Plant Carbohydrate Metabolism Research Articles

Effect of a drought period on the mobilisation of non-structural carbohydrates, photosynthetic efficiency and water status in an epiphytic orchid

Environmental stress induces modifications and adaptations, a marked characteristic of epiphytic orchids. The presence of the pseudobulb is associated with carbohydrate reserve functions and water maintenance in the plant. We evaluated the effects of drought on carbohydrate metabolism in plants subjected to drought for 0, 25 and 45 d by measuring the following parameters: F v/F m ratio, ethanol and water-soluble polysaccharides, acid invertase (β-D-fructofuranosidase; EC 3.2.1.25) activity, relative water content, concentration of osmotically active solutes and dry mass/fresh mass ratio. The carbohydrates present in leaf and pseudobulb were characterised by acid hydrolysis followed by paper chromatography and high performance anion exchange chromatography. Our results showed that a period of drought decreased the efficiency of the photochemical system (decrease in the ratio F v/F m) at the same time as the ratio of sucrose/monosaccharides was constant in the leaf, but increased in the pseudobulb. As a consequence of the changes in sucrose/monosaccharides ratio in the pseudobulb, plants under drought stress have decreased invertase activity and increased concentration of osmotically active solutes. The pseudobulb stores a glucomannan, thought to be a multifunctional carbohydrate involved in water stress and carbon reserve. Our results suggest that, under water stress, glucomannan in the pseudobulb is mobilised to moderate effects of drought.

The metabolic profile of the 14-3-3 repressed transgenic potato tubers

Recently potato three transgenes were created, all with 14-3-3 repression. Two different isoforms were repressed, those previously called 29G (J5 line) and 20R (J4 line), and additionally transgenic plants with repression of both isoforms (G1 line) were created. The plants were analysed for enzyme activities and metabolite levels. Detailed analysis of those plants suggested that a function of the isolated 14-3-3 isoform is in the control of plant carbohydrate metabolism and nitrogen fixation. Sucrose phosphate synthase and nitrate reductase were the most highly affected of the enzymes measured in transgenic tubers and leaves, respectively. In in vitro experiment, these enzymes were also significantly affected by 14-3-3 proteins. Thus, the results obtained are consistent with those from transgenic plants analysis and the in vitro data can be directly transferred to in vivo situation. Of many measured metabolites the significant increase in sucrose, starch, aspartic and glutamic acids, glutamine, malic and citric acids was detected which suggest the potential role of 14-3-3 in TCA cycle regulation.

Contrasting responses of photosynthesis and carbon metabolism to low temperatures in tall fescue and clovers.

Growth, photosynthesis and carbohydrate metabolism in plants of two grassland species, clover (Trifolium subterraneum L. cv. Areces and Gaitan) and tall fescue (Festuca arundinacea Schreb.), shifted from 25 to 12 degrees C for 1 day or developed at 12 degrees C were compared with controls kept at 25 degrees C. Cold development produced a larger inhibition of growth in fescue than in clovers. In contrast, transferring plants from high to low temperature inhibited photosynthesis to a lesser extent in fescue than in clovers, this difference being associated with an increase in the activation state of Calvin cycle enzymes in fescue, but not in the clovers, a decreased cytosolic fructose-1,6-bisphosphatase (cFBPase, EC 3.1.3.11) activity in clovers, and an accumulation of hexose phosphates only in fescue. Development at 12 degrees C partly relieved the inhibition of photosynthesis in clovers, in contrast with fescue, which correlated with increases in total ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco, EC 4.1.1.39) activity only in clovers, and with greater increases in total stromal FBPase (sFBPase) activity in clovers than in fescue. The activity of sucrose synthesis enzymes was increased in the two clovers and fescue developed in the cold, while carbohydrate accumulation was much bigger in cold-developed fescue than in clovers because of a 5-fold increase in fructan contents in the former. The contents of phosphorylated intermediates increased in clovers but decreased in fescue grown at 12 degrees C. Our results suggest that restricted ribulose-1,5-bisphosphate (RuBP) regeneration limited the recovery of photosynthetic capacity in cold-developed fescue.

Increase in lipid content in potato tubers modified by 14-3-3 gene overexpression.

Recently, transgenic potato plants were created with overexpression of the 14-3-3 protein derived from Cucurbita pepo. Detailed analysis of those plants suggested that the function of the isolated 14-3-3 isoform is in the control of carbohydrate and lipid metabolism in the plants. 14-3-3 protein overexpression gave rise to an increase in soluble sugar and catecholamine contents in both leaves and tubers. It is proposed that 14-3-3 protein affects carbohydrate metabolism in potato plants via regulation of catecholamine synthesis. Furthermore, genetically modified potato tubers with 14-3-3 protein overexpression showed changes in lipid content and composition. The transgenic potato tubers contained 69% more total fat compared to the wild-type plant. Separation of tuber lipids into polar and nonpolar fractions revealed that the transgenic potato tubers contained almost 3 times more nonpolar lipids than the control. Analysis of fatty acid composition, conducted by the means of gas chromatography, showed that linoleic acid was the main fatty acid present in the tubers of both modified and control potato plants. In the nonpolar fraction of the fat of the transgenic tubers the unsaturated fatty acids exhibited a higher participation in the sum of all fatty acids.

Fructose 2,6-bisphosphate activates pyrophosphate: fructose-6-phosphate 1-phosphotransferase and increases triose phosphate to hexose phosphate cycling in heterotrophic cells.

The aim of this work was to establish the influence of fructose 2,6-bisphosphate (Fru-2,6-P2) on non-photosynthetic carbohydrate metabolism in plants. Heterotrophic callus lines exhibiting elevated levels of Fru-2,6-P2 were generated from transgenic tobacco (Nicotiana tabacum L.) plants expressing a modified rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Lines containing increased amounts of Fru-2,6-P2 had lower levels of hexose phosphates and higher levels of 3-phosphoglycerate than the untransformed control cultures. There was also a greater redistribution of label into the C6 position of sucrose and fructose, following incubation with [1-13C]glucose, in the lines possessing the highest amounts of Fru-2,6-P2, indicating a greater re-synthesis of hexose phosphates from triose phosphates in these lines. Despite these changes, there were no marked differences between lines in the metabolism of 14C-substrates, the rate of oxygen uptake, carbohydrate accumulation or nucleotide pool sizes. These data provide direct evidence that physiologically relevant changes in the level of Fru-2,6-P2 can affect pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) activity in vivo, and are consistent with PFP operating in a net glycolytic direction in the heterotrophic culture. However, the results also show that activating PFP has little direct effect on heterotrophic carbohydrate metabolism beyond increasing the rate of cycling between hexose phosphates and triose phosphates.

A prokaryotic sucrose synthase gene (susA) isolated from a filamentous nitrogen-fixing cyanobacterium encodes a protein similar to those of plants.

Sucrose synthase (SS), a key enzyme in plant carbohydrate metabolism, has recently been isolated from Anabaena sp. strain PCC 7119, and biochemically characterized; two forms (SS-I and SS-II) were detected (Porchia et al. 1999, Planta 210: 34-40). The present study describes the first isolation and characterization of a prokaryotic SS gene, susA, encoding SS-II from that strain of Anabaena. A 7 kbp DNA fragment containing an open reading frame (EMBL accession number AJ010639) with about 30-40% amino acid identity with plant SSs was isolated from an Anabaena subgenomic library. The putative SS gene was demonstrated to encode an SS protein by expression in Escherichia coli. The biochemical properties of the recombinant enzyme were identical to those of the enzyme purified from the cyanobacterial cells. The deduced amino acid sequence of the Anabaena SS diverged from every plant SS reported. The occurrence of SS in cyanobacteria of different taxonomic groups was investigated. The enzyme occurs in several filamentous nitrogen-fixing cyanobacteria but not in two species of unicellular, non-diazotrophic cyanobacteria.

In vitro grown potato microtubers are a suitable system for the study of primary carbohydrate metabolism

The aim of this work was to determine the suitability of tissue culture microtubers for the study of primary carbohydrate metabolism in potato plants ( Solanum tuberosum L. cv. Desirée). We have determined the levels of key enzymes and intermediates in the pathways of sucrose metabolism, starch metabolism and glycolysis in wild type microtubers. Comparison of the metabolite levels in microtubers with a range of published studies on soil-grown developing tubers showed that the two systems were similar both in the absolute levels and in the ratios between metabolites, despite some differences in the maximum catalytic activities of some glycolytic enzymes. We conclude that in vitro grown microtubers are an adequate model system for studying primary carbohydrate metabolism in developing potato tubers.

Carbohydrate metabolism in plants

Tom ap Rees

Sensing trehalose biosynthesis in plants.

A most unexpected finding in research on plant carbohydrate metabolism is the recent discovery that angiosperms encode genes whose products are involved in trehalose metabolism. The presence and functionality of such genes has been elegantly shown by expressing Arabidopsis-derived trehalose phosphate synthase and trehalose phosphate phosphatase genes in yeast mutants lacking these enzymatic activities. Homologue sequences have now been cloned from a number of different plant species suggesting that the capacity to synthesise trehalose is ubiquitous in angiosperms. Except for Myrothamnus flabellifolius, trehalose biosynthesis has never been observed in tissues of higher plants, probably due to the presence of high levels of trehalase activity. The function of trehalose metabolism in plants is still a mystery. One of the postulated functions of trehalose metabolism in yeast is in the control of glucose repression and a similar function in sugar sensing can be proposed for plants as well.

Antisense expression of a sucrose non‐fermenting‐1‐related protein kinase sequence in potato results in decreased expression of sucrose synthase in tubers and loss of sucrose‐inducibility of sucrose synthase transcripts in leaves

SummaryThis report describes the analysis of transgenic potato plants stably transformed with chimeric genes comprising either a patatin (tuber‐specific) or a ST‐LS1 (leaf‐ and stem‐specific) gene promoter and a potato sucrose non‐fermenting‐1 (SNF1)‐related protein kinase gene (PKIN1) sequence in the antisense orientation. Presence of the transgene was confirmed by Southern analysis. The transformants were screened using a peptide kinase activity assay for SNF1‐related activity and/or Northern blot analysis, and two independent transgenic lines from each transformation, PAT 1.3, PAT 1.10, LS 1.4 and LS 1.11, were selected. Antisense PKIN1 transcripts were detectable in all four of the selected lines, and measurements made using the specific peptide phosphorylation assay showed that SNF1‐related protein kinase activity had decreased in both PAT 1.3 and PAT 1.10 compared with controls. SNF1 regulates the expression of many genes encoding enzymes of carbohydrate metabolism in yeast. In order to investigate an analogous role for PKIN1, the activities of fructokinase, glucokinase, neutral and acid invertase and sucrose synthase in the tubers of PAT 1.3 and PAT 1.10 were compared with those in wild‐type controls. Sucrose synthase activity was decreased to 36% of wild‐type activity in tubers of PAT 1.10, and sucrose synthase transcript levels were decreased in tubers of both PAT 1.3 and PAT 1.10. Activities of the other enzymes were unaffected. Leaves of lines LS 1.4, LS 1.11 and controls were then excised and cultured on a medium containing 250 mM sucrose. This treatment induced sucrose synthase gene expression in the control leaves but not those of the transgenic lines. This finding is the first demonstration of a role for SNF1‐related protein kinases in the regulation of carbohydrate metabolism of higher plants.

Effect of High Temperature on Plant Growth and Carbohydrate Metabolism in Potato.

This study was undertaken to determine the role of sucrose-metabolizing enzymes in altered carbohydrate partitioning caused by heat stress. Potato (Solanum tuberosum L.) genotypes characterized as susceptible and tolerant to heat stress were grown at 19/17[deg]C, and a subset was transferred to 31/29[deg]C. Data were obtained for plant growth and photosynthesis. Enzyme activity was determined for sucrose-6-phosphate synthase (SPS) in mature leaves and for sucrose synthase, ADP-glucose pyrophosphorylase, and UDP-glucose pyrophosphorylase in developing tubers of plants. High temperatures reduced growth of tubers more than of shoots. Photosynthetic rates were unaffected or increased slightly at the higher temperature. Heat stress increased accumulation of foliar sucrose and decreased starch accumulation in mature leaves but did not affect glucose. SPS activity increased significantly in mature leaves of plants subjected to high temperature. Changes in SPS activity were probably not due to altered enzyme kinetics. The activity of sucrose synthase and ADP-glucose pyrophosphorylase was reduced in tubers, albeit less quickly than leaf SPS activity. There was no interaction of temperature and genotype with regard to the enzymes examined; therefore, observed differences do not account for differences between genotypes in heat susceptibility.

The Use of Transgenic Plants to Study the Regulation of Plant Carbohydrate Metabolism

Transgenic plants with decreased expression of specific enzymes provide a powerful new tool to investigate metabolic regulation. Their use is discussed in the context of theories of metabolic regulation. It is argued that an enzyme is a key site for regulation, in the strict sense, when (i) natural mechanisms exist to alter the activity of the enzyme in vivo ('regulatability'), and (ii) a change in the activity of the enzyme is able to lead to a change in flux through the pathway ('regulatory capacity'). Previous approaches to the study of regulation allow the identification of enzymes with high 'regulatability', but they do not provide consistent or valid criteria to assess their 'regulatory capacity'. They therefore do not distinguish between enzymes which actually control metabolic fluxes, and enzymes which just respond to changes initiated elsewhere in the pathway. They may also underestimate the contribution of enzymes that catalyse reversible reactions. In contrast, mutants and transgenic plants can be used to directly test the importance of different aspects of an enzyme's regulatory properties in vivo. Even more importantly, they provide a method to determine flux control coefficients which provide a quantitative measure of an enzyme's 'regulatory capacity'. Recent results are surnrnarised, and potential practical problems in measuring control coefficients are reviewed.

Expression of α‐amylases, carbohydrate metabolism, and autophagy in cultured rice cells is coordinately regulated by sugar nutrient

A rice suspension cell culture system has been established to study how sugar depletion regulates alpha-amylase expression, carbohydrate metabolism, and other physiological and cellular changes. It is shown here that a group of 44 kDa alpha-amylases are constitutively expressed whether or not the cells are starved of sucrose. However, expression of a new group of alpha-amylases of 46 kDa is dramatically induced when cells are starved of sucrose. Cellular sugar and starch were rapidly consumed and metabolic activity was decreased in the starved cells. Extensive autophagy also occurred in the starved cells, which caused an increase in vacuolar volume and degradation of cytoplasmic constituents including amyloplasts. Immunocytochemical studies revealed that alpha-amylases are localized in starch granules within amyloplasts, in cell walls, and in some of the vacuoles. The presence of putative signal sequences in the N-termini of nine rice alpha-amylases suggests hitherto unidentified pathways for import of alpha-amylases into amyloplasts. The studies show that differential alpha-amylase expression, carbohydrate metabolism, metabolic activity, and vacuolar autophagy are coordinately regulated by the sugar level in the medium. As the starved suspension cells exhibit some sugar-regulated characteristics of alpha-amylase expression in germinating rice embryos as well as physiological changes similar to those in senescing cells, this system represents an ideal tool for studying cellular, biochemical, and molecular biological aspects of alpha-amylase gene regulation, carbohydrate metabolism, senescence, and protein targeting in plants.

Manipulation of carbohydrate partitioning

The reverse genetics approach is allowing the definition of specific reaction steps in plant carbohydrate metabolism. This is enabling the design of studies to alter sucrose and starch partitioning in the leaf, to inhibit phloem transport, or to increase/inhibit the rate of sucrose import into storage organs and its conversion to starch. These studies provide information about what kind of change should be made to biosynthetic pathways to achieve a desired aim. They also highlight the flexibility of plant primary metabolism and reveal a multi-faceted interaction between carbohydrate metabolism and many other aspects of plant growth and development.

MOLECULAR CHARACTERIZATION AND TRANSIENT EXPRESSION OF MAIZE SUS1 PROMOTER

Sucrose synthase (SS) is one of the key enzymes in plant carbohydrate metabolism. In maize, this enzyme is encoded by two genes, Sh1 and Sus1. We have isolated and determined the 5'-upstream sequence of maize Sus1 gene and compared it with the corresponding sequence in Sh1 gene. Sequence analysis revealed that there was a weak homology between the two promoters and no common sequence elements were found. To understand the differential regulation of the expression of the two genes, we constructed chimeric GUS fusions using the two promoters of SS genes. By using the biolistic system, we delivered these constructs into various plant tissues, and their transient expression was studied. Our results showed that the two promoters of SS genes directed tissue-specific expression in the same way that the two genes are expressed in vivo. The effectiveness of the expression of the constructs was recorded by counting the total blue expression units (blue spots) per shot and by fluorometric assays. High levels of GUS activity were detected in the immature embryos, young coleoptiles, and heterotrophic young leaves bombarded with the Sus–GUS construct. More than 100 expression units were observed in these tissues. Compared with the transient expression of the 35S promoter in the same tissue, Sus promoter activity was twice as high. Strong Sus–GUS expression was also detected in the aleurone cells of developing kernels. In contrast, the Sh-GUS construct was expressed only in the endosperm with an activity twice as high as that of Sus–GUS and 35S–GUS in the same tissue. The results will be discussed in terms of the physiological roles of the two SS isozymes in plant tissues.

Cyclitol production in transgenic tobacco

High levels of cyclic sugar alcohols (cyclitols) correlate with tolerance to osmotic stress in a number of plant species. A gene encoding a cyclitol biosynthesis enzyme from a halophyte, Mesembryanthemum crystallinum has been introduced into tobacco. The gene, lmt1, encodes a myo‐inositol O‐methyl transferase that, in M. crystallinum, catalyzes the first step in the stress‐induced accumulation of the cyclitol pinitol. Tobacco transformed with the lmt1 cDNA under the control of the CaMV 35S promoter appeared phenotypically normal and exhibited IMT1 enzyme activity. Transformants accumulated a carbohydrate product not detectable in non‐transformed control plants. This product was identified by HPLC and NMR as ononitol (1‐d‐4‐O‐methyl myo‐inositol). Ononitol was a major carbohydrate constituent in leaf tissue of plants expressing the lmt1 gene, accumulating to up to 25% the level of sucrose in transformant seedlings. The identification of ononitol as the IMT1 product and the specific accumulation of this compound in transformed tobacco support a role for ononitol as a stable intermediate in pinitol biosynthesis and indicate that an epimerization activity lacking in tobacco is responsible for the conversion of ononitol to pinitol in M. crystallinum. The production of ononitol in tobacco indicates that plant carbohydrate metabolism is flexible and can accommodate the synthesis and accumulation of non‐endogenous metabolites. The transgenic system described here will serve as a useful model to test the ability of cyclitols such as ononitol to confer tolerance to environmental stress in a normally glycophytic plant.

Comparative evaluation of the effects of gaseous pollutants, acidic deposition, and mineral deficiencies on the carbohydrate metabolism of trees

With a focus on conifers, recent literature on the interaction of pollution, acidic deposition and mineral (mainly Mg) deficiency on plant carbohydrate metabolism is reviewed. By combining most recent knowledge about the regulation of carbon partitioning and allocation of photoassimilate with data on symptom-related pool sizes of carbohydrates, biochemical indications for shifts in allocation are presented. In summary, the data, although in part conflicting, indicate that the impact of pollutants at moderate levels (up to three times ambient concentration) should not be responsible for the decline symptoms observed.

Structure and expression of a gene from Arabidopsis thaliana encoding a protein related to SNF1 protein kinase

The AKin10 gene from Arabidopsis thaliana encoding a putative Ser/Thr protein kinase (PK) has been isolated and characterized. The AKin10-encoding gene is located on a genomic 5.4-kb BamHI fragment and contains ten introns, one being located in the 5' untranslated region. The deduced amino acid sequence of AKin10 is 65% identical over the catalytic domain to the yeast PK (SNF1). SNF1 is essential for the derepression of many glucose-repressible genes, including Suc2 which encodes invertase. Southern blot hybridization experiments suggested the presence of one copy of the gene per haploid genome of A. thaliana. Northern hybridization experiments indicated that this gene is expressed in roots, shoots and leaves. AKin10 may play an important role in a signal transduction cascade regulating gene expression and carbohydrate metabolism in higher plants.

Expression of a bacterial mtlD gene in transgenic tobacco leads to production and accumulation of mannitol.

A bacterial gene encoding mannitol-1-phosphate dehydrogenase, mtlD, was engineered for expression in higher plants. Gene constructions were stably incorporated into tobacco plants. The mtlD gene was expressed and translated into a functional enzyme in tobacco, resulting in the synthesis and accumulation of mannitol, which was identified by NMR and mass spectroscopy. Mannitol concentrations exceeded 6 mumol/g (fresh weight) in the leaves and in the roots of some transformants, whereas this sugar alcohol was not detected in these organs of wild-type tobacco plants or of untransformed tobacco plants that underwent the same regeneration scheme. These experiments demonstrate that branch-points in plant carbohydrate metabolism can be generated by which novel gene products can utilize endogenous substrates to divert metabolic energy into novel compounds. Additionally, the system described here allows for physiological studies in which the responses of wild-type and transgenic tobacco to various environmental stimuli can be compared directly. Such studies will facilitate our understanding of the roles of sugar alcohols (e.g., in stress tolerance) in higher plants.

Metabolic derepression of alpha-amylase gene expression in suspension-cultured cells of rice.

We present evidence to show that the alpha-amylase gene family in rice is under two different modes of regulation: 1) hormonal regulation in germinating seeds, and 2) metabolic repression in cultured cells by available carbohydrate nutrients. Expression of alpha-amylase genes in deembryoed rice seeds is known to be induced by exogenous gibberellic acid. On the other hand, expression of alpha-amylase genes in suspension-cultured cells is induced by the deprivation of carbohydrate nutrient. A lag period of 2-4 h is required for the induction of alpha-amylase mRNA in sucrose-depleted medium. The induction of alpha-amylase expression is extraordinarily high and levels of alpha-amylase mRNA can be increased 8-20-folds after 24 h of sucrose starvation. The synthesis and secretion of alpha-amylase is also dependent upon the level of carbon source. The derepression or repression of alpha-amylase synthesis can be readily reversed by the deprivation or replenishment of sucrose in the medium, respectively. Glucose and fructose exert a repression on the alpha-amylase synthesis similar to that of sucrose. A hypothesis that explains the induction of alpha-amylase synthesis by carbohydrate starvation is proposed. Our data have suggested a hitherto undiscovered, potentially important control mechanism of carbohydrate metabolism in higher plants.