Changes in Starch Formation and Activities of Sucrose Phosphate Synthase and Cytoplasmic Fructose-1,6-bisphosphatase in Response to Source-Sink Alterations

Short term experiments were conducted with vegetative soybean plants (Glycine max L. Merr. `Ransom' or `Arksoy') to determine whether sourcesink manipulations, which rapidly changed the `demand' for sucrose and partitioning of photosynthetically fixed carbon into starch, were associated with alterations in activities of sucrose-P synthase and/or cytoplasmic fructose-1,6-bisphosphatase in leaf extracts. When demand for sucrose from a particular source leaf was increased by defoliation of other source leaves, starch accumulation was restricted and activities of both enzymes were markedly enhanced. When demand for sucrose from source leaves was limited by excision, starch accumulation in the detached leaves was increased while activity of sucrose-P synthase declined sharply. The consistent responsiveness of sucrose-P synthase activity to changes in demand for sucrose supports the contention that regulation of sucrose-P synthase is an integral component of the system which controls sucrose biosynthesis and partitioning of carbon between starch and sucrose biosynthesis in the light.     

Carbohydrate partitioning and metabolism during acclimatization of micropropagated Calathea

Regulation of carbohydrate metabolism and compartmentation were studied during the acclimatization of tissue cultured Calathea plantlets. At transplantation plants were characterised by a heterotrophic metabolism with roots and stems as the main storage organs for carbohydrates. As acclimatization proceeded, a switch to autotrophic growth was observed: leaves became source organs, which was among others reflected by significant increases of invertase, sucrose synthase and sucrose-P synthase activities. Mobilization of reserves in roots and stems was also observed during the same period. Sucrose and starch accumulation in leaves was positively correlated with increasing light intensity.     

Analysis of the Relationships between Growth, Photosynthesis and Carbohydrate Metabolism Using Quantitative Trait Loci (QTLs) in Young Maize Plants Subjected to Water Deprivation

One hundred to 120 maize recombinant inbred lines at the mature fourth leaf stage derived from F-2 and Io parental lines were grown in a glasshouse and were deprived of water for 9 days in order to detect pertinent markers of the physiological response to water stress which may be used for breeding. Carbohydrate metabolism QTLs were compared to photosynthesis gas exchange QTLs. The locations of these QTLs were further compared with those of morphological trait QTLs when water availability varied. The traits ranged from three enzyme activities (invertase, sucrose-P synthase, ADP glucose pyrophosphorylase) and hexose, sucrose, starch content to CO₂ uptake and stomatal conductance, water status, leaf size, root/shoot ratio, and ABA (leaf, root and xylem sap). Four main results were obtained (1) only 14 % of QTLs were common to both drought and watered treatments, confirming the existence of stress specific chromosome regions, (2) the QTLs tended to form clusters, frequently consisting of QTLs from different classes (growth, photosynthesis, water status, carbohydrate metabolism and ABA), (3) carbohydrate metabolism trait QTLs were more frequently co-located with growth trait QTLs than photosynthesis related ones, especially in control conditions, (4) one co-location was observed between the three enzyme activities implied in sucrose and starch metabolism and a corresponding structural gene, which can be considered as a candidate gene for explaining part of the variability of each enzymatic trait (invertase, sucrose-P synthase, ADPglucose pyrophosphorylase). It is concluded that, carbohydrate metabolism provides valuable traits for understanding and improving maize responses to water stress.     

Sucrose metabolism in lima bean seeds

Developing and germinating lima bean (Phaseolus lunatus var Cangreen) seeds were used for testing the sucrose synthase pathway, to examine the competition for uridine diphosphate (UDP) and pyrophosphate (PPi), and to identify adaptive and maintenance-type enzymes in glycolysis and gluconeogenesis. In developing seeds, sucrose breakdown was dominated by the sucrose synthase pathway; but in the seedling embryos, both the sucrose synthase pathway and acid invertase were active. UDPase activity was low and seemingly insufficient to compete for UDP during sucrose metabolism in seed development or germination. In contrast, both an acid and alkaline pyrophosphatase were active in seed development and germination. The set of adaptive enzymes identified in developing seeds were sucrose synthase, PPi-dependent phosphofructokinase, plus acid and alkaline pyrophosphatase; and, the adaptive enzymes identified in germinating seeds included the same set of enzymes plus acid invertase. The set of maintenance enzymes identified during development, in the dry seed, and during germination were UDP-glucopyrophosphorylase, neutral invertase, ATP and UTP-dependent fructokinase, glucokinase, phosphoglucomutase, ATP and UTP-dependent phosphofructokinase and sucrose-P synthase.     

Evidence for control of carbon partitioning by fructose 2,6-bisphosphate in spinach leaves

Excision of spinach (Spinacia oleracea L.) leaves had no effect on photosynthetic rates, but altered normal carbon partitioning to favor increased formation of starch and decreased formation of sucrose. The changes were evident within 2 hours after excision. Concurrently, leaf fructose-2,6-bisphosphate content increased about 5-fold (from 0.1 to 0.5 nanomoles per gram fresh weight). The activities of sucrose-P synthase and cytoplasmic fructose 1,6-bisphosphatase in leaf extracts remained constant during the time period tested. It is postulated that the rise in fructose 2,6-bisphosphate was responsible for the change in carbon partitioning.     

Sucrose Phosphate Synthetase from Various Plant Origins

Some properties of sucrose-P synthetases obtained from various plant tissues, including sweet potato roots, potato tubers and leaves of barley, rape and ladino clover were studied. The specific enzyme activity of the sucrose-P synthetase from sweet potato roots was much lower than that of the sucrose synthetase of the other tissues. The enzyme activity decreased gradually as the roots developed. The optimum pH did not differ between enzyme preparations from sweet potato roots and barley leaves. Manganese chloride exhibited a marked stimulative effect on the sucrose-P synthetase from sweet potato roots and potato tubers, whereas it was inhibited the barley leaf enzyme.

Kinetic studies of sucrose-P synthetase showed that the behavior of the enzyme to the substrates did not differ in the enzyme sources examined. The substrate saturation curve of the enzyme with respect to fructose-6-P was sigmodal in shape, giving a straight line with a slope of 1.35~1.5 (n value) in a plot of the data using the empirical Hill equation. On the other hand, enzymes from all the various tissues exhibited a hyperbolic substrate saturation curve for UDP-glucose, obeying the ordinary Michaelis-Menten type reaction. Manganese chloride had no effect on the Km for UDP-glucose, the S_0.5 for fructose-6-P and the n value of the enzyme from potato tuber tissues.     

Endonuclease-free, protoplast-forming enzyme preparation and its application in fungal transformation.

An endonuclease-free, protoplast-forming enzyme complex was prepared from the "snail enzyme." The purified preparation has high protoplast-forming activity comparable to the crude enzyme complex without destroying circular plasmid DNA. Furthermore, a higher transformation rate was achieved by the application of the endonuclease-free enzyme complex in both yeast and filamentous fungal vector-host systems.     

A chitinase indispensable for formation of protoplast of Schizophyllum commune in basidiomycete-lytic enzyme preparation produced by Bacillus circulans KA-304

KA-prep, a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune, has an activity to form protoplasts from S. commune mycelia. alpha-1,3-Glucanase, which was isolated from an ammonium sulfate fraction of 0-30% saturation of KA-prep, gave the protoplast-forming activity to an ammonium sulfate fraction of 30-50% saturation of KA-prep, which contained chitinase(s) and beta-glucanase(s) but was inactive in the protoplast formation. Chitinase(s) and beta-glucanase(s) in the ammonium sulfate fraction of 30-50% saturation were separated by DEAE-cellulofine A-500 column chromatography, and the protoplast-forming activity appeared when the chitinase preparation was mixed with the alpha-1,3-glucanase. The beta-glucanase preparation was not effective for the protoplast formation whereas its addition enhanced the protoplast-forming activity of the mixture of alpha-1,3-glucanase and the chitinase preparation. The chitinase preparation contained two chitinases (chitinase I and II). Chitinase I showed the protoplast-forming activity with alpha-1,3-glucanase, but chitinase II did not. Chitinase I, a monomeric protein with a molecular weight of 41,000, was active toward colloidal chitin and ethylene glycol chitin. Chitinase I produced predominantly N,N'-diacetylchitobiose and N,N',N"-triacetylchitotriose from colloidal chitin, and the enzyme was inactive to p-NP-beta-D-N-acetylglucosaminide, suggesting that it was an endo-type enzyme. The N-terminal amino acid sequence of chitinase I (A L A T P T L N V S A S S G M) had no sequential identity to those of known chitinases.     

Carbon and nitrogen metabolism in the seagrass, Zostera marina L.: Environmental control of enzymes involved in carbon allocation and nitrogen assimilation

This study experimentally examined influences of environmental variables on the activities of key enzymes involved in carbon and nitrogen metabolism of the submersed marine angiosperm, Zostera marina L. Nitrate reductase activity in leaf tissue was correlated with both water-column nitrate concentrations and leaf sucrose levels. Under elevated nitrate, shoot nitrate reductase activity increased in both light and dark periods if carbohydrate reserves were available. When water-column nitrate was low, glutamine synthetase activity in leaf tissue increased with environmental ammonium. In contrast, glutamine synthetase activity in belowground tissues was statistically related to both nitrate and temperature. At the optimal growth temperature for this species (ca. 25 °C), increased water-column nitrate promoted an increase in glutamine synthetase activity of belowground tissues. As temperatures diverged from the optimum, this nitrate effect on glutamine synthetase was no longer evident. Activities of both sucrose synthase and sucrose-P synthase were directly correlated with temperature. Sucrose-P synthase activity also was correlated with salinity, and sucrose synthase activity was statistically related to tissue ammonium. Overall, the enzymatic responses that were observed indicate a tight coupling between carbon and nitrogen metabolism that is strongly influenced by prevailing environmental conditions, especially temperature, salinity, and environmental nutrient levels.     

Effects of Water Stress on Photosynthesis and Carbon Partitioning in Soybean (Glycine max [L.] Merr.) Plants Grown in the Field at Different CO(2) Levels

The effects of water stress and CO₂ enrichment on photosynthesis, assimilate export, and sucrose-P synthase activity were examined in field grown soybean plants. In general, leaves of plants grown in CO₂-enriched atmospheres (300 microliters per liter above unenriched control, which was 349 ± 12 microliters per liter between 0500 and 1900 hours EST over the entire season) had higher carbon exchange rates (CER) compared to plants grown at ambient CO₂, but similar rates of export and similar activities of sucrose-P synthase. On most sample dates, essentially all of the extra carbon fixed as a result of CO₂ enrichment was partitioned into starch. CO₂-enriched plants had lower transpiration rates and therefore had a higher water use efficiency (milligrams CO₂ fixed per gram H₂O transpired) per unit leaf area compared to nonenriched plants. Water stress reduced CER in nonenriched plants to a greater extent than in CO₂-enriched plants. As CER declined, stomatal resistance increased, but this was not the primary cause of the decrease in assimilation because internal CO₂ concentration remained relatively constant. Export of assimilates was less affected by water stress than was CER. When CERs were low as a result of the imposed stress, export was supported by mobilization of reserves (mainly starch). Export rate and leaf sucrose concentration were related in a curvilinear manner. When sucrose concentration was above about 12 milligrams per square decimeter, obtained with nonstressed plants at high CO₂, there was no significant increase in export rate. Assimilate export rate was also correlated positively with SPS activity and the quantitative relationship varied with CER. Thus, export rate was a function of both CER and carbon partitioning.     

Acclimation of photosynthesis to low temperature in Spinacia oleracea L. I. Effects of acclimation on CO2 assimilation and carbon partitioning

Acclimation of spinach plants grown at 25C to a temperatureof 10C for 10 d resulted in an increased capacity for leafphotosynthesis in saturating light and CO₂ but not at ambientCO₂ concentrations. Gas exchange and chlorophyll fluorescencemeasurements indicated that acclimation was accompanied by anincreased capacity for the regeneration of ribulose-1,5-bisphosphate.Changes in starch, soluble carbohydrates and activities of sucrose-Psynthase and ADP-glucose pyrophosphorylase were measured duringthe acclimation process. There was an initial increase in starchand sucrose during the first 2 d, but these then declined. Therewas an increase in the capacity for sucrose synthesis duringlow temperature acclimation, evidenced by an increase in themaximum activity of sucrose-P synthase activity and an increasein partitioning of ¹⁴CO₂ into sucrose, but there was no increasein the activity of ADP-glucose pyrophosphorylase or carbon partitioninginto starch. Key words: Acclimation, carbon metabolism, gas exchange, low temperature, spinach, Spinacia oleracea     

Transformation of Intact Aspergillus niger by Electroporation

A new rapid transformation system for Aspergillus niger that uses electroporation to render intact germinating conidia permeable to DNA is described. The transformant colonies appeared earlier than transformants obtained by the protoplast-forming method. Without pretreatment of the conidia the transformation frequencies were 1.2 colonies per μg of integrative vector and 100 colonies per μg of plasmid DNA. When the conidia were treated with a dilute solution of fungal cell wall lytic enzyme, the frequency of transformation was increased by approx. 2-fold when using two vectors. Southern blot analysis of genomic DNA and restriction endonuclease-digested DNA from a random sample of transformants showed homologous and nonhomologous integration of the integrative vector into the genome, as is also observed with the protoplast-forming method. In transformation with the plasmid vector, the transformant DNA was shown to be mostly maintained in free form with minimal integration into the chromosome when transformed by either intact electroporation or the conventional method.     

Cloning and expression of an alpha-1,3-glucanase gene from Bacillus circulans KA-304: the enzyme participates in protoplast formation of Schizophyllum commune

A culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune has an activity to form protoplasts from S. commune mycelia, and a combination of alpha-1,3-glucanase and chitinase I, which were isolated from the filtrate, brings about the protoplast-forming activity. The gene of alpha-1,3-glucanase was cloned from B. circulans KA-304. It consists of 3,879 nucleotides, which encodes 1,293 amino acids including a putative signal peptide (31 amino acid residues), and the molecular weight of alpha-1,3-glucanase without the putative signal peptide was calculated to be 132,184. The deduced amino acid sequence of alpha-1,3-glucanase of B. circulans KA-304 showed approximately 80% similarity to that of mutanase (alpha-1,3-glucanase) of Bacillus sp. RM1, but no significant similarity to those of fungal mutanases.The recombinant alpha-1,3-glucanase was expressed in Escherichia coli Rosetta-gami B (DE 3), and significant alpha-1,3-glucanase activity was detected in the cell-free extract of the organism treated with isopropyl-beta-D-thiogalactopyranoside. The recombinant alpha-1,3-glucanase showed protoplast-forming activity when the enzyme was combined with chitinase I.     

Cloning and expression of a Bacillus circulans KA-304 gene encoding chitinase I, which participates in protoplast formation of Schizophyllum commune

KA-prep, a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune, has an activity to form protoplasts from S. commune mycelia, and a combination of alpha-1,3-glucanase and chitinase I, isolated from KA-prep, brings about the protoplast-forming activity. The gene of chitinase I was cloned from B. circulans KA-304 into pGEM-T Easy vector. The gene consists of 1,239 nucleotides, which encodes 413 amino acids including a putative signal peptide (24 amino acid residues). The molecular weight of 40,510, calculated depending on the open reading frame without the putative signal peptide, coincided with the apparent molecular weight of 41,000 of purified chitinase I estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The C-terminal domain of the deduced amino acid sequence showed high similarity to that of family 19 chitinases of actinomycetes and other organisms, indicating that chitinase I is the first example of family 19 chitinase in Bacillus species. Recombinant chitinase I without the putative signal peptide was expressed in Escherichia coli Rosetta-gami B (DE 3). The properties of the purified recombinant enzyme were almost the same as those of chitinase I purified from KA-prep, and showed the protoplast-forming activity when it was combined with alpha-1,3-glucanase from KA-prep. Recombinant chitinase I as well as the native enzyme inhibited hyphal extension of Trichoderma reesei.     

Purification and characterization of chitinase A of Streptomyces cyaneus SP-27: an enzyme participates in protoplast formation from Schizophyllum commune mycelia

A culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune has an activity to form protoplasts from S. commune mycelia. alpha-1,3-Glucanase and chitinase I, which were isolated from the filtrate, did not form the protoplast by itself while a mixture of them showed protoplast-forming activity. Streptomyces cyaneus SP-27 was isolated based on the productivity of chitinase. The culture filtrate of S. cyaneus SP-27 did not form S. commune protoplasts, but addition of it to alpha-1,3-glucanase of B. circulans KA-304 brought about protoplast-forming activity. Chitinase A isolated from the S. cyaneus SP-27 culture filtrate was more effective than chitinase I of B. circulans KA-304 for the protoplast formation in combination with alpha-1,3-glucanase. The N-terminal amino acid sequence of chitinase A (MW 29,000) has a sequential similarity to those of several Streptomycete family 19 chitinases. Chitinase A adsorbed to chitinous substrate and inhibited the growth of Trichoderma reesei mycelia. Anomer analysis of the reaction products also suggested that the enzyme is a family 19 chitinase.     

扬子鳄中脑视叶NOS、AChE阳性神经元的分布

目的观察扬子鳄中脑视叶一氧化氮合酶(nitric oxide synthase,NOS)和乙酰胆碱酯酶(acetylcholinesterase,AChE)阳性神经元的形态和分布,为扬子鳄脑的比较解剖学积累资料,为其机能研究提供形态学依据。方法采用还原型尼克酰胺腺嘌呤二核苷酸黄递酶(NADPH-d)法和亚铁氰化酮法观察扬子鳄中脑视叶NOS和AChE阳性神经元的分布和特征,并作统计学处理。结果扬子鳄中脑视叶有NOS和AChE阳性神经元分布,为大、中、小型细胞,以中、小型细胞为主,胞体呈椭圆形、三角形、圆形和梭形。结论扬子鳄中脑视叶有NOS和AChE阳性神经元分布。     

Rapid genetic characterization of poly(hydroxyalkanoate) synthase and its applications

Microorganisms containing short-chain-length (scl-) or medium-chain-length (mcl-) poly(hydroxyalkanoates) (PHAs) are commonly screened by applying rapid staining methods using lipophilic reagents. These methods provide powerful means for general screening of organisms actively producing and accumulating PHAs. The Southern blot hybridization method additionally allows the identification of potential PHA-producing microorganisms. Polymerase chain reaction (PCR)-based detection methods further afford rapid and sensitive means to screen for PHA biosynthesis genes. Specific PCR assays had been developed for the simultaneous or individual detection of the class II mcl-PHA synthase genes of Pseudomonas. The amplicons (approximately 0.54 kb) can be directly sequenced or used as probes for hybridization studies. The sequence information can further be used to initiate chromosome walking for an eventual cloning of the complete PHA biosynthesis operon. In addition, the amplification pattern and sequence data can be used to differentiate subgroups of organisms, as demonstrated for P. corrugata and P. mediterranea. Other researchers reported PCR methods for the detection of scl-PHA synthase genes and those of Bacillus spp., thus greatly expanding the types of PHA synthase gene and the organisms that can be characterized by this approach. The vast sequence information obtainable through PCR-based studies of various PHA synthase operons should facilitate the identification or construction of new PHA synthases capable of synthesizing novel PHAs.     

Digalactosyldiacylglycerol is required for better photosynthetic growth of Synechocystis sp. PCC6803 under phosphate limitation

Digalactosyldiacylglycerol (DGDG) is a typical membrane lipid of oxygenic photosynthetic organisms. Although DGDG synthase genes have been isolated from plants, no homologous gene has been annotated in the genomes of cyanobacteria and the unicellular red alga Cyanidioschyzon merolae. Here we used a comparative genomics approach and identified a non-plant-type DGDG synthase gene (designated dgdA) in Synechocystis sp. PCC6803. The enzyme produced DGDG in Escherichia coli when co-expressed with a cucumber monogalactosyldiacylglycerol synthase. A DeltadgdA knock-out mutant showed no obvious phenotype other than loss of DGDG when grown in a BG11 medium, indicating that DGDG is dispensable under optimal conditions. However, the mutant showed reduced growth under phosphate-limited conditions, suggesting that DGDG may be required under phosphate-limited conditions, such as those in natural niches of cyanobacteria.     

The seven deadly sins of comparative analysis

Phylogenetic comparative methods are extremely commonly used in evolutionary biology. In this paper, I highlight some of the problems that are frequently encountered in comparative analyses and review how they can be fixed. In broad terms, the problems boil down to a lack of appreciation of the underlying assumptions of comparative methods, as well as problems with implementing methods in a manner akin to more familiar statistical approaches. I highlight that the advent of more flexible computing environments should improve matters and allow researchers greater scope to explore methods and data.