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Abstract 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.

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 mediated 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

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]

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.

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.)

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.

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.

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]

Functional interactions of plastidial phosphorylase (Pho1) and starch branching enzymes (BEs) from the developing rice endosperm are the focus of this study. In the presence of both Pho1 and BE, the same branched primer molecule is elongated and further branched almost simultaneously even at very low glucan concentrations present in the purified enzyme preparations. By contrast, in the absence of any BE, glucans are not, to any significant extent, elongated by Pho1. Based on our in vitro data, in the developing rice endosperm, Pho1 appears to be weakly associated with any of the BE isozymes. By using fluorophore-labeled malto-oligosaccharides, we identified maltose as the smallest possible primer for elongation by Pho1. Linear dextrins act as carbohydrate substrates for BEs. By functionally interacting with a BE, Pho1 performs two essential functions during the initiation of starch biosynthesis in the rice endosperm: First, it elongates maltodextrins up to a degree of polymerization of at least 60. Second, by closely interacting with BEs, Pho1 is able to elongate branched glucans efficiently and thereby synthesizes branched carbohydrates essential for the initiation of amylopectin biosynthesis. [ABSTRACT FROM AUTHOR]

In a marine green starch-producing microalga Tetraselmis subcordiformis , the role of starch phosphorylase (SP) in the starch biosynthesis was disclosed by characterizing the enzyme properties and activity variations during the starch accumulation process. TsSP4, a SP isoform accounting for the major SP activity in T. subcordiformis , was unique to be active in a monomer form with a molecular weight of approximately 110 kDa. It resembled one of the chloroplast-located SPs (PhoA) in Chlamydomonas reinhardtii with a similarity of 63.3% in sequence, though it possessed the typical L78/80 domain found in the plastidial SPs (Pho1) of higher plants that was absent in PhoA. TsSP4 exhibited moderate sensitivity to ADP-Glc inhibition and had a high activity for longer-chain linear maltooligosacchride (MOS) and amylopectin against highly branched glycogen as the substrates. TsSP4 had 2-fold higher affinity for Glc-1-P in the synthetic direction than for Pi in the phosphorolytic direction, and the catalytic constant k cat for Glc-1-P was 2-fold of that for Pi. Collectively, TsSP4 preferred synthetic rather than phosphorolytic direction. TsSP4 could elongate MOSs even initially with Pi alone in the absence of Glc-1-P, which further supported its synthetic role in the starch biosynthesis. TsSP4 displayed increased activities in the developing and mature stage of starch biosynthesis under nitrogen-starvation conditions, indicating its possible contribution to the amylopectin amplification. [ABSTRACT FROM AUTHOR]

The effects of 4.5 kJ m −2 UV-C radiation on the quality of minimally processed lily bulb, such as browning, growth of microorganism, total phenolic content, starch, total soluble sugar, reducing sugar content, the activities of phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), peroxidase (POD), α-amylase, β-amylase, starch phosphorylase and the antioxidant activity were studied. Results showed that UV-C treatment inhibited browning index and microbial growth by approximately 37% and 32% respectively. Compared with the control, PPO and POD activities of UV-C treated minimally processed lily bulb were reduced by about 26% and 18% respectively, while total phenolic content was increased by 13%. UV-C treatment down-regulated the activities of α-amylase, β-amylase and starch phosphorylase, which resulted in higher content of starch, total soluble sugar content and lower content of reducing sugar. The antioxidant activity and PAL activity were higher than the control during storage. These results indicated UV-C treatment could effectively maintain quality of minimally processed lily bulb stored at 4 ± 1 °C. Therefore, UV-C radiation might be an effective and safe alternative technology for minimally processed lily bulb. [ABSTRACT FROM AUTHOR]

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.

Russet Burbank and Innovator are mid- to late-season frozen-processing cultivars with inherently different dormancy periods and susceptibilities to low temperature-induced sweetening (LTS). In contrast to Russet Burbank, which is highly prone to accumulation of reducing sugars (Glc + Fru) when stored below 8–9 °C, Innovator tubers exhibit moderate resistance to LTS and retain process quality longer at lower temperatures (4–6 °C). However, Innovator’s LTS resistance is not robust and often varies across production regions. Here we show that low O 2 storage modulates LTS to reveal metabolic differences intrinsic to these cultivars. Changes in tuber respiration, process quality, reducing sugars, sucrose, starch phosphorylase and invertase activities were compared at 4 and 8 °C in 2.5 and 21 kPa O 2 over a 212-d storage period. Tuber respiration declined rapidly as O 2 level decreased from 21 to 2.5 kPa during acclimation at 8 °C. Respiration rates then fell further as the temperature was lowered from 8 to 4 °C, but this response was greatly muted for tubers at 2.5 versus 21 kPa O 2 . Tubers at 21 kPa O 2 completed their cold-induced respiratory acclimation response (RAR) within 7 d compared with 13 d at 2.5 kPa O 2 , and the RAR was much greater for tubers at 2.5 kPa O 2 . While reducing sugars increased most rapidly in tubers over the first 30 d at 4 °C, Innovator tubers had lower invertase activity and sweetened less than Russet Burbank tubers, characterizing its LTS-resistant phenotype. Low O 2 greatly attenuated these initial LTS responses for both cultivars; however, the effect was only temporary in Innovator. LTS resumed in Innovator tubers from 93 to 212 d with reducing sugar levels increasing to equal that of Russet Burbank tubers stored at 4 °C and 21 kPa O 2 . Activities of α-1,4 glucan phosphorylase (SP) were higher in Russet Burbank than Innovator and increased progressively over the storage period regardless of temperature and oxygen concentration. Innovator tubers stored at 2.5 kPa O 2 had higher SP activities from 30 to 153 d at 4 and 8 °C compared with tubers stored at 21 kPa O 2 , which correlated well with increased sucrose buildup and earlier sprouting. The low O 2 -mediated inhibition of LTS was largely a consequence of reduced invertase activities. [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]

Starch synthesis in cereal grain endosperm is dependent on the concerted actions of many enzymes. The starch plastidial phosphorylase (Pho1) plays an important role in the initiation of starch synthesis and in the maturation of starch granule in developing rice seeds. Prior evidence has suggested that the rice enzyme, OsPho1, may have a physical/functional interaction with other starch biosynthetic enzymes. Pulldown experiments showed that OsPho1 as well as OsPho1 devoid of its L80 region, a peptide unique to higher plant phosphorylases, captures disproportionating enzyme (OsDpe1). Interaction of the latter enzyme form with OsDpe1 indicates that the putative regulatory L80 is not responsible for multienzyme assembly. This heterotypic enzyme complex, determined at a molar ratio of 1:1, was validated by reciprocal co-immunoprecipitation studies of native seed proteins and by co-elution chromatographic and comigration electrophoretic patterns of these enzymes in rice seed extracts. The OsPho1-OsDpe1 complex utilized a broader range of substrates for enhanced synthesis of larger maltooligosaccharides than each individual enzyme and significantly elevated the substrate affinities of OsPho1 at 30 °C. Moreover, the assembly with OsDpe1 enables OsPho1 to utilize products of transglycosylation reactions involving G1 and G3, sugars that it cannot catalyze directly. [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]

Protein–protein interactions between starch phosphorylase (SP) and other starch biosynthetic enzymes were investigated using isolated maize endosperm amyloplasts and a recombinant maize enzyme. Plastidial SP is a stromal enzyme existing as a multimeric protein in amyloplasts. Biochemical analysis of the recombinant maize SP indicated that the tetrameric form was catalytically active in both glucan-synthetic and phosphorolytic directions. Protein–protein interaction experiments employing the recombinant SP as an affinity ligand with amyloplast extracts showed that the multimeric state of SP determined interactions with other enzymes of the starch biosynthetic pathway. The monomeric form of SP interacts with starch branching enzyme I (SBEI) and SBEIIb, whereas only SBEI interacts with the tetrameric form of SP. In all cases, protein–protein interactions were broken when amyloplast lysates were dephosphorylated in vitro , and enhanced following pre-treatment with ATP, suggesting a mechanism of protein complex formation regulated by protein phosphorylation. In vitro protein phosphorylation experiments with [ γ - 32 P]-ATP show that SP is phosphorylated by a plastidial protein kinase. Evidence is presented which suggests SBEIIb modulates the catalytic activity of SP through the formation of a heteromeric protein complex. [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]