetworks analysis suggested that the two enzymes involved in starch metabolism, starch phosphorylase and trehalose-phosphate synthase may be the potential ideal genetic engineering targets. [ABSTRACT FROM AUTHOR]

Nickel (Ni) is a trace element essential for the growth and development of plants. Conversely, when in excess, Ni inhibits seed germination and reduces seedling growth. Therefore, we investigated the effect of Ni (5-50 μM; supplied as nickel sulfate: NiSO·6HO) on the activity of enzymes involved in sugar metabolism of wheat ( Triticum aestivum L.) seedlings after 96 h of exposure to the metal. Ni treatment reduced root and coleoptile length of emerging wheat seedlings and the effect was more pronounced on the root length. Ni (5-50 μM) treatment significantly enhanced carbohydrate content by 21-100 % over that of the control. In contrast, protein and reducing sugar contents declined by 17-43 and 22-69 %, respectively. The reduction in total protein content was confirmed by SDS-PAGE analysis. The activities of starch-metabolizing enzymes declined upon Ni stress in a concentration-dependent manner. Activities of α- and β-amylases, acid and alkaline invertases, acid and alkaline phosphatases, and starch phosphorylase declined by 18-74 and 24-85 %, 42-76 and 21-73 %, 15-54 and 28-72 %, and 50-83 %, respectively, when compared to the control. The study concludes that Ni impairs sugar metabolism as indicated by decline in the activity of sucrose and starch hydrolyzing enzymes. It resulted in decrease in the availability of biochemical energy and sugars required for the synthesis, leading to inhibition of radicle growth in germinating wheat seeds. [ABSTRACT FROM AUTHOR]

A soil–pot culture experiment was conducted to study the individual and interactive effects of zinc (0, 5, and 10 mg kg−1soil) and boron (0, 0.75, and 1.5 mg kg−1soil) on growth, enzymatic activity, nutrient uptake, seed reserve content, and yield in wheat (var. HD2285) in a soil deficient in available zinc and hot water–extractable boron. Although the application of zinc and boron alone significantly increased the plant height, grain yield, total dry-matter yield, tissue zinc, and boron content in maize, maximum increase was obtained with the combined application of zinc and boron. The activities of enzymes peroxidase and starch phosphorylase decreased as compared to untreated control but the activity of carbonic anhydrase increased. Protein, starch, and phytate content of grains also increased with the application of the micronutrients. Moreover, in the absence of zinc, application of boron decreased the starch content of grains. [ABSTRACT FROM AUTHOR]

The function of starch phosphorylase has long been debated on the regulation of starch metabolism during the growth and development of plants. In this study, we isolated starch phosphorylase genes ( Pho1 and Pho2) from barley, characterized their gene and protein structures, predicated their promoter’s cis-elements and analyzed expression patterns. Multiple alignments of these genes showed that (1) both Pho1 and Pho2 genes possess 15 exons and 14 introns in all but three of the species analyzed, A egilops tauschii (for Pho1 which contains 16 exons and 15 introns), potato (for Pho1b which contains 14 exons and 13 introns), and Triticum uraru (for Pho2 which contains 15 exons and 14 introns); (2) the exon–intron junctions of Pho1 and Pho2 flanking the ligand-binding sites are more conservative than the other regions. Analysis of protein sequences revealed that Pho1 and Pho2 were highly homologous except for two regions, the N terminal domain and the L78 insertion region. The results of real-time quantitative PCR (RT-qPCR) indicated that Pho2 is mainly expressed in germinating seeds, and the expression of Pho1 is similar to that of starch synthesis genes during seed development in barley. Microarray-based analysis indicated that the accumulation of Pho1 or Pho2 transcripts exhibited uniform pattern both in various tissues and various stages of seed development among species of barley, rice, and Arabidopsis. Pho1 of barley was significantly down-regulated under cold and drought treatments, and up-regulated under stem rust infection. Pho2 exhibited similar expression to Pho1 in barley. However, significant difference in expression was not detected for either Pho1 or Pho2 under any of the investigated abiotic stresses. In Arabidopsis, significant down-regulation was detected for Pho1 ( PHS1) under abscisic acid (ABA) and for Pho2 ( PHS2) under cold, salt, and ABA. Our results provide valuable information to genetically manipulate phosphorylase genes and to further elucidate their regulatory mechanism in the starch biosynthetic pathway. [ABSTRACT FROM AUTHOR]

Translucent tissue defect (TTD) is an undesirable postharvest disorder of potato tubers characterized by the development of random pockets of semi-transparent tissue containing high concentrations of reducing sugars. Translucent areas turn dark during frying due to the Maillard reaction. The newly released cultivar, Premier Russet, is highly resistant to low temperature sweetening, but susceptible to TTD. Symptoms appeared as early as 170 days after harvest and worsened with time in storage (4–9 °C, 95 % RH). In addition to higher concentrations of glucose, fructose and sucrose, TTD resulted in lower dry matter, higher specific activities of starch phosphorylase and glc-6-phosphate dehydrogenase, higher protease activity, loss of protein, and increased concentrations of free amino acids (esp. asparagine and glutamine). The mechanism of TTD is unknown; however, the disorder has similarities with the irreversible senescent sweetening that occurs in tubers during long-term storage, where much of the decline in quality is a consequence of progressive increases in oxidative stress with advancing age. The respiration rate of non-TTD ‘Premier Russet’ tubers was inherently higher (ca. 40 %) than that of ‘Russet Burbank’ tubers (a non-TTD cultivar). Moreover, translucent tissue from ‘Premier Russet’ tubers had a 1.9-fold higher respiration rate than the average of non-translucent tissue and tissue from non-TTD tubers. Peroxidation of membrane lipids during TTD development resulted in increased levels of malondialdehyde and likely contributed to a measurable increase in membrane permeability. Superoxide dismutase and catalase activities and the ratio of oxidized to total glutathione were substantially higher in translucent tissue. TTD tubers also contained twofold less ascorbate than non-TTD tubers. TTD appears to be a consequence of oxidative stress associated with accelerated aging of ‘Premier Russet’ tubers. [ABSTRACT FROM AUTHOR]

The present study examined the toxic effects of Cr(VI; 100, 250 and 500 μM) in maize seedlings by investigating the changes in carbohydrate metabolism after 48, 96, and 144 h of exposure. Cr-stress results in severe alterations in the contents of carbohydrates and reducing sugars and the activities of carbohydrate metabolizing enzymes, amylases, phosphatases and phosphorylases, and invertases in maize seedlings. Under Cr stress, the contents of carbohydrates and reducing sugars declined in roots, whereas an increase was noticed in leaves. The catalytic activity of carbohydrate metabolizing enzymes, except invertases, in roots declined in the presence of Cr(VI) in a concentration- and exposure time-dependent manner. In contrast, the activities of these enzymes were enhanced in leaves under Cr(VI) stress. The activity of invertases increased with increasing amount of Cr(VI) but declined with an increase in the time interval. In conclusion, our results show that carbohydrate metabolism is severely affected under Cr(VI) toxicity. The study suggests that Cr-induced perturbations in the carbohydrate metabolism are one of the factors resulting in growth inhibition under Cr(VI) stress. [ABSTRACT FROM AUTHOR]

Background Starch is synthesized in both leaves and storage tissues of plants. The role of starch syntheses and branching enzymes is well understood; however, the role of starch phosphorylase is not clear. Results A gene encoding Pho1 from barley was characterized and starch phosphorylases from both developing and germinating grain were characterized and purified. Two activities were detected: one with a molecular mass of 110 kDa and the other of 95 kDa. It was demonstrated through the use of antisera that the 110 kDa activity was located in the amyloplast and could correspond to the polypeptide encoded by the Pho1 gene cloned. The 95 kDa activity was localized to the cytoplasm, most strongly expressed in germinating grain, and was classified as a Pho2-type sequence. Using RNAi technology to reduce the content of Pho1 in the grain to less than 30% of wild type did not lead to any visible phenotype, and no dramatic alterations in the structure of the starch were observed. Conclusion Two starch phosphorylase activities were identified and characterized in barley grains, and shown to be present during starch synthesis. However, their role in starch synthesis still remains to be elucidated.© 2012 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Activities of enzymes presumably involved in starch biosynthesis (ADP-glucose pyrophosphorylase, AGPase) and/or breakdown (starch phosphorylase, STP; amylases) were determined during potato ( Solanum tuberosum L.) tuber dormancy and sprouting. Overall activities of all these enzymes decreased during the first stage of tuber dormancy. No clear changes were detected at the time of dormancy breaking and sprouting. However, when AGPase activity was monitored by in situ staining during the entire dormancy period, a clear decrease during the dormant period and a large increase before visible sprouting could be observed. This increase was especially evident near the vascular tissue and at the apical bud, which showed a very intensive staining. In situ staining of STP activity in sprouting tubers showed that the tissue distribution of STP was the same as for AGPase. As a possible explanation, direct starch cycling is suggested: STP produces glucose-1-phosphate during starch breakdown, which can be directly used as a substrate by AGPase for starch synthesis. Gene expression studies with the AGPaseS promoter coupled to the firefly luciferase reporter gene also clearly showed a higher activity in sprouting tubers as compared to dormant tubers, with the highest expression levels observed around the apical buds. The presence of amylase activity at dormancy initiation and AGPase activity persistent at the sprouting stage suggest that starch was cycling throughout the entire dormancy period. According to the in situ studies, the AGPase activity increased well before visible sprout growth and could therefore be one of the first physiological determinants of dormancy breakage. [ABSTRACT FROM AUTHOR]

The present study investigated the effect of Arsenic (As; 5, 10, 50 μM) on protein and sugar metabolism vis-à-vis oxidative damage during early germination process and radicle emergence (at 12, 24 and 48 h stage) in Phaseolus aureus. As-exposure (50 μM) significantly enhanced protein content (by 40-60%), whereas carbohydrate content declined (by 31-44%) over that in the control. It was associated with a decline in the activities of proteases (47-53%), and increase in the activities of α- and β-amylases, starch phosphorylases, and acid invertases by 3.0, 2.6, 4.8, and 1.7 times after 48 h exposure to 50 μM As. The alteration in protein and carbohydrate metabolic machinery was also accompanied by As-induced reactive oxygen species (ROS)-mediated oxidative damage. As treatment enhanced malondialdehyde and hydrogen peroxide content by 46-252% and 23-216%, and hydroxyl and superoxide ion generation by 15-104% and 17-278%, respectively. As-induced lipid peroxidation and membrane disruption was confirmed by enhanced electrolyte leakage (by 49%) and reduced cell viability (by 43%). Furthermore, in response to 50 μM As, the activities of superoxide dismutases, catalases, ascorbate peroxidases, guaiacol peroxidases, and glutathione reductases increased by 77%, 70%, 116%, 43% and 120%, respectively, in radicles at 48 h stage over that in the control. The study concludes that As inhibits radicle emergence and elongation in germinating P. aureus seeds by altering biochemical processes related to sugar metabolism and inducing an ROS-mediated oxidative damage. [ABSTRACT FROM AUTHOR]

The present study established the way in which plastidial α-glucan phosphorylase (Pho1) synthesizes maltodextrin (MD) which can be the primer for starch biosynthesis in rice endosperm. The synthesis of MD by Pho1 was markedly accelerated by branching enzyme (BE) isozymes, although the greatest effect was exhibited by the presence of branching isozyme I (BEI) rather than by isozyme IIa (BEIIa) or isozyme IIb (BEIIb). The enhancement of the activity of Pho1 by BE was not merely due to the supply of a non-reducing ends. At the same time, Pho1 greatly enhanced the BE activity, possibly by generating a branched carbohydrate substrate which is used by BE with a higher affinity. The addition of isoamylase to the reaction mixture did not prevent the concerted action of Pho1 and BEI. Furthermore, in the product, the branched structure was, at least to some extent, maintained. Based on these results we propose that the interaction between Pho1 and BE is not merely due to chain-elongating and chain-branching reactions, but occurs in a physically and catalytically synergistic manner by each activating the mutual capacity of the other, presumably forming a physical association of Pho1, BEI and branched MDs. This close interaction might play a crucial role in the synthesis of branched MDs and the branched MDs can act as a primer for the biosynthesis of amylopectin molecules. [ABSTRACT FROM AUTHOR]

amylose extender (ae−) starches characteristically have modified starch granule morphology resulting from amylopectin with reduced branch frequency and longer glucan chains in clusters, caused by the loss of activity of the major starch branching enzyme (SBE), which in maize endosperm is SBEIIb. A recent study with ae− maize lacking the SBEIIb protein (termed ae1.1 herein) showed that novel protein–protein interactions between enzymes of starch biosynthesis in the amyloplast could explain the starch phenotype of the ae1.1 mutant. The present study examined an allelic variant of the ae− mutation, ae1.2, which expresses a catalytically inactive form of SBEIIb. The catalytically inactive SBEIIb in ae1.2 lacks a 28 amino acid peptide (Val272–Pro299) and is unable to bind to amylopectin. Analysis of starch from ae1.2 revealed altered granule morphology and physicochemical characteristics distinct from those of the ae1.1 mutant as well as the wild-type, including altered apparent amylose content and gelatinization properties. Starch from ae1.2 had fewer intermediate length glucan chains (degree of polymerization 16–20) than ae1.1. Biochemical analysis of ae1.2 showed that there were differences in the organization and assembly of protein complexes of starch biosynthetic enzymes in comparison with ae1.1 (and wild-type) amyloplasts, which were also reflected in the composition of starch granule-bound proteins. The formation of stromal protein complexes in the wild-type and ae1.2 was strongly enhanced by ATP, and broken by phosphatase treatment, indicating a role for protein phosphorylation in their assembly. Labelling experiments with [γ-32P]ATP showed that the inactive form of SBEIIb in ae1.2 was phosphorylated, both in the monomeric form and in association with starch synthase isoforms. Although the inactive SBEIIb was unable to bind starch directly, it was strongly associated with the starch granule, reinforcing the conclusion that its presence in the granules is a result of physical association with other enzymes of starch synthesis. In addition, an Mn2+-based affinity ligand, specific for phosphoproteins, was used to show that the granule-bound forms of SBEIIb in the wild-type and ae1.2 were phosphorylated, as was the granule-bound form of SBEI found in ae1.2 starch. The data strongly support the hypothesis that the complement of heteromeric complexes of proteins involved in amylopectin synthesis contributes to the fine structure and architecture of the starch granule. [ABSTRACT FROM PUBLISHER]

Starch is globally important as a source of food and, in addition, has a wide range of industrial applications. Much of this agriculturally produced starch is synthesized in developing seeds, where its biological function is to provide energy for seedling establishment. Storage starch in developing seeds is synthesized in heterotrophic plastids called amyloplasts and is distinct from the transient synthesis of starch in chloroplasts. This article reviews our current understanding of storage starch biosynthesis occurring in these organelles and discusses recent advances in research in this field. The review discusses starch structure and granule initiation, emerging ideas on the evolution of the pathway, the enzymes of starch synthesis, and the post-translational modification and regulation of key enzymes of amylopectin biosynthesis. [ABSTRACT FROM AUTHOR]

The amylose extender (ae–) mutant of maize lacks starch branching enzyme IIb (SBEIIb) activity, resulting in amylopectin with reduced branch point frequency, and longer glucan chains. Recent studies indicate isozymes of soluble starch synthases form high molecular weight complexes with SBEII isoforms. This study investigated the effect of the loss of SBEIIb activity on interactions between starch biosynthetic enzymes in maize endosperm amyloplasts. Results show distinct patterns of protein–protein interactions in amyloplasts of ae– mutants compared with the wild type, suggesting functional complementation for loss of SBEIIb by SBEI, SBEIIa, and SP. Coimmunoprecipitation experiments and affinity chromatography using recombinant proteins showed that, in amyloplasts from normal endosperm, protein–protein interactions involving starch synthase I (SSI), SSIIa, and SBEIIb could be detected. By contrast, in ae– amyloplasts, SSI and SSIIa interacted with SBEI, SBEIIa, and SP. All interactions in the wild-type were strongly enhanced by ATP, and broken by alkaline phosphatase, indicating a role for protein phosphorylation in their assembly. Whilst ATP and alkaline phosphatase had no effect on the stability of the protein complexes from ae– endosperm, radiolabelling experiments showed SP and SBEI were both phosphorylated within the mutant protein complex. It is proposed that, during amylopectin biosynthesis, SSI and SSIIa form the core of a phosphorylation-dependent glucan-synthesizing protein complex which, in normal endosperm, recruits SBEIIb, but when SBEIIb is absent (ae–), recruits SBEI, SBEIIa, and SP. Differences in stromal protein complexes are mirrored in the complement of the starch synthesizing enzymes detected in the starch granules of each genotype, reinforcing the hypothesis that the complexes play a functional role in starch biosynthesis. [ABSTRACT FROM PUBLISHER]

His334 facilitates catalysis by Corynebacterium callunae starch phosphorylase through selective stabilization of the transition state of the reaction, partly derived from a hydrogen bond between its side chain and the C-6 hydroxy group of the glucosyl residue undergoing transfer to and from phosphate. We have substituted His334 by a Gly and measured the disruptive effects of the site-directed replacement on active site function using steady-state kinetics and NMR spectroscopic characterization of the cofactor pyridoxal 5′-phosphate and binding of carbohydrate ligands. Purified H334G showed 0.05% and 1.3% of wild-type catalytic center activity for phosphorolysis of maltopentaose ( kcat P = 0.033 s−1) and substrate binding affinity in the ternary complex with enzyme bound to phosphate ( Km = 280 mm), respectively. The 31P chemical shift of pyridoxal 5′-phosphate in the wild-type was pH-dependent and not perturbed by binding of arsenate. At pH 7.25, it was not sensitive to the replacement His334→Gly. Analysis of interactions of α-d-glucose 1-phosphate and α-d-xylose 1-phosphate upon binding to wild-type and H334G phosphorylase, derived from saturation transfer difference NMR experiments, suggested that disruption of enzyme–substrate interactions in H334G was strictly local, affecting the protein environment of sugar carbon 6. pH profiles of the phosphorolysis rate for wild-type and H334G were both bell-shaped, with the broad pH range of optimum activity in the wild-type (pH 6.5–7.5) being narrowed and markedly shifted to lower pH values in the mutant (pH 6.5–7.0). External imidazole partly restored the activity lost in the mutant, without, however, participating as an alternative nucleophile in the reaction. It caused displacement of the entire pH profile of H334G by + 0.5 pH units. A possible role for His334 in the formation of the oxocarbenium ion-like transition state is suggested, where the hydrogen bond between its side chain and the 6-hydroxyl polarizes and positions O-6 such that electron density in the reactive center is enhanced. [ABSTRACT FROM AUTHOR]

A 78-amino acid insertion (L78) is found in the low-affinity type (L-form) of starch phosphorylase (L-SP, EC 2.4.1.1). This insertion blocks the starch-binding site on the L-SP molecule, and it decreases the binding affinity of L-SP toward starch. The computational analysis of the amino acid sequence on L78 predicts several phosphorylation sites at its Ser residues. Indeed, from the immunoblotting results using antibodies against phosphoamino acids, we observed that the purified L-SP from mature sweet potato ( Ipomoea batatas) roots is phosphorylated. This observation led us to the detection of a protein kinase activity in the protein fraction of the crude extract from the sweet potato roots. The kinase was partially purified by liquid chromatography, and its native molecular mass was estimated as 338 kDa. An expressed peptide (L78P) containing the essential part of L78 was intensively phosphorylated by the kinase. However, H-SP (the high-affinity isomer of SP lacking the L78 insertion) and the proteolytic modified L-SP, which lost its L78 fragment, could not be phosphorylated. Furthermore, using L78P mutants by site-directed mutagenesis at Ser residues on L78, we demonstrate that only one Ser residue on L78 is phosphorylated by the kinase. These results imply that this kinase is specific to L-SP, or more precisely, to the L78 insertion. The in vitro phosphorylated L-SP shows higher sensitivity to proteolytic modification, but has no change in its kinetic parameters. [ABSTRACT FROM AUTHOR]

Arsenic (As) is a potential contaminant of groundwater as well as soil in many parts of the world. The effects of increasing concentration of As (25 µm and 50 µm As2O3) in the medium on the content of starch and sugars and activity levels of enzymes involved in starch and sugar metabolism i.e. α-amylase, β-amylase, starch phosphorylase and acid invertase were studied in germinating seeds of two rice cvs. Malviya-36 and Pant-12 during 0–120 h period. As toxicity in situ led to a marked decline in the activities of α-amylase, β-amylase in endosperms as well as embryoaxes of germinating rice seeds. The activity of acid invertase increased in endosperms as well as embryoaxes whereas starch phosphorylase activity declined in endosperms but increased in embryoaxes under As treatment. In endosperms a decline in starch mobilization was observed under As toxicity, however under similar conditions the content of total soluble sugars increased in embryoaxes. The observed inhibition in activities of amylolytic enzymes might contribute to delayed mobilization of endospermic starch which could affect germination of seeds in As polluted environment, while the induced acid invertase activity and increased sugar accumulation in embryoaxes could serve as a possible component for adaptation mechanism of rice seedlings grown under As containing medium. [ABSTRACT FROM AUTHOR]

This article reviews current knowledge of starch metabolism in higher plants, and focuses on the control and regulation of the biosynthetic and degradative pathways. The major elements comprising the synthetic and degradative pathways in plastids are discussed, and show that, despite present knowledge of the core reactions within each pathway, understanding of how these individual reactions are co-ordinated within different plastid types and under different environmental conditions, is far from complete. In particular, recently discovered aspects of the fine control of starch metabolism are discussed, which indicate that a number of key reactions are controlled by post-translational modifications of enzymes, including redox modulation and protein phosphorylation. In some cases, enzymes of the pathway may form protein complexes with specific functional significance. It is suggested that some of the newly discovered aspects of fine control of the biosynthetic pathway may well apply to many other proteins which are directly and indirectly involved in polymer synthesis and degradation. [ABSTRACT FROM PUBLISHER]

We have used alanine-scanning site-directed mutagenesis of the dimer contact region of starch phosphorylase from Corynebacterium callunae to explore the relationship between a protein conformational change induced by phosphate binding and the up to 500-fold kinetic stabilization of the functional quarternary structure of this enzyme when phosphate is present. Purified mutants (at positions Ser-224, Arg-226, Arg-234, and Arg-242) were characterized by Fourier transform-infrared (FT-IR) spectroscopy and enzyme activity measurements at room temperature and under conditions of thermal denaturation. Difference FT-IR spectra of wild type and mutants in 2H2O solvent revealed small changes in residual amide II band intensities at ≈ 1,550 cm–1, indicating that 1H/2H exchange in the wild type is clearly perturbed by the mutations. Decreased 1H/2H exchange in comparison to wild type suggests formation of a more compact protein structure in S224A, R234A, and R242A mutants and correlates with rates of irreversible thermal denaturation at 45°C that are up to 10-fold smaller for the three mutants than the wild type. By contrast, the mutant R226A inactivates 2.5-fold faster at 45°C and shows a higher 1H/2H exchange than the wild type. Phosphate (20 mM) causes a greater change in FT-IR spectra of the wild type than in those of S224A and 234A mutants and leads to a 5-fold higher stabilization of the wild type than the two mutants. Therefore, structural effects of phosphate binding leading to kinetic stability of wild-type starch phosphorylase are partially complemented in the S224A and R234A mutants. Infrared spectroscopic measurements were used to compare thermal denaturations of the mutants and the wild type in the absence and presence of stabilizing oxyanion. The broad denaturation transition of unliganded wild type in the range 40–50°C is reduced in the S224A and R234A mutants, and this reflects mainly a shift of the onset of denaturation to a 4–5°C higher value. [ABSTRACT FROM PUBLISHER]

Starch mobilization is well understood in cereal endosperms, but both the pathway and the regulation of the process are poorly characterized in other types of plant organs. Arabidopsis leaves offer the opportunity for rapid progress in this area, because of the genomic resources available in this species and the ease with which starch synthesis and degradation can be monitored and manipulated. Progress in understanding three aspects of starch degradation is described: the role of disproportionating enzyme, the importance of phosphorolytic degradation, and new evidence about the involvement of a starch-phosphorylating enzyme in the degradative process. Major areas requiring further research are outlined. [ABSTRACT FROM AUTHOR]

Two main forms of starch phosphorylase (EC 2.4.1.1) were identified and purified from banana (Musa acuminata Colla. cv. Nanicão) fruit. One of them, designated phosphorylase I, had a native molecular weight of 155 kDa and subunit of 90 kDa, a high affinity towards branched glucans and an isoelectric point around 5.0. The other, phosphorylase II, eluted at a higher salt concentration from the anion exchanger, had a low affinity towards branched glucans, a native molecular weight of 290 kDa and subunit of 112 kDa. Kinetic studies showed that both forms had typical hyperbolic curves for orthophosphate (Pi) and glucose-1-phosphate, and that they could not react with substrates with a blocked reducing end or α-1,6 glucosidic bonds. Antibodies prepared against the purified type-II form and cross-reacting with the type-I form showed that there was an increase in protein content during development and ripening of the fruit. The changes in protein level were parallel to those of phosphorylase activity, in both the phosphorolytic and synthetic directions. Considering the kinetics, indicating that starch phosphorylases are not under allosteric control, it can be argued that protein synthesis makes a contribution to regulating phosphorylase activity in banana fruit and that hormones, like gibberellic acid and indole-3-acetic acid, may play a regulating role. For the first time, starch phosphorylases isoforms were detected as starch-granule-associated proteins by immunostaining of SDS–PAGE gels. [ABSTRACT FROM AUTHOR]

In a synchronously grown Chlamydomonas reinhardtii (Chlorophyceae) culture the CO2-concentrating mechanism (CCM) was induced by lowering the CO2 level from 4% to 0.036% CO2 (culture HL). The effects of the reduced carbon supply on starch levels were studied over a period of up to 100 h and compared with control cultures kept either at 4% CO2 (culture H) or continuously at ambient air (0.036% CO2, culture L). Lowering the CO2 supply reduced culture growth as estimated by chlorophyll, protein and cell density. The starch level continued to show diurnal variations with an initially reduced rate of starch synthesis at reduced or abolished culture growth. Subsequently, starch maxima and minima increased. After 4 days the resulting pattern for culture HL was similar to that of culture L, which possessed higher minima but identical maxima to culture H. The intracellular starch localisation was examined on electron micrographs. Cell extracts were assayed for ADP-glucose pyrophosphorylase (EC 2.7.7.27) and starch phosphorylase (EC 2.4.1.1) activities. Over the assayed period of 2 days, there was a good correlation between the observed changes in the starch levels and the measured enzyme activities. The rate of CO2-dependent oxygen evolution of culture HL declined from 100% to 60% of the control over the day. This indicates that the diminished or abolished growth and the impairment of starch accumulation upon CO2 depletion are not simply consequences of the lowered level of the substrate CO2. The diminished growth and the peculiar starch accumulation pattern with higher positions of the starch minima in low-CO2 cells are interpreted as economised starch utilisation as long-term aspects of induction of the CCM. [ABSTRACT FROM AUTHOR]

Starch phosphorylase is a member of the GT35-glycogen-phosphorylase superfamily. Glycogen phosphorylases have been researched in animals thoroughly when compared to plants. Genetic evidence signifies the integral role of plastidial starch phosphorylase (PHO1) in starch biosynthesis in model plants. The counterpart of PHO1 is PHO2, which specifically resides in cytosol and is reported to lack L80 peptide in the middle region of proteins as seen in animal and maltodextrin forms of phosphorylases. The function of this extra peptide varies among species and ranges from the substrate of proteasomes to modulate the degradation of PHO1 in Solanum tuberosum to a non-significant effect on biochemical activity in Oryza sativa and Hordeum vulgare. Various regulatory functions, e.g., phosphorylation, protein–protein interactions, and redox modulation, have been reported to affect the starch phosphorylase functions in higher plants. This review outlines the current findings on the regulation of starch phosphorylase genes and proteins with their possible role in the starch biosynthesis pathway. We highlight the gaps in present studies and elaborate on the molecular mechanisms of phosphorylase in starch metabolism. Moreover, we explore the possible role of PHO1 in crop improvement. [ABSTRACT FROM AUTHOR]

Changes in the starch and sucrose contents, and the sucrose phosphate synthase, acid invertase, and starch phosphorylase activities were studied in the seedlings of salt sensitive and salt tolerant rice cultivars growing under two NaCl concentrations (7 and 14 dS m-1) for 20 d. Under salinity, the starch content in roots declined more in salt sensitive cvs. Ratna and Jaya than in salt tolerant cvs. CSR-1 and CSR-3 and was unchanged in shoots. The contents of reducing and non-reducing sugars, and the activity of sucrose phosphate synthase was increased more in the sensitive than in the tolerant cultivars. Acid invertase activity decreased in shoots of the salt tolerant cultivars, whereas increased in salt sensitive cultivars. Starch phosphorylase activity decreased in all cultivars. [ABSTRACT FROM AUTHOR]

The expression of the gene encoding the plastidic enzyme starch phosphorylase (EC 2.4.1.1) varies according to tissue carbohydrate status. Incubation of excised potato ( Solanum tuberosum L. cv. Désirée) leaves carrying a portion of the stem under a short photoperiod resulted in a drastic accumulation of starch, accompanied by a rapid increase in the level of phosphorylase mRNA and by a similar change in phosphorylase protein level. However, under the same incubation conditions, the transcriptional activity of the phosphorylase promoter in transgenic plants did not change markedly. Therefore, the increased expression of the phosphorylase gene in petioles of stem cuttings is not controlled by the level of initiation of transcription. Phosphorylase mRNA accumulated to a high level in petioles of detached leaves kept under constant light for 24 h, but not in petioles kept in the dark. The effect of light on the accumulation of the mRNA was appreciably reduced if the petioles were incubated in ethylendiaminetetraacetic acid (EDTA), a treatment known to increase phloem exudation in detached leaves. The inhibition by EDTA could be partially counteracted by the addition of sucrose to the incubation solution. Furthermore, incubation of petioles in darkness in solutions with high levels of sucrose resulted in enhanced expression of the gene. These results suggest that sucrose, the main compound transported by phloem in potato, is involved in the regulation of the starch phosphorylase gene. This also indicates that conditions favouring starch synthesis lead to increased expression of the phosphorylase gene. [ABSTRACT FROM AUTHOR]

Starch-degrading enzymes in isolated pea ( Pisum sativum L. cv. Laxton's Progress No. 9) chloroplasts were investigated and compared with those in crude pea leaf and stipule preparations. End-product analysis of amylopectin degradation by chloroplast and crude extracts indicates that maltose is the major product of both. Two multiforms of β-amylase (EC 3.2.1.2) were detected in pea chloroplasts using an electrophoretic transfer technique. A starch-debranching enzyme (EC 3.2.1.10) was detected in chloroplasts by electrophoretic transfer and the degradation of pullulan. A different multiform of debranching enzyme was found in crude preparations. α-Amylases (EC 3.2.1.1) were detected by electrophoretic transfer through gels containing starch and starch azure, and by change in viscosity of a starch solution, but were only found in crude preparations indicating an extrachloroplastic location. Incubation of maltotriose with chloroplast extracts gave high levels of glucose production and formation of oligosaccharides with degrees of polymerization larger than that of maltotriose indicating transglycosylase (EC 2.4.1.25) activity. Neither α-glucosidase (EC 3.2.1.20) nor maltose-phosphorylase (EC 2.4.1.1) activity were found in either chloroplast or crude preparations, whereas starch-phosphorylase (EC 2.4.1.1) activity was in both. The possible role of these enzymes in starch degradation by pea chloroplasts is discussed. [ABSTRACT FROM AUTHOR]

Phosphorylase was purified from young and senescent potato tubers. Antibodies raised against the enzyme from young tubers crossreacted with phosphorylase from old tissue, although the latter exhibited different physico-chemical properties. In polyacrylamide gel electrophoresis it migrated with higher mobility, its subunit molecular weight was determined in the range of 40,000 in contrast to 100,000 of the phosphorylase in young tubers. The enzyme of senescent tubers displayed an isoelectric point of 5.4 different from the one of young tubers with 5.0, and the diffusion coefficients of the two enzymes varied. The appearance of the phosphorylase form typical for senescent tissue is connected with changes in the intracellular localization as revealed by immunofluorescence. Before massive starch accumulation is initiated, non-vacuolated subepidermal cells contain antigenically active material in their cytoplasm. During starch accumulation in fully differentiated storage parenchyma, only amyloplasts fluoresce, indicating the presence of adsorbed phosphorylase protein. Cytoplasmic phosphorylase can be detected in the continuance of senescence and, finally, after 16 months of tuber storage, the particle-bound enzyme had mostly disappeared. Simultaneously, we observed membrane destruction and decomposition on the ultrastructural level. The phosphorylase from senescent potatoes is a converted molecule and seems to be formed by proteolytic cleavage. The location of phosphorylase in the amyloplasts during starch synthesis indicates that it also plays a role in starch synthesis and not only in its degradation. [ABSTRACT FROM AUTHOR]

Changes in the contents of starch, protein, DNA, RNA, total phosphorus., acid soluble phosphorus and inorganic phosphorus, and in the activities of some enzymes. of carbohydrate, amino acid, nucleic acid and phosphate metabolism were studied during the germination of Cuscuta campestris seeds. The results are expressed on per seed basis. Starch content in Cuscuta seeds showed: .a steady decline with most of it depleted by the end of the eighth day of germination. Protein content increased with germination up to 48 h and then decreased. RNA and DNA contents increased to a maximal level on the fourth day of germination aid then decreased, Total phosphorus in the seeds remained almost unchanged during the period of study, Both trichioroacetic add soluble and inorganic phosphorus increased until the third day and then decreased. Phytin was rapidly hydrolyzed with little being detectable by the seventh day of germination. Glucose-6-phosphate dehydrogenase increased with germination, while fructose bisphosphate aldolase which is indispensable for glycolysis, decreased with germination. Ribonuclease and ,deoxyribonuclease increased till the third and fourth day, respectively, and then decreased. Aspartate and alanine aminotransferases showed .a maximum on the second day and then decreased. Activities of alkaline fructose-1,6-bisphosphatase and phytase were absent in the dry seeds. and appeared only on the second day of germination. Both α- and β-amylase activities were present in the dry seed. [ABSTRACT FROM AUTHOR]

The transfer of potato (Solanum tuberosum) tubers from 10 to 1°C was associated with an initial decline in the rate of CO2 output followed by a rapid increase reaching, within some 12 days, a peak which was about 3-fold higher than at 10°C. Thereafter the rate of CO2 evolution declined gradually for the duration of the experiment. The specific rate of mitochondrial O2 uptake decreased initially, followed by a rise to a level similar to that of mitochondria prepared from tubers stored at 10°C. Low temperature decreased by 30% the capacity of the cytochrome pathway while it sharply increased the capacity of the alternative pathway. Sucrose was the first sugar to accumulate at 1°C, followed after a delay of 6-7 days by glucose and fructose. Low temperature induced within 4-5 days a rise in amylase activity which increased by 10-fold after 30 days. The increase was reflected in only two out of four existing isoforms. In addition a novel isoform of amylase was detected later in storage. The induction and the accumulation of invertase mRNA and extractable activity followed the increase in sucrose but preceded that of hexoses. The activity of starch phosphorylase isoforms was not affected by temperature. There was a 3-fold increase in chlorogenic acid at 1°C. Hypoxia strongly inhibited the accumulation of sugars and chlorogenic acid, the increase in the amylase activity, and the appearance of the novel isoform. Low O2 totally suppressed the induction of invertase mRNA and increased the capacity of the alternative oxidase. It did not, however, prevent the decrease in cytochrome capacity; neither did it affect the activity of starch phosphorylase isoforms. [ABSTRACT FROM AUTHOR]

Starch and sucrose metabolism of one- and two-year-old needles of Norway spruce (Picea abies [L.] Karst., about 30 years old) was investigated from three months before until three months after bud break at a natural site. We distinguish different metabolic states according to the extractable activities of enzymes (α-amylase [EC 3.2.1.1 ], ADP-glucose pyrophosphorylase [AGP, EC 2.7.7.27], D-enzyme [EC 2.4.1.25], starch phosphorylase [STP, EC 2.4.1.1]), sucrose phosphate synthase [SPS, EC 2.4.1.14], sucrose synthase [SS, EC 2.4.1.13], acid invertase [AI, EC 3.2.1.26]) and pool sizes of related metabolites (starch, glucose, fructose, sucrose, raffinose, stachyose, fructose 6-phosphate [F6P], glucose 6-phosphate [G6P], fructose 2,6-bisphosphate [F26BP], and inorganic phosphate [Pi]). The period ending with bud break was characterized by high rates of net photosynthesis, a pronounced decrease in the amount of soluble sugars, and a steep rise in starch (from the detection limit to approximately 600 nmol glycosyl units [mg dry weight]-1). In parallel, the extractable activity of AGP increased, while D-enzyme was on a relative high level when compared with the period after bud break. With respect to sucrose metabolism, F26BP, an inhibitor of sucrose synthesis, decreased from 1 to 0.4 pmol (mg dry weight)-1. This was complemented by SPS activity, which was due to both increased protein levels shown by immunoblotting and activation under metabolite control (high levels of G6P and a low Pi/G6P ratio). This indicates a high capacity of synthesis of starch and sucrose in the period before bud break. These observations are in accordance with estimates of photosynthetic carbon gain, which indicate that in early spring large amounts of carbon from current photosynthesis are exported out of the needles. In addition, the content of nonstructural carbohydrates (expressed as hexoses) increased in the bark of the stem. This could also be a consequence of an enhanced carbon export from the needles. After the onset of bud break, starch concentration decreased in all tissues under investigation. In contrast, the level of total nonstructural carbohydrates in the outermost sapwood nearly doubled from bud break until the end of sampling. In the needles, net photosynthesis was reduced by about 75% and a decrease in SPS activity and protein level were found together with lower G6P concentration, and an increased Pi/G6P ratio. These results suggest that during that period sucrose synthesis was reduced in the older needles. In addition, under conditions of reduced photosynthesis, carbon demand of current year needles was in part ensured by the mobilization of starch in the older needles. Taken together our data show that before bud break carbon metabolism of mature leaves is related with the sink demands of storage organs. After bud break the accumulated assimilate pools in needles and stem, mainly the bark, are mobilized and support carbon supply to new tissues. [ABSTRACT FROM AUTHOR]

Starch is a water-insoluble polymer of glucose synthesized as discrete granules inside the stroma of plastids in plant cells. Starch reserves provide a source of carbohydrate for immediate growth and development, and act as long term carbon stores in endosperms and seed tissues for growth of the next generation, making starch of huge agricultural importance. The starch granule has a highly complex hierarchical structure arising from the combined actions of a large array of enzymes as well as physicochemical self-assembly mechanisms. Understanding the precise nature of granule architecture, and how both biological and abiotic factors determine this structure is of both fundamental and practical importance. This review outlines current knowledge of granule architecture and the starch biosynthesis pathway in relation to the building block-backbone model of starch structure. We highlight the gaps in our knowledge in relation to our understanding of the structure and synthesis of starch, and argue that the building block-backbone model takes accurate account of both structural and biochemical data. [ABSTRACT FROM AUTHOR]

Durable response, inherent or acquired resistance, and dose-limiting toxicities continue to represent major barriers in the treatment of patients with advanced clear-cell renal cell carcinoma (ccRCC). The majority of ccRCC tumors are characterized by the loss of Von Hippel–Lindau tumor suppressor gene function, a stable expression of hypoxia-inducible factors 1α and 2α (HIFs), an altered expression of tumor-specific oncogenic microRNAs (miRNAs), a clear cytoplasm with dense lipid content, and overexpression of thymidine phosphorylase. The aim of this manuscript was to confirm that the downregulation of specific drug-resistant biomarkers deregulated in tumor cells by a defined dose and schedule of methylselenocysteine (MSC) or seleno-l-methionine (SLM) sensitizes tumor cells to mechanism-based drug combination. The inhibition of HIFs by selenium was necessary for optimal therapeutic benefit. Durable responses were achieved only when MSC was combined with sunitinib (a vascular endothelial growth factor receptor (VEGFR)-targeted biologic), topotecan (a topoisomerase 1 poison and HIF synthesis inhibitor), and S-1 (a 5-fluorouracil prodrug). The documented synergy was selenium dose- and schedule-dependent and associated with enhanced prolyl hydroxylase-dependent HIF degradation, stabilization of tumor vasculature, downregulation of 28 oncogenic miRNAs, as well as the upregulation of 12 tumor suppressor miRNAs. The preclinical results generated provided the rationale for the development of phase 1/2 clinical trials of SLM in sequential combination with axitinib in ccRCC patients refractory to standard therapies. [ABSTRACT FROM AUTHOR]