Computer, Simulated Evaluation of Possible Mechanisms for Sequestering Metal Ion Activity in Plant Vacuoles: II. Zinc

Various mechanisms have been suggested for sequestering Zn ion activity in vacuoles of Zn-tolerant plants. One of these mechanisms, complexation in the vacuole with organic acids, has received some support in the recent literature. However, the lack of experimental evidence for anticipated vacuolar compartmentation and concerning the nature of metal-ligand species occurring in the vacuole has been criticized. In this study we have used computer modeling of chemical equilibria to predict the metalligand species in vacuoles of tobacco (Nicotiana tabacum) cultured cells. Results of this thermodynamic evaluation support the conclusion that citrate in the concentration range encountered in tobacco cultured cells exposed to 300 or 2000 μm Zn has high potential for forming soluble complexes with Zn, over the entire probable range of vacuolar pH 4 to 7. Complexation of Zn with oxalate is also predicted, especially in cells exposed to high Zn levels. Malate, though the most abundant acid present, showed little potential for competing with other ligands for Zn. Overall, results suggest that vacuolar sequestration of Zn by high levels of vacuolar citrate may be a central mechanism in the accumulation of Zn in plants exposed to either low or high levels of this metal.     

Dependence of photosynthesis of sunflower and maize leaves on phosphate supply, ribulose-1,5-bisphosphate carboxylase/oxygenase activity, and ribulose-1,5-bisphosphate pool size

Sunflower (Helianthus annuus L. cv Asmer) and maize (Zea mays L. cv Eta) plants were grown under controlled environmental conditions with a nutrient solution containing 0, 0.5, or 10 millimolar inorganic phosphate. Phosphate-deficient leaves had lower photosynthetic rates at ambient and saturating CO₂ and much smaller carboxylation efficiencies than those of plants grown with ample phosphate. In addition, phosphate-deficient leaves contained smaller quantities of total soluble proteins and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) per unit area, although the relative proportions of these components remained unchanged. The specific activity of Rubisco (estimated in the crude extracts of leaves) was significantly reduced by phosphate deficiency in sunflower but not in maize. Thus, there was a strong dependence of carboxylation efficiency and CO₂-saturated photosynthetic rate on Rubisco activity only in sunflower. Phosphate deficiency decreased the 3-phosphoglycerate and ribulose-1,5-bisphosphate (RuBP) contents of the leaf in both species. The ratio of 3-phosphoglycerate to RuBP decreased in sunflower but increased in maize with phosphate deficiency. The calculated concentrations of RuBP and RuBP-binding sites in the chloroplast stroma decreased markedly with phosphate deficiency. The ratio of the stromal concentration of RuBP to that of RuBP-binding sites decreased in sunflower but was not affected in maize with phosphate deficiency. We suggest that a decrease in this ratio made the RuBP-binding sites more vulnerable to blockage or inactivation by tight-binding metabolites/inhibitors, causing a decrease in the initial specific activity of Rubisco in the crude extract from phosphate-deficient sunflower leaves. However, the decrease in Rubisco specific activity was much less than the decrease in the RuBP content in the leaf and its concentration in the stroma. A large ratio of RuBP to RuBP-binding sites may have maintained the Rubisco-specific activity in phosphate-deficient maize leaves. We conclude that the effect of phosphate deficiency is more on RuBP regeneration than on Rubisco activity in both sunflower and maize.     

Ethrel (Ethylene Releaser)-Induced Increases in the Adenylate Pool and Transtonoplast DeltapH within Hevea Latex Cells

The treatment of rubber tree (Hevea brasiliensis) bark with chloro-2-ethyl phosphonic acid (ethrel), an ethylene-releasing chemical, induced, after a lag period of 13 to 21 hours, a marked increase in the total adenine nucleotides (essentially ATP and ADP) of latex cells. This rise in the latex adenylate pool was concomitant with a marked decrease in the [ATP]/[ADP] ratio without significant changes in the adenylate energy charge. The apparent equilibrium constant for the adenylate kinase, which appeared to behave as a key enzyme in maintaining the adenylate energy charge in the latex, was considerably reduced, probably as a consequence of the alkalinization of the latex cytosol induced by the treatment with ethrel. To reduce the “sink effect” and activation of the metabolism induced in Hevea bark by regular tapping, the latex was collected by micropuncture (few drops) at increasing distance (5-50 centimeters) above and below an ethrel-treated area on the virgin bark of resting trees. The effect of ethrel was shown to spread progressively along the trunk. The increase in the adenylate pool (essentially ATP) was detectable as early as 24 hours after the bark treatment and was maximum after 6 or 8 days, 5 centimeters as well as 50 centimeters above and below the stimulated bark ring. The correlative vacuolar acidification and cytosolic alkalinization, i.e. the increase in the transtonoplast ΔpH, induced in the latex cells by ethrel were shown to be concomitant with the rise in ATP content of the latex. This suggests that the tonoplast H⁺-pumping ATPase, which catalyzes vacuolar acidification in the latex, is directly and essentially under the control of the availability of its substrate (i.e. ATP) in the latex. The results are discussed in relation to energy-dependent activation of metabolism, and increased rubber production, as induced by the stimulation of rubber trees with ethrel.     

Isolation and characterization of two safflower oleoyl-acyl carrier protein thioesterase cDNA clones

Oleoyl-acyl carrier protein (18:1-ACP) thioesterase has been partially purified from developing safflower (Carthamus tinctorius) seeds. Protein species with molecular masses of 34 and 40 kD associated with thioesterase activity were identified and partially sequenced. Analysis of amino-terminal and internal cyanogen bromide peptide sequences revealed no differences in the primary structure of the two species. Amino acid sequence was used to design degenerate oligonucleotides for primers in a polymerase chain reaction (PCR) using safflower embryo cDNA as a template. A 380-base pair PCR product was used to isolate two classes of cDNA clones, designated 2-1 and 5-2, from the embryo cDNA library. Clone 2-1 encodes a 389-amino acid protein including a 60-amino acid transit peptide, and contains all of the protein sequence determined from the 34- and 40-kD proteins. Clone 5-2 encodes a 385-amino acid protein with 80% identity to that encoded by 2-1. Expression of the two safflower cDNA clones in Escherichia coli resulted in a 50- to 100-fold increase in the level of 18:1-ACP thioesterase activity. Both thioesterases are most active on 18:1-ACP; however, the enzyme encoded by 5-2 shows less discrimination against saturated 16- and 18-carbon acyl-ACP substrates.     

The effect of heat shock on morphogenesis in barley : coordinated circadian regulation of mRNA levels for light-regulated genes and of the capacity for accumulation of chlorophyll protein complexes

The effect of daily heat-shock treatments on gene expression and morphogenesis of etiolated barley (Hordeum vulgare) was investigated. Heat-shock treatments in the dark induced shortening of the primary leaves and the coleoptiles to the length of those in light-grown plantlets. In addition, the mRNA levels of the light-induced genes that were investigated were raised under these conditions and showed distinct oscillations over a period of at least 3 d. While the mRNA levels for chlorophyll a/b binding protein (LHC II), plastocyanin, and the small subunit of ribulose-1,5-bisphosphate carboxylase had maxima between 8 and 12 pm (12-16h after the last heat-shock treatment), the mRNA levels for thionin oscillated with a phase opposed to that of LHC II. Etiolated barley, the circadian oscillator of which was synchronized by cyclic heatshock treatments, was illuminated for a constant interval at different times of the day; this led to the finding that greening was fastest at the time when the maximal levels of mRNA for LHC II were also observed. Whereas accumulation of chlorophyll a during a 4-h period of illumination oscillated by a factor of 3, chlorophyll b accumulation changed 10- to 15-fold. Similarly, accumulation of LHC II was highest when pigments accumulated maximally. Hence, greening or, in other words, thylakoid membrane assembly is under control of the circadian oscillator.     

Leaf Photosynthesis and Respiration of High CO(2)-Grown Tobacco Plants Selected for Survival under CO(2) Compensation Point Conditions

Four self-pollinated, doubled-haploid tobacco, (Nicotiana tabacum L.) lines (SP422, SP432, SP435, and SP451), selected as haploids by survival in a low CO₂ atmosphere, and the parental cv Wisconsin-38 were grown from seed in a growth room kept at high CO₂ levels (600-700 parts per million). The selected plants were much larger (especially SP422, SP432, and SP451) than Wisconsin-38 nine weeks after planting. The specific leaf dry weight and the carbon (but not nitrogen and sulfur) content per unit area were also higher in the selected plants. However, the chlorophyll, carotenoid, and alkaloid contents and the chlorophyll a/b ratio varied little. The net CO₂ assimilation rate per unit area measured in the growth room at high CO₂ was not higher in the selected plants. The CO₂ assimilation rate versus intercellular CO₂ curve and the CO₂ compensation point showed no substantial differences among the different lines, even though these plants were selected for survival under CO₂ compensation point conditions. Adult leaf respiration rates were similar when expressed per unit area but were lower in the selected lines when expressed per unit dry weight. Leaf respiration rates were negatively correlated with specific leaf dry weight and with the carbon content per unit area and were positively correlated with nitrogen and sulfur content of the dry matter. The alternative pathway was not involved in respiration in the dark in these leaves. The better carbon economy of tobacco lines selected for low CO₂ survival was not apparently related to an improvement of photosynthesis rate but could be related, at least partially, to a significantly reduced respiration (mainly cytochrome pathway) rate per unit carbon.     

Radial Turgor and Osmotic Pressure Profiles in Intact and Excised Roots of Aster tripolium: Pressure Probe Measurements and Nuclear Magnetic Resonance-Imaging Analysis

High-resolution nuclear magnetic resonance images (using very short spin-echo times of 3.8 milliseconds) of cross-sections of excised roots of the halophyte Aster tripolium showed radial cell strands separated by air-filled spaces. Radial insertion of the pressure probe (along the cell strands) into roots of intact plants revealed a marked increase of the turgor pressure from the outermost to the sixth cortical layer (from about 0.1-0.6 megapascals). Corresponding measurements of intracellular osmotic pressure in individual cortical cells (by means of a nanoliter osmometer) showed an osmotic pressure gradient of equal magnitude to the turgor pressure. Neither gradient changed significantly when the plants were grown in, or exposed for 1 hour to, media of high salinity. Differences were recorded in the ability of salts and nonelectrolytes to penetrate the apoplast in the root. The reflection coefficients of the cortical cells were approximately 1 for all the solutes tested. Excision of the root from the stem resulted in a collapse of the turgor and osmotic pressure gradients. After about 15 to 30 minutes, the turgor pressure throughout the cortex attained an intermediate (quasistationary) level of about 0.3 megapascals. This value agreed well with the osmotic value deduced from plasmolysis experiments on excised root segments. These and other data provided conclusions about the driving forces for water and solute transport in the roots and about the function of the air-filled radial spaces in water transport. They also showed that excised roots may be artifactual systems.     

Chloroplast Dedifferentiation in Mechanically Isolated Asparagus Cells during Culture Initiation

Mechanically isolated asparagus (Asparagus officinalis) mesophyll cells dedifferentiate and divide when cultured in the dark in a medium containing sucrose. A strong correlation was observed between the onset of cell division and a loss of photosynthetic capacity. For the first 8 to 9 d of culture, there was no change in chloroplast size or morphology. However, following this period, the chloroplasts divided to form smaller proplastid-like structures. The gross chlorophyll content of the cell population did not change, suggesting that the loss of photosynthetic potential was not by senescence. Northern analysis showed that mRNA of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase was undetectable within 1 d postisolation, which was quicker than in dark-treated plants. The mRNA of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase decreased to low levels within 2 d of cell isolation. Both the large and small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase protein showed a gradual reduction in abundance, falling to basal levels by days 6 to 7, which coincided with the onset of rapid cell division. A similar trend was observed with chloroplast rRNA molecules, which decreased to basal levels by day 6 in culture.     

Intracellular Localization of Three l-Glutamate Dehydrogenase Isozymes from Chlamydomonas reinhardtii

The intracellular localization of the activity and synthesis of three isozymes of NAD(P)⁺-glutamate dehydrogenase from the unicellular green alga Chlamydomonas reinhardtii cw-92 has been established. Isozyme activities have been located within mitochondria by using differential centrifugation techniques and discontinuous Percoll gradient separations. Experiments with protein synthesis inhibitors cycloheximide, rifampicin, chloramphenicol, and actinomycin D, under dark and carbon starvation conditions, revealed that synthesis of the three isozymes was likely to occur in cytosol as precursor proteins that are then transported and processed inside the mitochondria.     

Purification and characterization of cytosolic 6-phosphogluconate dehydrogenase isozymes from maize

Cytosolic isozymes of 6-phosphogluconate dehydrogenase were purified from roots of maize (Zea mays L.). The final preparation contained two 55-kD proteins. Affinity-purified dehydrogenases from a maize line that is null for both cytosolic 6-phosphogluconate dehydrogenase isozymes (Pgd1-null, Pgd2-null) lacked the 55-kD proteins. The substrate kinetics of the purified enzyme were determined.