Water stress decreases the transfer conductance of Douglas-fir (Pseudotsuga menziesii) seedlings.

We tested the hypothesis that transfer conductance (gi) of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings is reduced by water stress. Seedlings were irrigated with a solution of 25% polyethylene glycol so as to impose water stress rapidly, thereby limiting acclimatory responses. Transfer conductance was measured pre-treatment and post-treatment by two methods. Water stress reduced net photosynthesis by 20-50%. The initial slope of the rate of photosynthesis (A) over the intercellular carbon dioxide (CO2) concentration (Ci) response was reduced by water stress, indicating that reduced photosynthesis was not wholly accounted for by reduced stomatal conductance. The carbon isotope and chlorophyll fluorescence methods both indicated that water stress decreased gi. From isotopic measurements with 1% O2, gi was 0.076 +/- 0.009 (mean +/- SE) mol m(-2) s(-1) in well-watered seedlings and 0.044 +/- 0.004 mol m(-2) s(-1) in water-stressed seedlings. Fluorescence estimates of gi were 0.08 +/- 0.01 mol m(-2) s(-1) in well-watered seedlings and 0.044 +/- 0.004 mol m(-2) s(-1) in water-stressed seedlings. The drought-induced reduction in gi was responsible for the reduction in slope of the A/Ci response, and thus there was no difference in the slope of the A over the chloroplastic CO2 concentration (Cc) response between treatments and no indication of impaired mesophyll metabolism. These data illustrate that impairments of mesophyll metabolism can be revealed only from analysis of the A/Cc response.

Photosynthetic responses and N allocation in Douglas-fir needles following a brief pulse of nutrients.

The temporal distribution of soil nutrients is heterogeneous, and thus the uptake, storage and later remobilization of brief nutrient pulses may be critical for growth in nutrient-limited habitats. We investigated the response of photosynthesis and the major nitrogen (N) fractions in needles of 2-year-old Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings to a 15-day nutrient pulse (containing 250 ppm N). The nutrient pulse (N pulse) was imposed in late July, toward the end of the seedlings' third growing season, and subsequent changes in photosynthesis and needle N fractions were examined over the following 3 months. Needles are sites of photosynthesis and putative storage organs. Thus we tested two hypotheses: (1) N from the N pulse is quickly synthesized from soluble non-protein N into soluble proteins, especially Rubisco, and (2) the N pulse increases photosynthetic rates and thus growth. We also examined an alternative hypothesis that Rubisco functions also as a storage protein, in which case we would predict increases in amount of Rubisco in response to the N pulse without concomitant increases in photosynthesis. Soluble non-protein N was the most dynamic N pool and may have constituted a temporary storage reservoir; however, the quantitative significance of soluble non-protein N is questionable because this pool was at most only 7% of total N. Concentrations of Rubisco were unaffected by the N-pulse treatment and there was little evidence that Rubisco served as a storage protein. Nutrient-pulse seedlings added twice as much dry mass as controls during the 3 months post-treatment (Warren et al. 2003a). Over the same period, the maximum rate of light-saturated photosynthesis (A(max)) declined to low rates in control seedlings, whereas A(max) increased in N-pulse seedlings. Nevertheless, treatment and temporal trends in N and Rubisco content per unit area were poorly related to A(max), and it seems likely that photosynthesis was limited by additional factors, perhaps thylakoid proteins or an inadequate supply of other nutrients.

Response of Douglas-fir seedlings to a brief pulse of 15N-labeled nutrients.

The temporal distribution of soil nutrients is heterogeneous, and thus the uptake, storage and later remobilization of brief nutrient pulses may be critical for growth in nutrient-limited habitats. We investigated the response of 2-year-old Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings receiving a low nutrient supply to a 15-day nutrient pulse (containing 250 ppm nitrogen (N) as 10 atom % 15NH4 15NO3). The nutrient pulse was imposed in late July, toward the end of the seedlings' third growing season, and subsequent changes in dry mass and N content over the following 3 months were determined from destructive harvests. We tested three hypotheses: (1) N from the nutrient pulse is rapidly assimilated and accumulated primarily in needles and roots; (2) this accumulated N is later remobilized to support new growth; and (3) the nutrient pulse leads to a larger second flush of shoot growth. Seedlings increased their N content by 175 mg (67%) in response to the nutrient pulse. Nitrogen was taken up preferentially into younger tissues, especially the secondary flush and current-year roots. Immediately after the nutrient pulse, tissue N concentrations were high and supported subsequent increases in dry mass. Over 3 months, seedlings receiving the nutrient pulse added twice as much dry mass as control seedlings, and even after 3 months of growth, N concentrations remained greater than in controls. Current-year and older needles were the only components whose dry mass did not increase over this period. The nutrient pulse increased the size of the second flush, but it was still a minor component of increments in dry mass (approximately 10% of the total dry mass increment) and N content (23%). The relatively modest increases in N content during autumn could be accounted for by soil uptake and there was no evidence that N was remobilized to support growth of new tissues. Short-term (15 days) elevated N uptake led to sustained growth in the long term (> 3 months), and thus growth rate was to a large extent decoupled from current nutrient supply.

Responses of gas exchange to reversible changes in whole-plant transpiration rate in two conifer species.

This study examined the autonomy of branches with respect to the control of transpiration (E) in Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) and western red cedar (Thuja plicata Donn) seedlings. Experiments were conducted on whole seedlings in a gas exchange system with a dual-cuvette that permitted independent manipulation and measurement of E in the upper and lower cuvettes. The value of E in one cuvette was manipulated by varying vapor pressure deficit (D) between 2.2 and 0.2 kPa, whereas D in the other cuvette was held at 2.2 kPa. Reducing D, while increasing stomatal conductance (gs), resulted in an overall decrease in E. In western red cedar, this decrease was almost threefold, and in Douglas-fir, approximately fourfold. In well-watered western red cedar, a reduction of whole-plant E by 46% (brought about by reducing D in the upper cuvette) resulted in a 12% increase in gs, a 12% increase in E and a 7% increase in net assimilation (A) of untreated foliage in the lower cuvette. Responses of gs, E and A of untreated foliage were similar irrespective of whether foliage was at the top or bottom of the seedling. When D in the treatment cuvette was restored to 2.2 kPa, gs, E and A of foliage in the untreated cuvette returned to pretreatment values. In contrast, in well-watered Douglas-fir, there was almost no change in gs, E or A of untreated foliage in one cuvette when D in the other cuvette was reduced, causing a 52% reduction in whole-plant E. However, similar manipulations on drought-stressed Douglas-fir led to 7-19% increases in gs, E and A of untreated foliage. In well-watered western red cedar, daytime leaf water potential (Psil) was maintained near -0.9 MPa over a wide range of D, whereas Psil of Douglas-fir decreased from -1.2 to -1.5 MPa as D increased. The tighter (isohydric) regulation of Psil in western red cedar may partly explain its greater stomatal response to D and variation in whole-plant E compared with Douglas-fir. In response to a reduction in E, measured increases in Psil and gs of unmanipulated foliage were less than predicted by a model assuming complete hydraulic connectivity of foliage. Our results suggest the foliage of both species is partially autonomous with respect to water.

Whole-plant gas exchange and reductive biosynthesis in white lupin.

Simultaneous measurements of CO(2) (CER) and O(2) (OER) exchange in roots and shoots of vegetative white lupin (Lupinus albus) were used to calculate the flow of reducing power to the synthesis of biomass that was more reduced per unit of carbon than carbohydrate. On a whole-plant basis, the diverted reductant utilization rate (DRUR which is: 4 x [CER + OER]) of shoot tissue was consistently higher than that of roots, and values obtained in the light were greater than those in the dark. An analysis of the biomass being synthesized over a 24-h period provided an estimate of whole-plant DRUR (3.5 mmol e(-) plant(-1) d(-1)), which was similar to that measured by gas exchange (3.2 mmol e(-) plant(-1) d(-1)). Given that nitrate reduction to ammonia makes up about 74% of whole-plant DRUR, root nitrate reduction in white lupin was estimated to account for less than 43% of whole-plant nitrate reduction. The approach developed here should offer a powerful tool for the noninvasive study of metabolic regulation in intact plants or plant organs.

Two unrelated phosphoenolpyruvate carboxylase polypeptides physically interact in the high molecular mass isoforms of this enzyme in the unicellular green alga Selenastrum minutum.

In the chlorophyte Selenastrum minutum, phosphoenolpyruvate carboxylase (PEPC) exists as two kinetically distinct classes of isoforms sharing the same 102-kDa catalytic subunit (p102). Class 1 PEPC is homotetrameric, whereas Class 2 PEPCs consist of three large protein complexes. The different Class 2 PEPCs contain p102 and 130-, 73-, and 65-kDa polypeptides in different stoichiometric combinations. Immunoblot, immunoprecipitation, and chemical cross-linking studies indicated that p102 physically interacts with the 130-kDa polypeptide (p130) in Class 2 PEPCs. Immunological data and mass spectrometric and sequence analyses revealed that p102 and p130 are not closely related even if a p130 tryptic peptide had significant similarity to a conserved PEPC C-terminal domain from several sources. Evidence supporting the hypothesis that p130 has PEPC activity includes the following. (i) Specific activity expressed relative to the amount of p102 was lower in Class 1 than in Class 2 PEPCs; (ii) reductive pyridoxylation of both p102 and p130 was inhibited by magnesium-phosphoenolpyruvate; and (iii) biphasic phosphoenolpyruvate binding kinetics were observed with Class 2 PEPCs. These data support the view that unicellular green algae uniquely express, regulate, and assemble divergent PEPC polypeptides. This probably serves an adaptive purpose by poising these organisms for survival in different environments varying in nutrient content.

Purification and characterization of high- and low-molecular-mass isoforms of phosphoenolpyruvate carboxylase from Chlamydomonas reinhardtii. Kinetic, structural and immunological evidence that the green algal enzyme is distinct from the prokaryotic and higher plant enzymes.

Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme in the supply of carbon skeletons for the assimilation of nitrogen by green algae. Two PEPC isoforms with respective native molecular masses of 400 (PEPC1) and 650 (PEPC2) kDa have been purified from Chlamydomonas reinhardtii CW-15 cc1883 (Chlorophyceae). SDS/PAGE, immunoblot and CNBr peptide-mapping analyses indicate the presence of the same 100 kDa PEPC catalytic subunit in both isoforms. PEPC1 is a homotetramer, whereas PEPC2 seems to be a complex between the PEPC catalytic subunit and other immunologically unrelated polypeptides of 50-70 kDa. Kinetic analyses indicate that these PEPC isoforms are (1) differentially regulated by pH, (2) activated by glutamine and dihydroxyacetone phosphate and (3) inhibited by glutamate, aspartate, 2-oxoglutarate and malate. These results are consistent with the current model for the regulation of anaplerotic carbon fixation in green algae, and demonstrate that green algal PEPCs are uniquely regulated by glutamine. Several techniques were used to assess the structural relationships between C. reinhardtii PEPC and the higher plant or prokaryotic enzyme. Immunoblot studies using anti-(green algal or higher plant PEPC) IgGs suggested that green algal (C. reinhardtii, Selenastrum minutum), higher plant (maize, banana fruit, tobacco) and prokaryotic (Synechococcus leopoliensis, Escherichia coli) PEPCs have little or no immunological relatedness. Moreover, the N-terminal amino acid sequence of the C. reinhardtii PEPC subunit did not have significant similarity to the highly conserved corresponding region in enzymes from higher plants, and CNBr cleavage patterns of green algal PEPCs were distinct from those of higher plant and cyanobacterial PEPCs. These results point to significant evolutionary divergence between green algal, higher plant and prokaryotic PEPCs.

In Vivo and in Vitro Studies of Glucose-6-Phosphate Dehydrogenase from Barley Root Plastids in Relation to Reductant Supply for NO2- Assimilation.

Pyridine nucleotide pools were measured in intact plastids from roots of barley (Hordeum vulgare L.) during the onset of NO2- assimilation and compared with the in vitro effect of the NADPH/NADP ratio on the activity of plastidic glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) from N-sufficient or N-starved roots. The NADPH/NADP ratio increased from 0.9 to 2.0 when 10 mM glucose-6-phosphate was supplied to intact plastids. The subsequent addition of 1 mM NaNO2 caused a rapid decline in this ratio to 1.5. In vitro, a ratio of 1.5 inactivated barley root plastid G6PDH by approximately 50%, suggesting that G6PDH could remain active during NO2- assimilation even at the high NADPH/NADP ratios that would favor a reduction of ferredoxin, the electron donor of NO2- reductase. Root plastid G6PDH was sensitive to reductive inhibition by dithiothreitol (DTT), but even at 50 mM DTT the enzyme remained more than 35% active. In root plastids from barley starved of N for 3 d, G6PDH had a substantially reduced specific activity, had a lower Km for NADP, and was less inhibited by DTT than the enzyme from N-sufficient root plastids, indicating that there was some effect of N starvation on the G6PDH activity in barley root plastids.

Interactions between Photosynthesis and Respiration in the Green Alga Chlamydomonas reinhardtii (Characterization of Light-Enhanced Dark Respiration).

The rate of respiratory O2 consumption by Chlamydomonas reinhardtii cell suspensions was greater after a period of photosynthesis than in the preceding dark period. This "light-enhanced dark respiration" (LEDR) was a function of both the duration of illumination and the photon fluence rate. Mass spectrometric measurements of gas exchange indicated that the rate of gross respiratory O2 consumption increased during photosynthesis, whereas gross respiratory CO2 production decreased in a photon fluence rate-dependent manner. The rate of postillumination O2 consumption provided a good measure of the O2 consumption rate in the light. LEDR was substantially decreased by the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea or glycolaldehyde, suggesting that LEDR was photosynthesis-dependent. The onset of photosynthesis resulted in an increase in the cellular levels of phosphoglycerate, malate, and phosphoenolpyruvate, and a decrease in whole-cell ATP and citrate levels; all of these changes were rapidly reversed upon darkening. These results are consistent with a decrease in the rate of respiratory carbon flow during photosynthesis, whereas the increase in respiratory O2 consumption during photosynthesis may be mediated by the export of photogenerated reductant from the chloroplast. We suggest that photosynthesis interacts with respiration at more than one level, simultaneously decreasing the rate of respiratory carbon flow while increasing the rate of respiratory O2 consumption.

Purification and properties of four phosphoenolpyruvate carboxylase isoforms from the green alga Selenastrum minutum: evidence that association of the 102-kDa catalytic subunit with unrelated polypeptides may modify the physical and kinetic properties of the enzyme.

Four isoforms of phosphoenolpyruvate carboxylase (PEPC1, PEPC2, PEPC3, PEPC4) have been purified from the green alga Selenastrum minutum. PEPC1 is a homotetramer with a subunit M(r) of 102 kDa. PEPC2, PEPC3, and PEPC4 have respective native M(r)S of approximately 984, 1186, and 1590 kDa. SDS/PAGE analysis revealed that the latter three isoforms contain polypeptides having M(r)S of 102, 73, 70, 65, and 61 kDa. Immunoblot analyses and CNBr cleavage patterns suggest that the 102-kDa polypeptide present in all four isoforms is the same PEPC catalytic subunit. Our data suggest that the three high M(r)S PEPC isoforms are heteromeric protein complexes consisting of the 102-kDa PEPC1 catalytic subunit and immunologically unrelated polypeptides. Attempts to measure other enzyme activities associated with the protein complexes gave negative results. However, PEPC1 had immunological, physical, and kinetic properties very different from those of the larger M(r) PEPC isoforms: (i) the anti-PEPC1 immune-serum was relatively inefficient for immunoprecipitating PEPC2, PEPC3, or PEPC4; (ii) immune-serum raised against a mixture of PEPC2, PEPC3, and PEPC4 had relatively weak immunoprecipitating activity toward PEPC1; (iii) PEPC1 was more heat sensitive than the other three isoforms; (iv) PEPC1 had a pH optimum of 9 versus 8.5 for the PEPC protein complexes; (v) the high Mr PEPCs had greater apparent affinity for phosphoenolpyruvate compared to PEPC1 and (vi) PEPC1 activity was far more sensitive to metabolite activators (Gln and dihydroxyacetone phosphate) and inhibitors (Asp, Glu, 2-oxoglutarate and malate). We conclude that the interaction of the PEPC catalytic subunit with unrelated polypeptides is responsible for the observed differences between PEPC1 and the high molecular mass isoforms. We propose that this interaction possibly regulates PEPC activity in vivo.

Appearance of Novel Glucose-6-Phosphate Dehydrogenase Isoforms in Chlamydomonas reinhardtii during Growth on Nitrate.

Extractable glucose-6-phosphate dehydrogenase activity is higher from N-limited Chlamydomonas reinhardtii cells than from N-sufficient cells. Native gels reveal that the isoform complexity varies depending on the form of N supplied. The isoforms associated with NO3- growth appear within 2 h of switching cells from NH4+ to NO3-.

Immediate Activation of Respiration in Petroselinum crispum L. in Response to the Phytophthora megasperma f. sp. Glycinea Elicitor.

Treatment of parsley (Petroselinum crispum L.) cell cultures with the Phytophthora megasperma elicitor isolated from the fungus Phytophthora megasperma f. sp. Glycinea caused an immediate increase in the rate of respiratory CO2 evolution in the dark. The respiratory response was biphasic, showing a rapid enhancement in the first 20 min and then a slower increase until a steady rate was attained 60 min posttreatment. The enhanced rate of CO2 evolution corresponded to the activation of phosphofructokinase and glucose-6-phosphate dehydrogenase, key enzymes in the regulation of carbohydrate flow to glycolysis and the oxidative pentose phosphate (OPP) pathway, respectively. The increased rate of CO2 evolution and the activation of phosphofructokinase and glucose-6-phosphate dehydrogenase were maintained for the duration of the experiments, indicating long-term stimulation of respiration through both glycolysis and the OPP pathway. A 23% decrease in the C6:C1 ratio of 14CO2 evolution from labeled glucose 60 min after the addition of Phytophthora megasperma elicitor is consistent with an increased contribution of the OPP pathway to cellular respiration. Long-term activation of the OPP pathway following elicitation could serve to maintain the pools of substrates necessary during activation of the shikimic acid pathway, leading to the production of defensive compounds.

Coordination of Chloroplastic Metabolism in N-Limited Chlamydomonas reinhardtii by Redox Modulation (I. The Activation of Phosphoribulosekinase and Glucose-6-Phosphate Dehydrogenase Is Relative to the Photosynthetic Supply of Electrons).

Extraction of Chlamydomonas reinhardtii CW-15 cells by rapid freezing and thawing demonstrates that the in vivo activity of the algal glucose-6-phosphate dehydrogenase (G6PDH) is inhibited by the presence of light and activated in the dark, whereas phosphoribulosekinase (PRK) is light activated and inhibited in the dark. The effects of darkening are reversed by incubation with dithiothreitol (DTT) and mimicked by chemical oxidants, indicating that, as in higher plants, reduction via the ferredoxin-thioredoxin system likely regulates these enzymes. The two enzymes varied in their sensitivity to reduction; the inclusion of 0.5 mM DTT during extraction inhibited G6PDH, whereas PRK required treatment with 40 mM DTT for 1 h to reach maximum activation. The activation change for both enzymes was nearly complete within the 1st min after cells were transferred between light and dark, but the level of activation was relative to the incident light at low intensities; G6PDH activity decreased with increasing light, whereas PRK became more active. The reductive inhibition of G6PDH saturated at very low light, whereas PRK activation kinetics closely followed the increase in photosynthetic oxygen evolution. These results indicate that light-driven redox modulation of G6PDH and PRK is more than an on/off switch, but acts to optimize the reduction and oxidation of carbon in the chloroplast in accordance with the supply of electrons.

Coordination of Chloroplastic Metabolism in N-Limited Chlamydomonas reinhardtii by Redox Modulation (II. Redox Modulation Activates the Oxidative Pentose Phosphate Pathway during Photosynthetic Nitrate Assimilation).

The onset of photosynthetic NO3- assimilation in N-limited Chlamydomonas reinhardtii increased the initial extractable activity of the glucose-6-phosphate dehydrogenase (G6PDH), the key regulatory step of the oxidative pentose phosphate pathway. The total activated enzyme activity did not change upon NO3- resupply. The higher activity, therefore, represents activation of existing enzyme. No activation occurred during NH4+ assimilation. Incubation of extracts with DTT reversed the NO3- stimulation of G6PDH activity, indicating that the activation involved redox modulation of G6PDH. Phosphoribulosekinase, an enzyme activated by thioredoxin reduction, was inhibited at the onset of NO3- assimilation. A 2-fold stimulation of O2 evolution and a 70% decrease in the rate of photosynthetic CO2 assimilation accompanied the enzyme activity changes. There was an immediate drop in the NADPH and an increase in NADP upon addition of NO3-, whereas NH4+ caused only minor fluctuations in these pools. The response of C. reinhardtii to NO3- indicates that the oxidative pentose phosphate pathway was activated to oxidize carbon upon the onset of NO3- assimilation, whereas reduction of carbon via the reductive pentose phosphate pathway was inhibited. This demonstrates a possible role for the Fd-thioredoxin system in coordinating enzyme activity in response to the metabolic demands for reducing power and carbon during NO3- assimilation.

Inorganic Phosphate (Pi) Enhancement of Dark Respiration in the Pi-Limited Green Alga Selenastrum minutum (Interactions between H+/Pi Cotransport, the Plasmalemma H+-ATPase, and Dark Respiratory Carbon Flow).

Inorganic phosphate (Pi) enrichment of the Pi-limited green alga Selenastrum minutum in the dark caused a 2.5-fold increase in the rate of O2 consumption. Alkalization of the media during Pi assimilation was consistent with a H+/Pi cotransport mechanism with a stoichiometry of at least 2 H+ cotransported per Pi. Dark O2 consumption remained enhanced beyond the period of Pi assimilation and did not recover until the medium was reacidified. This result, coupled with an immediate decrease in adenylate energy charge following Pi enrichment, suggested that respiration is regulated by the ATP requirements of a plasmalemma H+-ATPase that is activated to maintain intracellular pH and provide proton motive force to power Pi uptake. Concentrations of tricarboxylic acid cycle intermediates decreased following Pi enrichment and respiratory CO2 efflux increased, indicating that the tricarboxylic acid cycle was activated to supply reductant to the mitochondrial electron transport chain. These results are consistent with direct inhibition of electron transport by ADP limitation. Enhanced rates of starch breakdown and increases in glycolytic metabolites indicated that respiratory carbon flow was activated to supply reductant to the electron transport chain and to rapidly assimilate Pi into metabolic intermediates. The mechanism that initiates glycolytic carbon flow could not be clearly identified by product:substrate ratios due to the complex nature of Pi assimilation. High levels of triose-P and low levels of phosphoenolpyruvate were the primary regulators of pyruvate kinase and phosphofructokinase, respectively.

Evidence for Activation of the Oxidative Pentose Phosphate Pathway during Photosynthetic Assimilation of NO(3) but Not NH(4) by a Green Alga.

Addition of NO(3) (-) to N-limited Selenastrum minutum during photosynthesis resulted in an immediate drop in the NADPH/NADP ratio and a slower increase of the NADH/NAD ratio. These changes were accompanied by a rapid decrease in glucose-6-phosphate and increase in 6-phosphogluconate, indicating activation of glucose-6-phosphate dehydrogenase and a role for the oxidation pentose phosphate pathway during photosynthetic NO(3) (-) assimilation. In contrast, the short-term changes in pyridine nucleotides and metabolites during photosynthetic assimilation of NH(4) (+) were not consistent with a stimulation of the oxidative pentose phosphate pathway.

Normal growth of transgenic tobacco plants in the absence of cytosolic pyruvate kinase.

The coding sequence of the cytosolic isozyme of potato tuber pyruvate kinase (PK) was attached to the transit peptide of the small subunit of pea ribulose-1,5-bisphosphate carboxylase oxygenase and placed under the control of the cauliflower mosaic virus 35S promoter. This construct was transformed into Nicotiana tabacum. Unexpectedly, two primary transformants were recovered in which PK activity in leaves was greatly reduced. The reduction in PK activity appeared to result from the complete absence of the cytosolic form of the enzyme (PK(c)). In addition, no PK(c) could be detected on western blots of leaf extracts. Metabolite analyses indicated that the levels of phosphoenolpyruvate are substantially higher in PK(c)-deficient leaves than in wild-type leaves, consistent with a block in glycolysis at the step catalyzed by PK. PK(c) deficiency in the leaves does not appear to adversely affect plant growth. Analysis of progeny indicates that PK(c) deficiency is a heritable trait. The leaves of PK(c)-deficient transformants have normal rates of photosynthetic O(2) evolution and respiratory O(2) consumption, indicating that these plants are using alternative pathways to bypass PK.

Activation of Respiration to Support Dark NO(3) and NH(4) Assimilation in the Green Alga Selenastrum minutum.

Short-term changes in pyridine nucleotides and other key metabolites were measured during the onset of NO(3) (-) or NH(4) (+) assimilation in the dark by the N-limited green alga Selenastrum minutum. When NH(4) (+) was added to N-limited cells, the NADH/NAD ratio rose immediately and the NADPH/NADP ratio followed more slowly. An immediate decrease in glutamate and 2-oxoglutarate indicates an increased flux through the glutamine synthase/glutamate oxoglutarate aminotransferase. Pyruvate kinase and phosphoenolpyruvate carboxylase are rapidly activated to supply carbon skeletons to the tricarboxylic acid cycle for amino acid synthesis. In contrast, NO(3) (-) addition caused an immediate decrease in the NADPH/NADP ratio that was accompanied by an increase in 6-phosphogluconate and decrease in the glucose-6-phosphate/6-phosphogluconate ratio. These changes show increased glucose-6-phosphate dehydrogenase activity, indicating that the oxidative pentose phosphate pathway supplies some reductant for NO(3) (-) assimilation in the dark. A lag of 30 to 60 seconds in the increase of the NADH/NAD ratio during NO(3) (-) assimilation correlates with a slow activation of pyruvate kinase and phosphoenolpyruvate carboxylase. Together, these results indicate that during NH(4) (+) assimilation, the demand for ATP and carbon skeletons to synthesize amino acid signals activation of respiratory carbon flow. In contrast, during NO(3) (-) assimilation, the initial demand on carbon respiration is for reductant and there is a lag before tricarboxylic acid cycle carbon flow is activated in response to the carbon demands of amino acid synthesis.

Malate- and pyruvate-dependent Fatty Acid synthesis in leucoplasts from developing castor endosperm.

Leucoplasts were isolated from the endosperm of developing castor (Ricinis communis) endosperm using a discontinuous Percoll gradient. The rate of fatty acid synthesis was highest when malate was the precursor, at 155 nanomoles acetyl-CoA equivalents per milligram protein per hour. Pyruvate and acetate also were precursors of fatty acid synthesis, but the rates were approximately 4.5 and 120 times less, respectively, than when malate was the precursor. When acetate was supplied to leucoplasts, exogenous ATP, NADH, and NADPH were required to obtain maximal rates of fatty acid synthesis. In contrast, the incorporation of malate and pyruvate into fatty acids did not require a supply of exogenous reductant. Further, the incorporation of radiolabel into fatty acids by leucoplasts supplied with radiolabeled malate, pyruvate, or acetate was reduced upon coincubation with cold pyruvate or malate. The data suggest that malate and pyruvate may be good in vivo sources of carbon for fatty acid synthesis and that, in these preparations, leucoplast fatty acid synthesis may be limited by activity at or downstream of the acetyl-CoA carboxylase reaction.