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The cooperation of the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways, operating in parallel in plants to generate isoprenoid precursors, has been studied extensively. Elucidation of the isoprenoid metabolic pathways is indispensable for the rational design of plant and microbial systems for the production of industrially valuable terpenoids. Here, we describe a new method, based on numerical modeling of mass spectra of metabolically labeled dolichols (Dols), designed to quantitatively follow the cooperation of MVA and MEP reprogrammed upon osmotic stress (sorbitol treatment) in Arabidopsis (Arabidopsis thaliana). The contribution of the MEP pathway increased significantly (reaching 100%) exclusively for the dominating Dols, while for long-chain Dols, the relative input of the MEP and MVA pathways remained unchanged, suggesting divergent sites of synthesis for dominating and long-chain Dols. The analysis of numerically modeled Dol mass spectra is a novel method to follow modulation of the concomitant activity of isoprenoid-generating pathways in plant cells; additionally, it suggests an exchange of isoprenoid intermediates between plastids and peroxisomes.

Plastid isoprenoids, a diverse group of compounds that includes carotenoids, chlorophylls, tocopherols, and multiple hormones, are essential for plant growth and development. Here, we identified and characterized SEED CAROTENOID DEFICIENT (SCD), which encodes an enzyme that functions in the biosynthesis of plastid isoprenoids in maize (Zea mays). SCD converts 2C-methyl-d-erytrithol 2,4-cyclodiphosphate to 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate in the penultimate step of the methylerythritol phosphate (MEP) pathway. In scd mutants, plant growth and development are impaired and the levels of MEP-derived isoprenoids, such as carotenoids, chlorophylls, and tocopherols, as well as abscisic and gibberellic acids, are reduced in leaves and seeds. This scd metabolic alteration varies among plant tissues and under different light conditions. RNA-sequencing of the scd mutant and wild type identified a limited number of differentially expressed genes in the MEP pathway, although isoprenoid levels were significantly reduced in scd seeds and dark-grown leaves. Furthermore, SCD-overexpressing transgenic lines showed little or no differences in isoprenoid levels, indicating that SCD may be subject to posttranslational regulation or not represent a rate-limiting step in the MEP pathway. These results enhance our understanding of the transcriptomic and metabolic regulatory roles of enzymes in the MEP pathway and of their effects on downstream isoprenoid pathways in various plant tissues and under different light conditions.

Trehalose 6-P (T6P) is a sugar signal in plants that inhibits SNF1-related protein kinase, SnRK1, thereby altering gene expression and promoting growth processes. This provides a model for the regulation of growth by sugar. However, it is not known how this model operates under sink-limited conditions when tissue sugar content is uncoupled from growth. To test the physiological importance of this model, T6P, SnRK1 activities, sugars, gene expression, and growth were measured in Arabidopsis (Arabidopsis thaliana) seedlings after transfer to cold or zero nitrogen compared with sugar feeding under optimal conditions. Maximum in vitro activities of SnRK1 changed little, but T6P accumulated up to 55-fold, correlating with tissue Suc content in all treatments. SnRK1-induced and -repressed marker gene expression strongly related to T6P above and below a threshold of 0.3 to 0.5 nmol T6P g−1 fresh weight close to the dissociation constant (4 µm) of the T6P/ SnRK1 complex. This occurred irrespective of the growth response to Suc. This implies that T6P is not a growth signal per se, but through SnRK1, T6P primes gene expression for growth in response to Suc accumulation under sink-limited conditions. To test this hypothesis, plants with genetically decreased T6P content and SnRK1 overexpression were transferred from cold to warm to analyze the role of T6P/SnRK1 in relief of growth restriction. Compared with the wild type, these plants were impaired in immediate growth recovery. It is concluded that the T6P/SnRK1 signaling pathway responds to Suc induced by sink restriction that enables growth recovery following relief of limitations such as low temperature.

Trehalose-6-phosphate (T6P) is a proposed signaling molecule in plants, yet how it signals was not clear. Here, we provide evidence that T6P functions as an inhibitor of SNF1-related protein kinase1 (SnRK1; AKIN10/AKIN11) of the SNF1-related group of protein kinases. T6P, but not other sugars and sugar phosphates, inhibited SnRK1 in Arabidopsis (Arabidopsis thaliana) seedling extracts strongly (50%) at low concentrations (1–20 μm). Inhibition was noncompetitive with respect to ATP. In immunoprecipitation studies using antibodies to AKIN10 and AKIN11, SnRK1 catalytic activity and T6P inhibition were physically separable, with T6P inhibition of SnRK1 dependent on an intermediary factor. In subsequent analysis, T6P inhibited SnRK1 in extracts of all tissues analyzed except those of mature leaves, which did not contain the intermediary factor. To assess the impact of T6P inhibition of SnRK1 in vivo, gene expression was determined in seedlings expressing Escherichia coli otsA encoding T6P synthase to elevate T6P or otsB encoding T6P phosphatase to decrease T6P. SnRK1 target genes showed opposite regulation, consistent with the regulation of SnRK1 by T6P in vivo. Analysis of microarray data showed up-regulation by T6P of genes involved in biosynthetic reactions, such as genes for amino acid, protein, and nucleotide synthesis, the tricarboxylic acid cycle, and mitochondrial electron transport, which are normally down-regulated by SnRK1. In contrast, genes involved in photosynthesis and degradation processes, which are normally up-regulated by SnRK1, were down-regulated by T6P. These experiments provide strong evidence that T6P inhibits SnRK1 to activate biosynthetic processes in growing tissues.Suc and trehalose are widespread nonreducing disaccharides that function as translocated carbon sources and stress protection compounds. Plants and cyanobacteria are the only organisms in which the pathways of trehalose and Suc synthesis coexist. In the majority of plants, trehalose occurs in trace amounts only, prohibiting a function as a carbon source. This raises the question of the role of the trehalose pathway in plants, given the large number and ubiquity of putative genes encoding enzymes for trehalose synthesis (Avonce et al., 2006; Lunn, 2007). Many of these genes are subject to a high level of regulation at the gene (Paul et al., 2008) and posttranslational (Harthill et al., 2006) levels, suggesting an important function. This is confirmed in transgenic and mutant plants with modified trehalose pathway gene expression, which show a range of phenotypes. For example, a trehalose-6-P synthase (TPS1) has been shown to be essential for embryo development (Eastmond et al., 2002) and for normal vegetative growth and the transition to flowering (Van Dijken et al., 2004). Overexpression of TPS from different species, for example, otsA encoding Escherichia coli TPS, produces effects on sugar utilization in seedlings (Schluepmann et al., 2003) and on vegetative and photosynthetic phenotypes (Pellny et al., 2004; Almeida et al., 2007; Stiller et al., 2008), opposite in nature to seedling and photosynthetic phenotypes of plants expressing E. coli otsB encoding trehalose-6-P phosphatase (TPP; Schluepmann et al., 2003; Pellny et al., 2004). The phenotype of the maize (Zea mays) ramosa3 mutant has been attributed to a knockout of a TPP gene normally expressed in discrete domains subtending axillary meristems (Satoh-Nagasawa et al., 2006). This gives rise to an inflorescence architecture that aids the efficient packing and harvesting of seeds. Trehalose-6-P (T6P) has been found to stimulate starch synthesis via redox activation of ADP-Glc pyrophosphorylase (Kolbe et al., 2005), and T6P responds to light and sugar in relation to carbon status (Lunn et al., 2006). This supports a role for T6P in signaling the sugar status of the cytosol to the chloroplast and thereby activating starch synthesis. However, most of the phenotypes produced where the pathway has been genetically modified in mutant and transgenic lines, which also extend to effects on abiotic stress resistance (Garg et al., 2002; Avonce et al., 2004), cell division, cell walls (Gómez et al., 2006), and cell shape (Chary et al., 2008), cannot be explained simply in terms of the effect on starch metabolism but rather support a more central function. In some fungi, T6P inhibits hexokinase, an enzyme implicated in sugar signaling in plants and other organisms (Moore et al., 2003), but there is no evidence that T6P affects plant hexokinases (Eastmond et al., 2002). Another important signaling route in plants is through SNF1-related protein kinase1 (SnRK1) of the family of calcium-independent Ser/Thr protein kinases that includes AMPK of mammals and SNF1 of yeast (Hardie, 2007; Polge and Thomas, 2007). These conserved kinases perform a fundamental role in transcriptional, metabolic, and developmental regulation in response to energy limitation and starvation of the carbon source (Hardie, 2007). SnRK1 in plants is thought to consist of a heterotrimeric complex, as in AMPK and SNF1, composed of an AKIN10 or AKIN11 catalytic α-subunit and β- and γ-subunits together with a number of additional interacting and regulatory factors (Pierre et al., 2007; Polge and Thomas, 2007; Ananieva et al., 2008). Recent work established that AKIN10 catalytic activity regulates 1,000 or so target genes involved in the response of metabolism and growth to starvation (Baena-González et al., 2007). It was shown that SnRK1 activates genes involved in degradation processes and photosynthesis and inhibits those involved in biosynthetic processes and, by so doing, regulates metabolism and growth in response to available carbon (Baena-González et al., 2007). SnRK1 can phosphorylate class II TPSs (Glinski and Weckwerth, 2005; Harthill et al., 2006) and regulate their transcription (Baena-González et al., 2007), although the importance of this is not clear. SnRK1 transcript increased in response to 100 mm trehalose feeding (Schluepmann et al., 2004) and was decreased 2-fold in tps1 mutants (Gómez et al., 2006). Given a possible interaction between the two pathways, we went on to determine whether T6P affects the catalytic activity of SnRK1. Here, we provide evidence for a function of T6P as an inhibitor of SnRK1 activity. First, we show that low micromolar concentrations of T6P inhibit SnRK1 activity in Arabidopsis (Arabidopsis thaliana) seedling extracts and other young plant material, but not in mature leaves. Second, we show that T6P inhibits SnRK1 at a site distinct and separable from the SnRK1 catalytic site via an intermediary factor. This as yet unknown intermediary factor was not found in mature leaves. Third, we establish effects on gene expression in seedlings with elevated T6P consistent with inhibition of SnRK1 in vivo. Overall, the data provide strong evidence for a function of T6P as an inhibitor of SnRK1 to promote biosynthetic reactions in growing tissues.

Plants respond to low carbon supply by massive reprogramming of the transcriptome and metabolome. We show here that the carbon starvation-induced NAC (for NO APICAL MERISTEM/ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR/CUP-SHAPED COTYLEDON) transcription factor Arabidopsis (Arabidopsis thaliana) Transcription Activation Factor1 (ATAF1) plays an important role in this physiological process. We identified TREHALASE1, the only trehalase-encoding gene in Arabidopsis, as a direct downstream target of ATAF1. Overexpression of ATAF1 activates TREHALASE1 expression and leads to reduced trehalose-6-phosphate levels and a sugar starvation metabolome. In accordance with changes in expression of starch biosynthesis- and breakdown-related genes, starch levels are generally reduced in ATAF1 overexpressors but elevated in ataf1 knockout plants. At the global transcriptome level, genes affected by ATAF1 are broadly associated with energy and carbon starvation responses. Furthermore, transcriptional responses triggered by ATAF1 largely overlap with expression patterns observed in plants starved for carbon or energy supply. Collectively, our data highlight the existence of a positively acting feedforward loop between ATAF1 expression, which is induced by carbon starvation, and the depletion of cellular carbon/energy pools that is triggered by the transcriptional regulation of downstream gene regulatory networks by ATAF1. ispartof: Plant Physiology vol:169 issue:1 pages:379-90 ispartof: location:United States status: published

More volatile organic carbon is lost from plants as isoprene than any other molecule. This flux of carbon to the atmosphere affects atmospheric chemistry and can serve as a substrate for ozone production in polluted air. Isoprene synthesis may help leaves cope with heatflecks and active oxygen species. Isoprene synthase, an enzyme related to monoterpene synthases, converts dimethylallyl diphosphate derived from the methylerythritol 4-phosphate pathway to isoprene. We used dideuterated deoxyxylulose (DOX-d2) to study the regulation of the isoprene biosynthetic pathway. Exogenous DOX-d2 displaced endogenous sources of carbon for isoprene synthesis without increasing the overall rate of isoprene synthesis. However, at higher concentrations, DOX-d2 completely suppressed isoprene synthesis from endogenous sources and increased the overall rate of isoprene synthesis. We interpret these results to indicate strong feedback control of deoxyxylulose-5-phosphate synthase. We related the emission of labeled isoprene to the concentration of labeled dimethylallyl diphosphate in order to estimate the in situ K m of isoprene synthase. The results confirm that isoprene synthase has a K m 10- to 100-fold higher for its allylic diphosphate substrate than related monoterpene synthases for geranyl diphosphate.

The genomic response to low levels of nitrate was studied in Arabidopsis using the Affymetrix ATH1 chip containing more than 22,500 probe sets. Arabidopsis plants were grown hydroponically in sterile liquid culture on ammonium as the sole source of nitrogen for 10 d, then treated with 250 μm nitrate for 20 min. The response to nitrate was much stronger in roots (1,176 genes showing increased or decreased mRNA levels) than in shoots (183 responding genes). In addition to known nitrate-responsive genes (e.g. those encoding nitrate transporters, nitrate reductase, nitrite reductase, ferredoxin reductase, and enzymes in the pentose phosphate pathway), genes encoding novel metabolic and potential regulatory proteins were found. These genes encode enzymes in glycolysis (glucose-6-phosphate isomerase and phosphoglycerate mutase), in trehalose-6-P metabolism (trehalose-6-P synthase and trehalose-6-P phosphatase), in iron transport/metabolism (nicotianamine synthase), and in sulfate uptake/reduction. In many cases, only a few select genes out of several in small gene families were induced by nitrate. These results show that the effect of nitrate on gene expression is substantial (affecting almost 10% of the genes with detectable mRNA levels) yet selective and affects many genes involved in carbon and nutrient metabolism.

The discovery of the 2-C-methyl-d-erythritol-4-phosphate pathway for the biosynthesis of isoprenoids raises the important question of the nature and regulation of the enzymes involved in this pathway.CLA1, a gene previously isolated from Arabidopsis, encodes the first enzyme of the 2-C-methyl-d-erythritol-4-phosphate pathway, 1-deoxy-d-xylulose-5-phosphate synthase. We demonstrate this enzyme activity by complementation of the cla1-1mutant phenotype and by direct enzymatic assays. Based on mRNA and protein expression patterns this enzyme is expressed mainly in developing photosynthetic and non-photosynthetic tissues. The β-glucuronidase expression pattern driven from theCLA1 gene regulatory region supports the northern and protein data while also showing that this gene has some level of expression in most tissues of the plant. A mutation in theCLA1 gene interferes with the normal development of chloroplasts and etioplasts, but does not seem to affect amyloplast structure. Microscopic analysis also shows a pleiotropic effect of theCLA1 gene mutation in mesophyll tissue formation.

L-myo-inositol 1-phosphate synthase (EC 5.5.1.4) from cyanobacterial (Spirulina platensis), algal (Euglena gracilis), and higher plant (Oryza sativa, Vigna radiata) sources was purified to electrophoretic homogeneity, biochemically characterized, and compared. Both chloroplastic and cytosolic forms of the enzyme were detected in E. gracilis, O. sativa, and V. radiata, whereas only the cytosolic form was detected in streptomycin-bleached or chloroplastic mutants of E. gracilis and in S. platensis. Both the chloroplastic and cytosolic forms from different sources could be purified following the same three-step chromatographic protocol. L-myo-inositol 1-phosphate synthases purified from these different sources do not differ significantly with respect to biochemical and kinetic parameters except for the molecular mass of the chloroplastic and cytosolic native holoenzymes, which appear to be homotetrameric and homotrimeric associations of their constituent subunits, respectively. Monovalent and divalent cations, sugar alcohols, and sugar phosphates are inhibitory to the enzyme activity. N-ethylmaleimide inhibition of synthase activity could be protected by the combined presence of the substrate glucose-6-phosphate and cofactor NAD+. Antibody raised against the cytosolic enzyme from E. gracilis immunoprecipitates and cross-reacts with both chloroplastic and cytosolic forms from the other sources studied.

Comparative studies of the stress-tolerant Arabidopsis (Arabidopsis thaliana) halophytic relative, Eutrema salsugineum, have proven a fruitful approach to understanding natural stress tolerance. Here, we performed comparative phenotyping of Arabidopsis and E. salsugineum vegetative development under control and salt-stress conditions, and then compared the metabolic responses of the two species on different growth platforms in a defined leaf developmental stage. Our results reveal both growth platform-dependent and -independent phenotypes and metabolic responses. Leaf emergence was affected in a similar way in both species grown in vitro but the effects observed in Arabidopsis occurred at higher salt concentrations in E. salsugineum. No differences in leaf emergence were observed on soil. A new effect of a salt-mediated reduction in E. salsugineum leaf area was unmasked. On soil, leaf area reduction in E. salsugineum was mainly due to a fall in cell number, whereas both cell number and cell size contributed to the decrease in Arabidopsis leaf area. Common growth platform-independent leaf metabolic signatures such as high raffinose and malate, and low fumarate contents that could reflect core stress tolerance mechanisms, as well as growth platform-dependent metabolic responses were identified. In particular, the in vitro growth platform led to repression of accumulation of many metabolites including sugars, sugar phosphates, and amino acids in E. salsugineum compared with the soil system where these same metabolites accumulated to higher levels in E. salsugineum than in Arabidopsis. The observation that E. salsugineum maintains salt tolerance despite growth platform-specific phenotypes and metabolic responses suggests a considerable degree of phenotypic and metabolic adaptive plasticity in this extremophile.

Trehalose-6-phosphate (T6P) is a signaling metabolite that regulates carbon metabolism, developmental processes, and growth in plants. In Arabidopsis (Arabidopsis thaliana), T6P signaling is, at least in part, mediated through inhibition of the SNF1-related protein kinase SnRK1. To investigate the role of T6P signaling in a heterotrophic, starch-accumulating storage organ, transgenic potato (Solanum tuberosum) plants with altered T6P levels specifically in their tubers were generated. Transgenic lines with elevated T6P levels (B33-TPS, expressing Escherichia coli osmoregulatory trehalose synthesis A [OtsA], which encodes a T6P synthase) displayed reduced starch content, decreased ATP contents, and increased respiration rate diagnostic for high metabolic activity. On the other hand, lines with significantly reduced T6P (B33-TPP, expressing E. coli OtsB, which encodes a T6P phosphatase) showed accumulation of soluble carbohydrates, hexose phosphates, and ATP, no change in starch when calculated on a fresh weight basis, and a strongly reduced tuber yield. [14C]Glucose feeding to transgenic tubers indicated that carbon partitioning between starch and soluble carbohydrates was not altered. Transcriptional profiling of B33-TPP tubers revealed that target genes of SnRK1 were strongly up-regulated and that T6P inhibited potato tuber SnRK1 activity in vitro. Among the SnRK1 target genes in B33-TPP tubers, those involved in the promotion of cell proliferation and growth were down-regulated, while an inhibitor of cell cycle progression was up-regulated. T6P-accumulating tubers were strongly delayed in sprouting, while those with reduced T6P sprouted earlier than the wild type. Early sprouting of B33-TPP tubers correlated with a reduced abscisic acid content. Collectively, our data indicate that T6P plays an important role for potato tuber growth.

The activation of purified ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) has been studied in the presence of sugar phosphates, and the effect of rubisco activase on this process determined. During an 11-minute time course at pH 7.7 and 11 micromolar CO(2), the activation of rubisco was strongly inhibited by ribulose-1,5-bisphosphate (4 millimolar), fructose-1,6-bisphosphate (1 millimolar) and ribose 5-phosphate (5 millimolar), but this inhibition was overcome by the addition of rubisco activase and activation then proceeded to a greater extent than spontaneous activation of rubisco. Glycerate 3-phosphate (20 millomolar) slowed the initial rate but not the extent of activation and rubisco activase had no effect on this. The activation of rubisco was shown to be affected by phosphoenolpyruvate (3 millimolar) but not by creatine phosphate (3 millimolar) or ATP (3 millimolar), and the creatine-phosphate/creatine phosphokinase system was used to generate the high ATP/ADP quotients required for rubisco activase to function. ATP was shown to be required for the rubisco activase-dependent rubisco activation in the presence of fructose-1,6-bisphosphate (1 millimolar). It is concluded that rubisco activase has a mixed specificity for some sugar phosphate-bound forms of rubisco, but has low or no activity with others. Some possible bases for these differences among sugar phosphates are discussed but remain to be established.

The extent of phosphate uptake measured by the relative changes in cytoplasmic Pi, vacuolar Pi, ATP, glucose-6-phosphate, and UDPG was determined using in vivo31P nuclear magnetic resonance spectroscopy. Maize (Zea mays) root tips were perfused with a solution containing 0.5 or 1.0 millimolar phosphate at pH ∼6.5 under different conditions. In the aerated state, phosphate uptake resulted in a significant increase (>80%) in vacuolar Pi, but cytoplasmic Pi only transiently increased by 10%. Under N2, the cytoplasmic Pi increased ∼150% which could be attributed to a large extent to the breakdown of ATP, sugar phosphates and UDPG. Vacuolar Pi increased but only to the extent of ∼10% of that seen under aerobic conditions. 2-deoxyglucose pretreatment was utilized to decrease the level of cytoplasmic Pi. When pretreated with the 2-deoxyglucose, the excised maize roots absorbed phosphate from the perfusate with a significant increase in the cytoplasmic Pi. The increase could only be traced to external phosphate since the concentrations of other phosphorus containing species remained constant during the uptake period. With 2-deoxyglucose pretreatment, phosphate uptake under anaerobic conditions was substantially inhibited with only the vacuolar phosphate showing a slight increase. When roots were treated with carbonyl cyanide m-chlorophenyl hydrazone, no detectable Pi uptake was found. These results were used to propose a H+-ATPase related transport mechanism for phosphate uptake and compartmentation in corn root cells.

To function, the catalytic sites of Rubisco (EC 4.1.1.39) need to be activated by the reversible carbamylation of a lysine residue within the sites followed by rapid binding of magnesium. The activation of Rubisco in vivo requires the presence of the regulatory protein Rubisco activase. This enzyme is thought to aid the release of sugar phosphate inhibitors from Rubisco's catalytic sites, thereby influencing carbamylation. In C3 species, Rubisco operates in a low CO2 environment, which is suboptimal for both catalysis and carbamylation. In C4 plants, Rubisco is located in the bundle sheath cells and operates in a high CO2 atmosphere close to saturation. To explore the role of Rubisco activase in C4 photosynthesis, activase levels were reduced in Flaveria bidentis, a C4 dicot, by transformation with an antisense gene directed against the mRNA for Rubisco activase. Four primary transformants with very low activase levels were recovered. These plants and several of their segregating T1 progeny required high CO2 (>1 kPa) for growth. They had very low CO2 assimilation rates at high light and ambient CO2, and only 10% to 15% of Rubisco sites were carbamylated at both ambient and very high CO2. The amount of Rubisco was similar to that of wild-type plants. Experiments with the T1 progeny of these four primary transformants showed that CO2 assimilation rate and Rubisco carbamylation were severely reduced in plants with less than 30% of wild-type levels of activase. We conclude that activase activity is essential for the operation of the C4 photosynthetic pathway.

Isoprene synthase converts dimethylallyl diphosphate, derived from the methylerythritol 4-phosphate (MEP) pathway, to isoprene. Isoprene is made by some plants in substantial amounts, which affects atmospheric chemistry, while other plants make no isoprene. As part of our long-term study of isoprene synthesis, the genetics of the isoprene biosynthetic pathway of the isoprene emitter, kudzu (Pueraria montana), was compared with similar genes in Arabidopsis (Arabidopsis thaliana), which does not make isoprene. The MEP pathway genes in kudzu were similar to the corresponding Arabidopsis genes. Isoprene synthase genes of kudzu and aspen (Populus tremuloides) were cloned to compare their divergence with the divergence seen in MEP pathway genes. Phylogenetic analysis of the terpene synthase gene family indicated that isoprene synthases are either within the monoterpene synthase clade or sister to it. In Arabidopsis, the gene most similar to isoprene synthase is a myrcene/ocimene (acyclic monoterpenes) synthase. Two phenylalanine residues found exclusively in isoprene synthases make the active site smaller than other terpene synthase enzymes, possibly conferring specificity for the five-carbon substrate rather than precursors of the larger isoprenoids. Expression of the kudzu isoprene synthase gene in Arabidopsis caused Arabidopsis to emit isoprene, indicating that whether or not a plant emits isoprene depends on whether or not it has a terpene synthase capable of using dimethylallyl diphosphate.

In order to identify nuclear genes required for early chloroplast development, a collection of photosynthetic pigment mutants of Arabidopsis was assembled and screened for lines with extremely low levels of chlorophyll. Nine chloroplast biogenesis (clb) mutants that affect proplastid growth and thylakoid membrane formation and result in an albino seedling phenotype were identified. These mutations identify six new genes as well as a novel allele of cla1. clb mutants have less than 2% of wild-type chlorophyll levels, and little or no expression of nuclear and plastid-encoded genes required for chloroplast development and function. In all but one mutant, proplastids do not differentiate enough to form elongated stroma thylakoid membranes. Analysis of mutants during embryogenesis allows differentiation between CLB genes that act noncell autonomously, where partial maternal complementation of chloroplast development is observed in embryos, and those that act cell autonomously, where complementation during embryogenesis is not observed. Molecular characterization of the noncell autonomous clb4 mutant established that the CLB4 gene encodes for hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS), the next to the last enzyme of the methylerythritol 4-phosphate (MEP) pathway for the synthesis of plastidic isoprenoids. The noncell autonomous nature of the clb4 mutant suggests that products of the MEP pathway can travel between tissues, and provides in vivo evidence that some movement of MEP intermediates exists from the cytoplasm to the plastid. The isolation and characterization of clb mutants represents the first systematic study of genes required for early chloroplast development in Arabidopsis.

The acidophilic and thermophilic red alga Galdieria sulphuraria is able to grow heterotrophically on at least six different pentoses. These pentoses are reduced in the cell to pentiols by an NADP-dependent aldose reductase. The pentiols are then introduced into the oxidative pentose phosphate pathway via NAD-dependent polyol dehydrogenases and pentulokinases. The aldose reductase was purified 130-fold to apparent homogeneity by column chromatography. The enzyme is a homodimer of about 80 kD, as estimated by size-exclusion chromatography and from the sedimentation behavior. The Michaelis constant values for D-xylose (27 mM), D-ribose (29 mM), D-lyxose (30 mM), and D-arabinose (38 mM) were about three to five times lower than for the L-forms of the sugars. The activity of the enzyme with hexoses, deoxysugars, and sugar phosphates was only about 5 to 10% of the rate with pentoses. In the reverse reaction the activity was low and only detectable with pentiols. No activity was measured with NAD(H) as the cosubstrate in either direction.

Changes in photosynthesis, carbon partitioning, and growth following resupply of orthophosphate (Pi) to moderately P-deficient plants (low-P) were determined for sugar beets (Beta vulgaris L. cv F58-554H1) cultured hydroponically in growth chambers. One set of plants was supplied with 1.0 mM Pi in half-strength Hoagland solution (control plants), and a second set (low-P plants) was supplied with 0.05 mM Pi. At the end of 2 weeks, the low-P plants were resupplied with 1.0 mM Pi. Low-P plants rapidly accumulated large amounts of Pi, and the photosynthesis rate increased to control values within 4 to 6 h. The rate of photosynthesis appeared to be controlled by ribulose-1,5-bisphosphate (RuBP); low P reduced photosynthesis and RuBP levels, and P resupply increased photosynthesis and RuBP in a manner parallel with time. Low-P treatment reduced adenylate levels substantially but not nicotinamide nucleotides; adenylate levels recovered to control values over 3 to 6 h. With low P, more photosynthate is allocated to non-P carbon compounds (e.g. starch, sucrose) than to sugar phosphates. When P is resupplied, sugar phosphates increase as starch and sucrose pools decrease; this increase in leaf (chloroplast) sugar phosphates was most likely responsible for the increases in RuBP and photosynthesis and may have increased adenylate levels (through enhanced levels of ribose-5-phosphate).

The in vivo activity of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) is modulated in response to light intensity by carbamylation of the active site and by the binding of sugar phosphate inhibitors such as 2'-carboxyarabinitol-1-phosphate (CA 1P). These changes are influenced by the regulatory protein Rubisco activase, which facilitates the release of sugar phosphates from Rubisco's catalytic site. Activase levels in Nicotiana tabacum were reduced by transformation with an antisense gene directed against the mRNA for Rubisco activase. Activase-deficient plants were photosynthetically impaired, and their Rubisco carbamylation levels declined upon illumination. Such plants needed high CO2 concentrations to sustain reasonable growth rates, but the level of carbamylation was not increased by high CO2. The antisense plants had, on average, approximately twice as much Rubisco as the control plants. The maximum catalytic turnover rate (k cat) of Rubisco decreases in darkened tobacco leaves because of the binding of CA 1P. The dark-to-light increase in k cat that accompanies CA 1P release occurred to similar extents in antisense and control plants, indicating that normal levels of activase were not essential for CA 1P release from Rubisco in the antisense plants. However, CA 1P was released in the antisense plants at less than one-quarter of the rate that it was released in the control plants, indicating a role for activase in accelerating the release of CA 1P.

Activities of glucose 6-phosphate, 6-phosphogluconate, and isocitrate dehydrogenases, together with intermediate levels of the glycolytic pathway and the pentose phosphate cycle, were measured throughout a year in the living bark of poplar ( Populus gelrica ). Shoots, immediately after budding (early May), contained very high levels of the three enzyme activities, which fell gradually by early or mid-July to a level, roughly equivalent to budding (May) or growing (July) 2-year-old twigs. In September, the former two dehydrogenase activities of the new shoots and 2-year-old twigs began to rise, while the latter activity started to decrease. The rise of the two dehydrogenase activities continued until late November (or early December). The high level of the two dehydrogenase activities lasted until early in April of the following year and then the decrease in the activities began prior to the onset of budding, reaching a low, basal level in early May. The profile of changes in the two dehydrogenase activities appeared to coincide with the increase and decrease of soluble proteins. Normal concentrations of total hexose phosphates in the glycolytic pathway plus 6-phosphogluconate were found to be 288 to 895 μmoles/kilogram dry weight. During the metabolism transition (September and April), a transient and striking increase of 6-phosphogluconate was observed. In September, 6-phosphogluconate reached a level on the order of 10 −4 m and was 4 times that of fructose 6-phosphate. The increase in 6-phosphogluconate coincided with the increase in the glucose 6-phosphate dehydrogenase activity. Coincidentally, with the change of 6-phosphogluconate level, a large deviation of the in vivo ratio of fructose 6-phosphate to glucose 6-phosphate from the known equilibrium constant was observed, showing the relation of pentose phosphate cycle enzyme activity to the control of glycolysis. The ratio of glucose 6-phosphate to glucose 1-phosphate deviated from that predicted. These ratios fluctuated throughout the year and were affected by the growth phases. The levels of pentose phosphate cycle metabolites, except for 6-phosphogluconate, in the bark were extremely low. The level of inorganic phosphate in the living bark throughout the year was dependent upon the growth phases, suggesting the presence of a regulatory mechanism to maintain inorganic phosphate at a given level as the growth phase changed. Sugar phosphate levels of popular twigs or potato tubers ( Solanum tuberosum ) remained constant after they were stored for 2 weeks at low temperatures, whereas in sweet potato roots ( Ipomoea batatas ), the level rose to about 9-fold of the control, indicating the presence of a strict regulatory system for the synthesis and catabolism of sugar phosphate in the former two.

Existence of a group translocator for sucrose transfer into vacuoles of sugarcane (Saccharum sp.) cells has been further confirmed by the use of tonoplast vesicles isolated from intact vacuoles. The group translocator depends on external UDP-Glucose (Glc) and, via a series of enzymic reactions within the tonoplast, sucrose phosphate and sucrose are deposited inside the vesicles. Fructose-6-phosphate was not required for UDP-Glc uptake, nor was it taken up. None of the other sugar phosphates tested were taken up nor were the nucleotide sugars, UDP-Galactose and ADP-Glc. The uptake of UDP-Glc was concentration-dependent with a K(m) of 1.2 millimolar and a V(max) of 83.3 nanomoles per minute per milligram protein. The optimum pH for UDP-Glc uptake was 7.0. Uptake of UDP-Glc was inhibited by para-chloromercuribenzene-sulfonic acid, UDP, and GDP; carbonyl cyanide m-chlorophenylhydrazone inhibited to a lesser extent.

The effects of salinity on corn plants (Zea mays L.) are influenced by the concentration of nutrient orthophosphate. Salinity (-2 bars each of NaCl and CaCl(2)) was more injurious in combination with a high concentration of orthophosphate (2 mm) (that gave optimum yields in the absence of salinity) than it was with a lower concentration (0.1 mm). With 2 mm orthophosphate, salinity seemed to damage the plant mechanisms that normally regulate the internal concentration of orthophosphate resulting in excessive accumulation and P toxicity. On the other hand, with 0.1 mm orthophosphate, salinity decreased orthophosphate concentration in mature leaves. This effect was paralleled by decreases in the concentration of adenosine 5'-triphosphate and in the energy charge of the adenylate system, indicating an orthophosphate deficit. Even so, plants survived salinity better under these conditions than in the presence of 2 mm orthophosphate. The data indicated that salinity affected the phosphorylated state of the adenine nucleotides only indirectly through its effect on the concentration of orthophosphate in the cells.Salinity, especially in the presence of 2 mm orthophosphate, resulted in an increase in the concentrations of sugar phosphates in mature photosynthesizing leaves, suggesting that translocation rather than photosynthesis was a limiting process. Decreased translocation could be a secondary effect of decreased growth. However, a decreased translocation rate could cause decreased growth by limiting the supply of essential metabolites reaching growing tissues.

Sugar Beets (Beta vulgaris L. cv F58-554H1) were cultured hydroponically in growth chambers. Leaf orthophosphate (Pi) levels were varied nutritionally. The effect of decreased leaf phosphate (low-P) status was determined on the diurnal changes in the pool sizes of leaf ribulose 1,5-bisphosphate (RuBP), 3-phosphoglycerate (PGA), triose phosphate, fructose 1,6-bisphosphate, fructose-6-phosphate, glucose-6-phosphate, adenylates, nicotinamide nucleotides, and Pi. Except for triose phosphate, low-P treatment caused a marked reduction in the levels of leaf sugar phosphates (on a leaf area basis) throughout the diurnal cycle. Low-P treatment decreased the average leaf RuBP levels by 60 to 69% of control values during the light period. Low-P increased NADPH levels and NADPH/NADP(+) ratio but decreased ATP; the ATP/ADP ratio was unaffected. Low P treatment caused a marked reduction in RuBP regeneration (RuBP levels were half the RuBP carboxylase binding site concentration) but did not depress PGA reduction to triose phosphate. These results indicate that photosynthesis in low-P leaves was limited by RuBP regeneration and that RuBP formation in low-P leaves was not limited by the supply of ATP and NADPH. We suggest that RuBP regeneration was limited by the supply of fixed carbon, an increased proportion of which was diverted to starch synthesis.

When seven crop species were grown under identical environmental conditions, decreased sink:source ratio led to a decreased photosynthetic rate within 1 to 3 days in Cucumis sativus L., Gossypium hirsutum L., and Raphanus sativus L., but not in Capsicum annuum L., Solanum melongena L., Phaseolus vulgaris L., or Ricinus communis L. The decrease was not associated with stomatal closure. In cotton and cucumber, sink removal led to an increase in starch and sugar content, in glucose 6-phosphate and fructose 6-phosphate pools, and in the proportion of (14)C detected in sugar phosphates and UDPglucose following (14)CO(2) supply. When mannose was supplied to leaf discs to sequester cytoplasmic inorganic phosphate, promotion of starch synthesis, and inhibition of CO(2) fixation, were observed in control discs, but not in discs from treated plants. Phosphate buffer reduced starch synthesis in the latter, but not the former discs. The findings suggest that sink removal led to a decreased ratio inorganic phosphate:phosphorylated compounds. In beans (14)C in sugar phosphates increased following sink removal, but without sucrose accumulation, suggesting tighter feedback control of sugar level. Starch accumulated to higher levels than in the other plants, but CO(2) fixation rate was constant for several days.

Four forms of hexose kinase activity from developing maize (Zea mays L.) kernels have been separated by ammonium sulfate precipitation, gel filtration chromatography, blue-agarose chromatography, and ion exchange chromatography. Two of these hexose kinases utilized d-glucose most effectively and are classified as glucokinases (EC 2.7.1.2). The other two hexose kinases utilized only d-fructose and are classified as fructokinases (EC 2.7.1.4). All hexose kinases analyzed had broad pH optima between 7.5 and 9.5 with optimal activity at pH 8.5. The two glucokinases differed in substrate affinities. One form had low K(m) values [K(m)(glucose) = 117 micromolar, K(m)(ATP) = 66 micromolar] whereas the other form had much higher K(m) values [K(m)(glucose) = 750 micromolar, K(m)(ATP) = 182 micromolar]. Both fructokinases had similar substrate saturation responses. The K(m)(fructose) was about 130 micromolar and the K(m)(ATP) was about 700 micromolar. Both exhibited uncompetitive substrate inhibition by fructose [K(i)(fructose) = 1.40 to 2.00 millimolar]. ADP inhibited all four hexose kinase activities, whereas sugar phosphates had little effect on their activities. The data suggest that substrate concentrations are an important factor controlling hexose kinase activity in situ.

The effects of methionine sulfoximine and ammonium chloride on [(14)C] glutamate metabolism in excised leaves of Triticum aestivum were investigated. Glutamine was the principal product derived from [U(14)C]glutamate in the light and in the absence of inhibitor or NH(4)Cl. Other amino acids, organic acids, sugars, sugar phosphates, and CO(2) became slightly radioactive. Ammonium chloride (10 mm) increased formation of [(14)C] glutamine, aspartate, citrate, and malate but decreased incorporation into 2-oxoglutarate, alanine, and (14)CO(2). Methionine sulfoximine (1 mm) suppressed glutamine synthesis, caused NH(3) to accumulate, increased metabolism of the added radioactive glutamate, decreased tissue levels of glutamate, and decreased incorporation of radioactivity into other amino acids. Methionine sulfoximine also caused most of the (14)C from [U-(14)C]glutamate to be incorporated into malate and succinate, whereas most of the (14)C from [1-(14)C]glutamate was metabolized to CO(2) and sugar phosphates. Thus, formation of radioactive organic acids in the presence of methionine sulfoximine does not take place indirectly through "dark" fixation of CO(2) released by degradation of glutamate when ammonia assimilation is blocked. When illuminated leaves supplied with [U-(14)C] glutamate without inhibitor or NH(4)Cl were transferred to darkness, there was increased metabolism of the glutamate to glutamine, aspartate, succinate, malate, and (14)CO(2). Darkening had little effect on the labeling pattern in leaves treated with methionine sulfoximine.

The Emerson enhancement effect was evaluated in the intact spinach (Spinacia oleracea var. Long Standing Bloomsdale) chloroplast by monitoring the uptake of (14)CO(2) during illumination by 640 nm and 720 nm lights. Low levels (about 10 mum) of fructose 1,6-diphosphate, ribose 5-phosphate, and glycerate 3-phosphate stimulated the rate of photosynthesis and abolished enhancement values observed in their absence. Concentrations of the two sugar phosphates at levels of 1 mm responded similarly. In contrast, 1 mm glycerate 3-phosphate inhibited the rate of photosynthesis and increased enhancement. The exchange of glycerate 3-phosphate for glyceraldehyde 3-phosphate was speculated to be a factor underlying the decrease in photosynthesis and the increase in enhancement. Glucose 6-phosphate, NADPH, and l-malate did not influence photosynthesis or enhancement.The uncoupler, p-trifluoromethoxyphenylhydrazone, decreased the rate of photosynthesis but did not change the enhancement values. ATP (0.2 to 1 mm) had an occasional stimulating effect on CO(2) fixation but no effect on enhancement. Magnesium ions inhibited photosynthesis and decreased the enhancement values. It was concluded that the enhancement phenomenon reflects events of the photosynthetic carbon reduction cycle as well as the photochemical act.

Although d-galactose is normally toxic to sugarcane (Saccharum sp.) cells, a cell line that grows on 100 mm galactose has been propagated. Nonadapted cells in a medium containing galactose instead of sucrose accumulate UDP-galactose; these cells also have much lower UDP-galactose 4-epimerase (EC 5.1.3.2) activity than do adapted cells. This enzyme may determine whether or not galactose will cause toxicity symptoms to develop. The growth rate of galactose-adapted cells is similar to most cell lines on several other carbohydrates. The galactose-adapted cells are also similar to sucrose stock cells in cell wall composition and sugar phosphate concentrations, but, like the nonadapted cells, accumulate free galactose.

The effect of various sugar phosphates on CO 2 fixation in Anabaena flos-aquae was investigated and found to be very similar to that found for isolated spinach chloroplasts. One exception, glucose 6-phosphate, has a stimulatory effect on CO 2 fixation in Anabaena but not in isolated chloroplasts. Further examination of the role of glucose 6-phosphate metabolism in Anabaena indicates that: ( a ) this sugar phosphate can be taken up; ( b ) its uptake is greater in the light than the dark; ( c ) turnover of glucose 6-phosphate is inhibited in the light; and ( d ) glucose 6-phosphate can support dark CO 2 fixation. These results are discussed with reference to photosynthesis-related control of glucose 6-phosphate metabolism and the role of glucose 6-phosphate as a source for reducing equivalents and ATP in blue-green algae.

d-Lactate accumulation in Chlamydomonas reinhardtii was dependent on anaerobic conditions. As much as 50% of the 14 C after 2 minutes of photosynthetic 14 CO 2 fixation moved into d-lactate from sugar phosphates if the cells became anaerobic for short time periods. No lactate accumulated in the dark until the O 2 concentration decreased to less than 0.1%. Lactate was determined to be of the d-configuration using stereospecific lactate dehydrogenases. d-Lactate produced anaerobically by algae grown on 5% CO 2 was only slowly metabolized aerobically in the light or dark, and in the dark, only a trace of the lactate was excreted. A pyruvate reductase (d-lactate: diphosphopyridine nucleotide oxidoreductase, EC 1.1.1.28) was partially purified 47-fold from Chlamydomonas. Because this enzyme catalyzes an essentially irreversible reaction in the direction of pyruvate reduction, it is considered to be a pyruvate reductase. The reductase activity in extracts of Chlamydomonas was 30 micromoles per hour per milligram chlorophyll. For the partially purified enzyme, the apparent K m (pyruvate) was 0.5 millimolar, and the pH optimum was 7.0. Studies with cycloheximide and chloramphenicol indicated that the enzyme was constitutive in aerobic cells. Potassium phosphate stimulated the reductase, and high salt and dithiothreitol were required for stability. The enzyme demonstrated substrate inhibition and was inhibited by ATP. Pyruvate reductase was separated from a hydroxypyruvate reductase by gel filtration chromatography, indicating the presence of separate reductases for these two substrates in Chlamydomonas.

The products of short time photosynthesis and of enhanced dark 14 CO 2 fixation (illumination in helium prior to addition of 14 CO 2 in dark) by Chlorella pyrenoidosa and Anacystis nidulans were compared. Glycerate 3-phosphate, phosphoenolpyruvate, alanine, and aspartate accounted for the bulk of the 14 C assimilated during enhanced dark fixation while hexose and pentose phosphates accounted for the largest fraction of isotope assimilated during photosynthesis. During the enhanced dark fixation period, glycerate 3-phosphate is carboxyl labeled and glucose 6-phosphate is predominantly labeled in carbon atom 4 with lesser amounts in the upper half of the C 6 chain and traces in carbon atoms 5 and 6. Tracer spread throughout all the carbon atoms of photosynthetically synthesized glycerate 3-phosphate and glucose 6-phosphate. During the enhanced dark fixation period, there was a slow formation of sugar phosphates which subsequently continued at 5 times the initial rate long after the cessation of 14 CO 2 uptake. To explain the kinetics of changes in the labelling patterns and in the limited formation of the sugar phosphates during enhanced dark CO 2 fixation, the suggestion is made that most of the reductant mediating these effects did not have its origin in the preillumination phase. It is concluded that a complete photosynthetic carbon reduction cycle operates to a limited extent, if at all, in the dark period subsequent to preillumination.

The effects of kinetin on starch and sugar levels and on (14)CO(2) and (32)P-orthophosphate labeling patterns of floated Chinese cabbage (Brassica pekinensis) leaf discs were investigated. Kinetin caused gross starch degradation. Neutral sugars were depressed by 30 to 40% in leaf tissue treated with kinetin for 24 hours. (14)CO(2) labeling of leaf discs pretreated with kinetin for 24 hours showed increased radioactivity in chloroform-soluble material and most sugar phosphates, and a 35 to 40% decrease in radioactivity in the neutral sugars, glucose, sucrose, and fructose. Incorporation into ATP was increased by 40% by kinetin. (32)P-Orthophosphate uptake was inhibited 30% by kinetin. When corrected for uptake, kinetin stimulated incorporation into chloroform-soluble material but had little effect on other cell fractions. These results indicate that kinetin mobilizes starch reserves and increases the flow of sugars required for the synthesis of lipids and structural materials in floated discs.

The activities of glucose 6-phosphate and 6-phosphogluconate dehydrogenases, transketolase, phosphoglucose isomerase, and fructose 6-phosphate kinase were studied in extracts of wintering poplar (Populus gelrica) xylem. The xylem of wintering poplar showed high levels of transketolase, glucose 6-phosphate, and 6-phosphogluconate dehydrogenases. On recommencement of growth, the two dehydrogenase activities decreased. The three remaining enzymes appeared to be unchanged. In spring and early summer, glucose 6-phosphate dehydrogenase of the xylem was extremely low. On the other hand, 6-phosphogluconate dehydrogenase, which also became lower during the metabolic shift from winter to spring, was readily detected, and was several times higher than glucose 6-phosphate dehydrogenase throughout the year. The low dehydrogenase activities lasted into late October and then appeared to resume their original activity. A shift of metabolism at the beginning of growth was also observed by measuring the amount of sugar phosphates, soluble amino acids and amides, and proteins in the xylem. In contrast to the decrease of the two dehydrogenases and soluble proteins at the time of budding, incorporation of lysine-U-(14)C into the xylem protein ramained constant. A method to transfuse radioactive compounds into a section of stem was described.

With Chlorella ellipsoidea cells, the effect of oxygen was investigated on the products of enhanced dark (14)CO(2) fixation immediately following preillumination in the absence of CO(2). When the reaction mixture was made aerobic by bubbling air (CO(2)-free) throughout preillumination and the following dark (14)CO(2) fixation periods, the initial fixation product was mainly 3-phosphoglyceric acid. When nitrogen gas had been used instead of air, only about one-half of the total radioactivity in the initial fixation products was in 3-phosphoglyceric acid and the rest in aspartic, phosphoenolpyruvic, and malic acids. The percentage distribution of radioactivity incorporated in these initial products rapidly decreased during the rest of the dark period. Concurrent with the decrease in the initial (14)CO(2) fixation products, some increase was observed in the radioactivities of the sugar phosphates. The maximal radioactivity incorporated in sugar mono- and diphosphates accounted for only 10% of total (14)C, under either the aerobic or anaerobic conditions. Under anaerobic conditions most of the (14)C incorporated was eventually transferred to alanine, whereas the main end products under aerobic conditions were aspartate and glutamate. The pattern of (14)CO(2) fixation products was unaffected by the atmospheric condition during the period of preillumination. The preferential flow of the fixed carbon atom to alanine or aspartate depended on the presence or absence of oxygen during the period of dark CO(2) fixation.

The products of C(14)O(2) fixation by Chlamydomonas and Chlorella were studied under conditions most favorable for glycolate synthesis. The highest percentage of the C(14) was incorporated into glycolate in the pH range of 8 to 9. After 1 to 2 minutes as much as 40% of the C(14) was found in glycolate products and only a trace of C(14) was present as phosphoglycerate. Below pH 8 the rate of photosynthesis was much faster, but only a small percent of the C(14) was incorporated into glycolate in 1 or 2 minutes, while a high percent of the C(14) accumulated in phosphoglycerate. C(14) labeling of glycolate even at pH 8 or above did not occur at times shorter than 10 seconds. During the first seconds of photosynthesis, nearly all of the C(14) was found in phosphoglycerate and sugar phosphates. Thus glycolate appears to be formed after the phosphate esters of the photosynthetic carbon cycle.Washing Chlamydomonas with water 2 or 3 times resulted in the loss of most of their free phosphate. When a small aliquot of NaHC(14)O(3) was added to washed algae in the absence of this buffering capacity, the pH of the algal medium became 8 or above and much of the fixed C(14) accumulated in glycolate.

The variation in starch content in healthy and Puccinia striiformsi-infected wheat leaves was measured from 5 to 15 days after inoculation. The starch content of diseased leaves relative to healthy leaves decreased from 5 to 9 days, increased from 9 to 12 days to twice that of healthy leaves, and decreased from 12 to 15 days after inoculation. Electron micrographs of plant tissues indicated that the starch accumulated in the chloroplasts of host cells adjacent to fungal hyphae. Variations in sugar phosphates, ATP, and inorganic phosphate were measured during the infection process. ADP-glucose pyrophosphorylase was extracted and partially purified from healthy and diseased leaves. When proportionate concentrations of sugar phosphates and inorganic phosphate found in healthy and diseased leaves during the infection process were placed in the assay mixture, ADP-glucose pyrophosphorylase activity was similar to the pattern of starch accumulation and was almost the inverse of the variation observed in inorganic phosphate in diseased leaves during the infection process. A mechanism to explain the accumulation of starch is presented and discussed. This mechanism is based on the regulation of ADP-glucose pyrophosphorylase by changes in effector molecule concentrations during the infection process. Reasons for these changes are presented.

Acid extracts from tissues of two solanaceous plants were found to contain a heat-labile, nondialyzable factor which hydrolyzes nucleoside di- and triphosphates to nucleoside monophosphates. This acid-resistant factor shows optimal ATP-hydrolyzing activity at pH 5, whereas practically no activity was detected below pH 3 and above pH 9. It does not hydrolyze sugar phosphates, nucleoside monophosphates, uridine diphosphoglucose, and phosphoenolpyruvate. In order to estimate quantitatively the amount of nucleoside di- and triphosphates in a plant extract, care must be taken to circumvent possible interference by this factor. This is achieved by carefully maintaining the extract below pH 3.

A method for rapid separation of the chloroplast and cytoplasmic fractions from isolated leaf protoplasts of wheat and spinach has been used to determine the distribution of (14)C-labeled products during photosynthesis. In the dark, CO(2) fixation was only 1 to 2% of that in the light and the products were mainly in the cytoplasmic fraction suggesting fixation by phosphoenolpyruvate carboxylase. Label appeared rapidly in the chloroplast fraction following illumination but the amount leveled off after 4 to 5 minutes reflecting the buildup of intermediates to steady state levels. There was only a slight lag before label appeared in the cytoplasmic fraction and it continued to increase at a constant rate reflecting synthesis of neutral products. In the light, the percentage of label in the chloroplast fraction decreased rapidly in the first minute of illumination and was only 10 to 20% in the steady-state. It is suggested that the chloroplast phosphate transporter promotes a rapid transfer of sugar phosphates from the chloroplast to the cytoplasm, even during the induction phase of photosynthesis.

Increasing levels of CO(2) have been shown to stimulate the rate of photosynthesis, eliminate the oxygen inhibition of photosynthesis (Warburg effect), and decrease glycolate formation in isolated spinach chloroplasts. Ribose 5-phosphate and fructose 1,6-diphosphate at concentrations of 5 to 10 mum also stimulate the rate of plastid photosynthesis and eliminate the Warburg effect. In contrast to the effect of high CO(2) levels, these sugar phosphates have little effect on glycolate formation. Evidence is presented to show that the level of intermediates of the photosynthetic carbon reduction cycle may influence the Warburg effect in vivo. It is postulated that the formation of glycolate is not the causal factor of the Warburg effect.

The levels of glucose, sugar phosphates, and adenosine phosphates were determined in primary leaves of intact bean plants during normal senescence and compared to leaves in which senescence was delayed by application of benzyladenine (BA). In both cases there was a rise with time in the levels of glucose 1-phosphate, glucose 6-phosphate, and fructose 6-phosphate, and a decline in 2-phosphoglyceric acid, inorganic phosphate, and the adenosine phosphates (AMP, ADP, ATP). The levels of fructose 1,6-diphosphate remained fairly constant. Although the levels of hexose phosphates, adenosine phosphates, and inorganic phosphate were lower in the BA-treated leaves, the incorporation of (32)P into these compounds by 3- and 6-week-old plants was higher than in the controls. These results suggest that the retardation of leaf senescence by BA in intact bean plants is associated with increased utilization of metabolites, indicating a more rapid turnover of the adenosine phosphates. It is concluded that this effect is brought about by a regulatory coordination of metabolic processes in relation to energy production and utilization.

6-Phosphogluconate is a much more effective inhibitor of the photosynthetic carboxylation enzyme, ribulose-1, 5-diphosphate carboxylase, than other sugar phosphates and sugar acids of the reductive and oxidative pentose phosphate cycles. The inhibition appears to be noncompetitive with ribulose 1,5-diphosphate. Since 6-phosphogluconate is unique to the oxidative cycle and inhibits at concentrations comparable to those found in vivo, it is proposed that its inhibition of the carboxylase may be a regulatory factor. If so, it would operate during darkness as a different control factor from those factors postulated to activate the carboxylase during photosynthesis.

Photosynthetic (14)CO(2) fixation and the accumulation of photosynthetic products and the response of each process to both 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) and ascorbate were investigated in the intact spinach chloroplast.Ascorbate increased the rate of CO(2) uptake with an increase in all photosynthetic products, but, proportionally, there was a much larger increase in glycolate formation. CO(2) fixation inhibited by DCMU was partially restored on addition of ascorbate. Under conditions not optimal for glycolate formation, such as saturating levels of CO(2) and an anaerobic atmosphere, ascorbate in the presence of DCMU restored the formation of all photosynthetic products excluding glycolate. This effect of ascorbate on glycolate synthesis in the presence of DCMU was diminished under conditions which favored glycolate formation. Externally added glycerate 3-phosphate and fructose 1,6-diphosphate depressed the appearance of radioactivity in glycolate.The data are interpreted to indicate that glycolate is produced during photosynthesis as a result of a reaction between a 2-carbon piece derived from a sugar phosphate and an oxidant generated by the photochemical act. The oxidant may be an intermediate of photosystem 2 or a peroxide generated by a mechanism of the Mehler type involving molecular oxygen.

The uptake of P 32 and its incorporation into phosphorylated compounds was examined in the roots of barley seedlings which had been pretreated with aluminum. The rate at which phosphorus increased in Al-roots was greater than in controls, especially during the first 15 minutes of incubation. It was shown that the increased phosphorus in Al-roots was P i and that it was almost completely exchangeable. Similar increases over controls were found when root segments were incubated in phosphorus solutions containing 10 −3 m DNP and at low temperature. The increased P i in Al-roots did not result in an increase in the total amount of phosphorus incorporated into phosphorylated compounds. Aluminum treatment markedly decreased the incorporation of P 32 into sugar phosphates but increased the pool size of ATP and other nucleotide triphosphates present in the roots. The specific activities of P 32 in ATP in Al-roots and controls were similar indicating that the rates of ATP synthesis were similar in each case. Preliminary investigations showed that aluminum citrate inhibited both purified yeast hexokinase and phosphorylated sugar production by crude mitochondrial extracts from barley roots. The results suggest that there are 2 reactions between aluminum and phosphorus: 1) at the cell surface or in the free space which results in the fixation of phosphate by an adsorption-precipitation reaction; 2) within the cell, possibly within the mitochondria, which results in a marked decrease in the rate of sugar phosphorylation, probably effected by the inhibition of hexokinase. The evidence does not support the view that aluminum enhances phosphorus uptake or that the superficial reaction between aluminum and phosphate interferes with phosphorus transport.

Sugar uptake by sugarcane cells in suspension culture was measured over short incubation time spans (5 seconds to 4 minutes), and membrane transport rates were calculated. A relatively high proportion of labeled products in cell extracts after incubation of cells with (14)C-glucose for 5 seconds was sugar phosphates (56%); fructose and sucrose began to appear after 15 and 30 seconds, respectively. Galactose and 3-O-methylglucose competed appreciably with glucose uptake, but ketohexoses and pentoses did not; there was no detectable uptake of sucrose. It is postulated that besides endogenous phosphorylation and further metabolism of glucose the configuration of the hydroxyl on the carbon-2 may be important for efficient membrane transport. The cells had a particularly high affinity for glucose and 3-O-methylglucose (Km = 15 and 16 mum, respectively).