Biochemical Mechanism for Regulation of Sucrose Accumulation in Leaves during Photosynthesis

It is not known why some species accumulate high concentrations of sucrose in leaves during photosynthesis while others do not. To determine the possible basis, we have studied 10 species, known to differ in the accumulation of sucrose, in terms of activities of sucrose hydrolyzing enzymes. In general, acid invertase activity decreased as leaves expanded; however, activities remaining in mature, fully expanded leaves ranged from low (<10 micromoles per gram fresh weight per hour) to very high (>100 micromoles per gram fresh weight per hour). In contrast, sucrose synthase activities were low and relatively similar among the species (4-10 micromoles per gram fresh weight per hour). Importantly, leaf sucrose concentration, measured at midafternoon, was negatively correlated with acid invertase activity. We propose that sucrose accumulation in vacuoles of species such as soybean and tobacco is prevented by acid invertase-mediated hydrolysis. Initial attempts were made to characterize the relatively high activity of acid invertase from mature soybean leaves. Two apparent forms of the enzyme were resolved by Mono Q chromatography. The two forms had similar affinity for substrate (apparent K_m [sucrose] = 3 millimolar) and did not interconvert upon rechromatography. It appeared that the loss of whole leaf invertase activity during expansion was largely the result of changes in one of the enzyme forms. Overall, the results provide a mechanism to explain why some species do not accumulate sucrose in their leaves. Some futile cycling between sucrose and hexose sugars is postulated to occur in these species, and thus, the energy cost of sucrose production may be higher than is generally thought.     

Effect of phosphinothricin (glufosinate) on photosynthesis and photorespiration of C3 and C4 plants

Phosphinothricin (glufosinate), an irreversible inhibitor of glutamine synthetase, causes an inhibition of photosynthesis in C₃ (Sinapis alba) and C₄ (Zea mays) plants under atmospheric conditions (400 ppm CO₂, 21% O₂). This photosynthesis inhibition is proceeding slower in C₄ leaves. Under non-photorespiratory conditions (1000 ppm CO₂, 2% O₂) there is no inhibition of photosynthesis. The inhibition of glutamine synthetase by phosphinothricin results in an accumulation of NH₄ ⁺. The NH₄ ⁺-accumulation is lower in C₄ plants than in C₃ plants. The inhibition of glutamine synthetase through phosphinothricin in mustard leaves results in a decrease in glutamine, glutamate, aspartate, asparagine, serine, and glycine. In contrast to this, a considerable increase in leucine and valine following phosphinothricin treatment is measured. With the addition of either glutamine, glutamate, aspartate, glycine or serine, photosynthesis inhibition by phosphinothricin can be reduced, although the NH₄ ⁺-accumulation is greatly increased. This indicates that NH₄ ⁺-accumulation cannot be the primary cause for photosynthesis inhibition by phosphinothricin. The investigations demonstrate the inhibition of transmination of glyoxylate to glycine in photorespiration through the total lack of amino donors. This could result in a glyoxylate accumulation inhibiting ribulose-1,5-bisphosphate-carboxylase and consequently CO₂-fixation.Abbreviations GOGAT glutamine-2-oxoglutarate-amidotransferase - GS glutamine synthetase - PPT phosphinothricin - MSO methionine sulfoximine - RuBP ribulose-1,5-bisphosphate     

Ureide metabolism in leaves of nitrogen-fixing soybean plants

In leaf pieces from nodulated soybean (Glycine max [L] Merr cv Maple Arrow) plants, [¹⁴C]urea-dependent NH₃ and ¹⁴CO₂ production in the dark showed an approximately 2:1 stoichiometry and was decreased to less than 11% of the control (12-19 micromoles NH₃ per gram fresh weight per hour) in the presence of 50 millimolar acetohydroxamate, a urease inhibitor. NH₃ and CO₂ production from the utilization of [2-¹⁴C] allantoin also exhibited a 2:1 stoichiometry and was reduced to a similar extent by the presence of acetohydroxamate with a concomitant accumulation of urea which entirely accounted for the loss in NH₃ production. The almost complete sensitivity of NH₃ and CO₂ production from allantoin and urea metabolism to acetohydroxamate, together with the observed stoichiometry, indicated a path of ureide assimilation (2.0 micromoles per gram leaf fresh weight per hour) via allantoate, ureidoglycolate, and glyoxylate with the production of two urea molecules yielding, in turn, four molecules of NH₃ and two molecules of CO₂.     

CO(2) Concentrating Mechanism of C(4) Photosynthesis: Permeability of Isolated Bundle Sheath Cells to Inorganic Carbon

Diffusion of inorganic carbon into isolated bundle sheath cells from a variety of C₄ species was characterized by coupling inward diffusion of CO₂ to photosynthetic carbon assimilation. The average permeability coefficient for CO₂ (P_CO2) for five representatives from the three decarboxylation types was approximately 20 micromoles per minute per milligram chlorophyll per millimolar, on a leaf chlorophyll basis. The average value for the NAD-ME species Panicum miliaceum (10 determinations) was 26 with a standard deviation of 6 micromoles per minute per milligram chlorophyll per millimolar, on a leaf chlorophyll basis. A P_CO2 of at least 500 micromoles per minute per milligram chlorophyll per millimolar was determined for cells isolated from the C₃ plant Xanthium strumarium. It is concluded that bundle sheath cells are one to two orders of magnitude less permeable to CO₂ than C₃ photosynthetic cells. These data also suggest that CO₂ diffusion in bundle sheath cells may be made up of two components, one involving an apoplastic path and the other a symplastic (plasmodesmatal) path, each contributing approximately equally.     

Regulation of Ribulose-1,5-Bisphosphate Carboxylase Activity in Response to Light Intensity and CO(2) in the C(3) Annuals Chenopodium album L. and Phaseolus vulgaris L

The light and CO₂ response of (a) photosynthesis, (b) the activation state and total catalytic efficiency (k_cat) of ribulose-1,5-bisphosphate carboxylase (rubisco), and (c) the pool sizes of ribulose 1,5-bisphosphate, (RuBP), ATP, and ADP were studied in the C₃ annuals Chenopodium album and Phaseolus vulgaris at 25°C. The initial slope of the photosynthetic CO₂ response curve was dependent on light intensity at reduced light levels only (less than 450 micromoles per square meter per second in C. album and below 200 micromoles per square meter per second in P. vulgaris). Modeled simulations indicated that the initial slope of the CO₂ response of photosynthesis exhibited light dependency when the rate of RuBP regeneration limited photosynthesis, but not when rubisco capacity limited photosynthesis. Measured observations closely matched modeled simulations. The activation state of rubisco was measured at three light intensities in C. album (1750, 550, and 150 micromoles per square meter per second) and at intercellular CO₂ partial pressures (C₁) between the CO₂ compensation point and 500 microbars. Above a C₁ of 120 microbars, the activation state of rubisco was light dependent. At light intensities of 550 and 1750 micromoles per square meter per second, it was also dependent on C₁, decreasing as the C₁ was elevated above 120 microbars at 550 micromoles per square meter per second and above 300 microbars at 1750 micromoles per square meter per second. The pool size of RuBP was independent of C₁ only under conditions when the activation state of rubisco was dependent on C₁. Otherwise, RuBP pool sizes increased as C₁ was reduced. ATP pools in C. album tended to increase as C₁ was reduced. In P. vulgaris, decreasing C₁ at a subsaturating light intensity of 190 micromoles per square meter per second increased the activation state of rubisco but had little effect on the k_cat. These results support modelled simulations of the rubisco response to light and CO₂, where rubisco is assumed to be down-regulated when photosynthesis is limited by the rate of RuBP regeneration.     

Inorganic Carbon Diffusion between C(4) Mesophyll and Bundle Sheath Cells: Direct Bundle Sheath CO(2) Assimilation in Intact Leaves in the Presence of an Inhibitor of the C(4) Pathway

Photosynthesis rates of detached Panicum miliaceum leaves were measured, by either CO₂ assimilation or oxygen evolution, over a wide range of CO₂ concentrations before and after supplying the phosphoenolpyruvate (PEP) carboxylase inhibitor, 3,3-dichloro-2-(dihydroxyphosphinoyl-methyl)-propenoate (DCDP). At a concentration of CO₂ near ambient, net photosynthesis was completely inhibited by DCDP, but could be largely restored by elevating the CO₂ concentration to about 0.8% (v/v) and above. Inhibition of isolated PEP carboxylase by DCDP was not competitive with respect to HCO₃⁻, indicating that the recovery was not due to reversal of enzyme inhibition. The kinetics of ¹⁴C-incorporation from ¹⁴CO₂ into early labeled products indicated that photosynthesis in DCDP-treated P. miliaceum leaves at 1% (v/v) CO₂ occurs predominantly by direct CO₂ fixation by ribulose 1,5-bisphosphate carboxylase. From the photosynthesis rates of DCDP-treated leaves at elevated CO₂ concentrations, permeability coefficients for CO₂ flux into bundle sheath cells were determined for a range of C₄ species. These values (6-21 micromoles per minute per milligram chlorophyll per millimolar, or 0.0016-0.0056 centimeter per second) were found to be about 100-fold lower than published values for mesophyll cells of C₃ plants. These results support the concept that a CO₂ permeability barrier exists to allow the development of high CO₂ concentrations in bundle sheath cells during C₄ photosynthesis.     

Influence of Protein Synthesis on NO(3) Reduction, NH(4) Accumulation, and Amide Synthesis in Suspension Cultures of Paul's Scarlet Rose

Changes in the concentrations of NH₄⁺ and amides during the growth of suspension cultures of rose (Rosa cv. Paul's Scarlet) cells were examined. When cells were grown in medium possessing only NO₃⁻ as a nitrogen source, the concentrations of NH₄⁺ and amides increased to 4.0 × 10⁻¹ and 5.9 micromoles per gram fresh weight, respectively. The amounts of both constituents declined during the later stages of growth. When a trace amount of NH₄⁺ was added to the NO₃⁻ base starting medium, the concentration of NH₄⁺ in the cells was increased to 7.0 × 10⁻¹ micromoles per gram fresh weight.     

Effect of Glyoxylate on the Sensitivity of Net Photosynthesis to Oxygen (the Warburg Effect) in Tobacco

The addition of glyoxylate to tobacco (Nicotiana tabacum) leaf discs inhibited glycolate synthesis and photorespiration and increased net photosynthetic ¹⁴CO₂ fixation. This inhibition of photorespiration was investigated further by studying the effect of glyoxylate on the stimulation of photosynthesis that occurs when the atmospheric O₂ level was decreased from 21 to 3% (the Warburg effect). The Warburg effect is usually ascribed to the increased glycolate synthesis and metabolism that occurs at higher O₂ concentrations. Photosynthesis in control discs increased from 59.1 to 94.7 micromoles of CO₂ per gram fresh weight per hour (a 60% increase) when the O₂ level was lowered from 21 to 3%, while the rate for discs floated on 15 millimolar glyoxylate increased only from 82.0 to 99.7 micromoles of CO₂ per gram fresh weight per hour (a 22% increase). The decrease in the O₂ sensitivity of photosynthesis in the presence of glyoxylate was explained by changes in the rate of glycolate synthesis under the same conditions.

The rate of metabolism of the added glyoxylate by tobacco leaf discs was about 1.35 micromoles per gram fresh weight per hour and was not dependent on the O₂ concentration in the atmosphere. This rate of metabolism is about 10% the amount of stimulation in the rate of CO₂ fixation caused by the glyoxylate treatment on a molar carbon basis. Glyoxylate (10 millimolar) had no effect on the carboxylase/oxygenase activity of isolated ribulose diphosphate carboxylase. Although the biochemical mechanism by which glyoxylate inhibits glycolate synthesis and photorespiration and thereby decreases the Warburg effect is still uncertain, these results show that cellular metabolites can regulate the extent of the Warburg effect.

     

Inhibition of 3-Phosphoglycerate-Dependent O(2) Evolution by Phosphoenolpyruvate in C(4) Mesophyll Chloroplasts of Digitaria sanguinalis (L.) Scop

The effects of phosphoenolpyruvate (PEP), inorganic phosphate (Pi), and ATP on 3-phosphoglycerate (PGA)-dependent O₂ evolution by chloroplasts of Digitaria sanguinalis (L.) Scop. (crabgrass) were evaluated relative to possible mechanisms of PEP transport by the C₄ mesophyll chloroplast. Crude and Percoll purified chloroplast preparations exhibited rates of PGA-dependent O₂ evolution in the range of 90 to 135 micromoles O₂ per milligram chlorophyll per hour, and up to 180 micromoles O₂ per milligram chlorophyll per hour at optimal Pi concentrations (approximately 0.2 millimolar at 9 millimolar PGA). Higher concentrations of Pi were inhibitory. PEP inhibited O₂ evolution (up to 70%) in both chloroplast preparations when the PEP to PGA ratio was high (i.e. 9 millimolar PEP to 0.36 millimolar PGA). Usually no inhibition was seen when the PEP to PGA ratio was less than 2. PEP acted as a competitive inhibitor and, at a concentration of 9 millimolar, increased the apparent K_m (PGA) from 0.15 to 0.53 millimolar in Percoll purified chloroplasts. A low concentration of PGA and high ratio of PEP to PGA, which are considered unphysiological, were required to detect any inhibition of O₂ evolution by PEP. Similar results were obtained from crude versus Percoll purified preparations. Neither the addition of Pi nor ATP could overcome PEP inhibition. As PEP inhibition was competitive with respect to PGA concentration, and as addition of ATP or Pi could not prevent PEP inhibition of PGA-dependent O₂ evolution, the inhibition was not due to PEP exchange of adenylates or Pi out of the chloroplast. Analysis of the effect of Pi and PEP, separately and in combination, on PGA-dependent O₂ evolution suggests interactions between PEP, Pi, and PGA on the same translocator in the C₄ mesophyll chloroplast. C₃ spinach chloroplasts were also found to be sensitive to PEP, but to a lesser extent than crabgrass chloroplasts. The apparent K_i values (PEP) were 3 and 21 millimolar for crabgrass and spinach, respectively.     

Effects of the Phosphoenolpyruvate Carboxylase Inhibitor 3,3-Dichloro-2-(Dihydroxyphosphinoylmethyl)propenoate on Photosynthesis: C(4) Selectivity and Studies on C(4) Photosynthesis

The effect of 3,3-dichloro-2-(dihydroxyphosphinoylmethyl)-propenoate (DCDP), an analog of phosphoenolpyruvate (PEP), on PEP carboxylase activity in crude leaf extracts and on photosynthesis of excised leaves was examined. DCDP is an effective inhibitor of PEP carboxylase from Zea mays or Panicum miliaceum; 50% inhibition was obtained at 70 or 350 micromolar, respectively, in the presence of 1 millimolar PEP and 1 millimolar HCO₃⁻. When fed to leaf sections via the transpiration stream, DCDP at 1 millimolar strongly inhibited photosynthesis in C₄ species (79-98% inhibition for a range of seven C₄ species), but only moderately in C₃ species (12-46% for four C₃ species), suggesting different mechanisms of inhibition for each photosynthetic type. The response of P. miliaceum (C₄) net photosynthesis to intercellular pCO₂ showed that carboxylation efficiency, as well as the CO₂ saturated rate, are lowered in the presence of DCDP and supported the view that carboxylation efficiency in C₄ species is directly related to PEP carboxylase activity. A fivefold increase in intercellular pCO₂ over that occurring in P. miliaceum under normal photosynthesis conditions only increased net photosynthesis rate in the presence of 1 millimolar DCDP from zero to about 5% of the maximal uninhibited rate. Therefore, it seems unlikely that direct fixation of atmospheric CO₂ by the bundle sheath cells makes any significant contribution to photosynthetic CO₂ assimilation in C₄ species. The results support the concept that C₄-selective herbicides may be developed based on inhibitors of C₄ pathway reactions.     

Photosynthetic Characteristics of the C(3)-C(4) Intermediate Parthenium hysterophorus

The weedy species Parthenium hysterophorus (Asteraceae) possesses a Kranz-like leaf anatomy. The bundle sheath cells are thick-walled and contain numerous granal chloroplasts, prominent mitochondria, and peroxisomes, all largely arranged in a centripetal position. Both mesophyll and bundle sheath chloroplasts accumulate starch. P. hysterophorus exhibits reduced photorespiration as indicated by a moderately low CO₂ compensation concentration (20-25 microliters per liter at 30°C and 21% O₂) and by a reduced sensitivity of net photosynthesis to 21% O₂. In contrast, the related C₃ species P. incanum and P. argentatum (guayule) lack Kranz anatomy, have higher CO₂ compensation concentrations (about 55 microliters per liter), and show a greater inhibition of photosynthesis by 21% O₂. Furthermore, in P. hysterophorus the CO₂ compensation concentration is relatively less sensitive to changes in O₂ concentrations and shows a biphasic response to changing O₂, with a transition point at about 11% O₂. Based on these results, P. hysterophorus is classified as a C₃-C₄ intermediate. The activities of diagnostic enzymes of C₄ photosynthesis in P. hysterophorus were very low, comparable to those observed in the C₃ species P. incanum (e.g. phosphoenolpyruvate carboxylase activity of 10-29 micromoles per milligram of chlorophyll per hour). Exposures of leaves of each species to ¹⁴CO₂ (for 8 seconds) in the light resulted in 3-phosphoglycerate and sugar phosphates being the predominant initial ¹⁴C products (77-84%), with ≤4% of the ¹⁴C-label in malate plus aspartate. These results indicate that in the C₃-C₄ intermediate P. hysterophorus, the reduction in leaf photorespiration cannot be attributed to C₄ photosynthesis.     

Whole Leaf Carbon Exchange Characteristics of Phosphate Deficient Soybeans (Glycine max L.)

Low phosphate nutrition results in increased chlorophyll fluorescence, reduced photosynthetic rate, accumulation of starch and sucrose in leaves, and low crop yields. This study investigated physiological responses of soybean (Glycine max [L.] Merr.) leaves to low inorganic phosphate (Pi) conditions. Responses of photosynthesis to light and CO₂ were examined for leaves of soybean grown at high (0.50 millimolar) or low (0.05 millimolar) Pi. Leaves of low Pi plants exhibited paraheliotropic orientation on bright sunny days rather than the normal diaheliotropic orientation exhibited by leaves of high Pi soybeans. Leaves of plants grown at high Pi had significantly higher light saturation points (1000 versus 630 micromole photons [400-700 nanometers] per square meter per second) and higher apparent quantum efficiency (0.062 versus 0.044 mole CO₂ per mole photons) at ambient (34 pascals) CO₂ than did low Pi leaves, yet stomatal conductances were similar. High Pi leaves also had significantly higher carboxylation efficiency (2.90 versus 0.49 micromole CO₂ per square meter per second per pascal), a lower CO₂ compensation point (6.9 versus 11.9 pascals), and a higher photosynthetic rate at 34 pascals CO₂ (19.5 versus 6.7 micromoles CO₂ per square meter per second) than did low Pi leaves. Soluble protein (0.94 versus 0.73 milligram per square centimeter), ribulose-1,5-bisphosphate carboxylase/oxygenase content (0.33 versus 0.25 milligram per square centimeter), and ribulose-1,5-bisphosphate carboxylase/oxygenase specific activity (25.0 versus 16.7 micromoles per square meter per second) were significantly greater in leaves of plants in the high Pi treatment. The data indicate that Pi stress alters the plant's CO₂ reduction characteristics, which may in turn affect the plant's capacity to accommodate normal radiation loads.     

Environmental Effects on Photorespiration of C(3)-C(4) Species : I. Influence of CO(2) and O(2) during Growth on Photorespiratory Characteristics and Leaf Anatomy

The possibility of altering CO₂ exchange of C₃-C₄ species by growing them under various CO₂ and O₂ concentrations was examined. Growth under CO₂ concentrations of 100, 350, and 750 micromoles per mole had no significant effect on CO₂ exchange characteristics or leaf anatomy of Flaveria pringlei (C₃), Flaveria floridana (C₃-C₄), or Flaveria trinervia (C₄). Carboxylation efficiency and CO₂ compensation concentrations in leaves of F. floridana developed under the different CO₂ concentrations were intermediate to F. pringlei and F. trinervia. When grown for 12 days at an O₂ concentration of 20 millimoles per mole, apparent photosynthesis was strongly inhibited in Panicum milioides (C₃-C₄) and to a lesser degree in Panicum laxum (C₃). In P. milioides, acute starch buildup was observed microscopically in both mesophyll and bundle sheath cells. Even after only 4 days exposure to 20 millimoles per mole O₂, the presence of starch was more pronounced in leaf cross-sections of P. milioides compared to those at 100 and 210 millimoles per mole. Even though this observation suggests that P. milioides has a different response to low O₂ with respect to translocation of photosynthate or sink activity than C₃ species, the concentration of total available carbohydrate increased in shoots of all species by 33% or more when grown at low O₂. This accumulation occurred even though relative growth rates of Festuca arundinacea (C₃) and P. milioides grown for 4 days at 210 millimoles per mole O₂, were inhibited 83 and 37%, respectively, when compared to plants grown at 20 millimoles per mole O₂.     

Utilization of Ammonium as a Nitrogen Source : Effects of Ambient Acidity on Growth and Nitrogen Accumulation by Soybean

Dry matter accumulation of plants utilizing NH₄⁺ as the sole nitrogen source generally is less than that of plants receiving NO₃⁻ unless acidity of the root-zone is controlled at a pH of about 6.0. To test the hypothesis that the reduction in growth is a consequence of nitrogen stress within the plant in response to effects of increased acidity during uptake of NH₄⁺ by roots, nonnodulated soybean plants (Glycine max [L.] Merr. cv Ransom) were grown for 24 days in flowing nutrient culture containing 1.0 millimolar NH₄⁺ as the nitrogen source. Acidities of the culture solutions were controlled at pH 6.1, 5.1, and 4.1 ± 0.1 by automatic additions of 0.01 n H₂SO₄ or Ca(OH)₂. Plants were sampled at intervals of 3 to 4 days for determination of dry matter and nitrogen accumulation. Rates of NH₄⁺ uptake per gram root dry weight were calculated from these data. Net CO₂ exchange rates per unit leaf area were measured on attached leaves by infrared gas analysis. When acidity of the culture solution was increased from pH 6.1 to 5.1, dry matter and nitrogen accumulation were reduced by about 40% within 14 days. Net CO₂ exchange rates per unit leaf area, however, were not affected, and the decreased growth was associated with a reduction in rates of appearance and expansion of new leaves. The uptake rates of NH₄⁺ per gram root were about 25% lower throughout the 24 days at pH 5.1 than at 6.1. A further increase in solution acidity from pH 5.1 to 4.1 resulted in cessation of net dry matter production and appearance of new leaves within 10 days. Net CO₂ exchange rates per unit leaf area declined rapidly until all viable leaves had abscised by 18 days. Uptake rates of NH₄⁺, which were initially about 50% lower at pH 4.1 than at 6.1, continued to decline with time of exposure until net uptake ceased at 10 days. Since these responses also are characteristic of the sequence of responses that occur during onset and progression of a nitrogen stress, they corroborate our hypothesis.     

Changes in Levels of Intermediates of the C(4) Cycle and Reductive Pentose Phosphate Pathway under Various Light Intensities in Maize Leaves

The rate of CO₂ assimilation and levels of metabolites of the C₄ cycle and reductive pentose phosphate pathway in attached leaves of maize (Zea mays L.) were measured over a range of light intensity from 0 to 1,900 microEinsteins per square meter per second under a saturated CO₂ concentration of 350 microliters per liter and a limiting CO₂ concentration of 133 microliters per liter. The level of ribulose 1,5-bisphosphate (RuBP) stayed almost constant (around 60 nanomoles per milligram chlorophyll [Chl]) from low to high light intensities under 350 microliters per liter. Levels of 3-phosphoglycerate (PGA) increased from 100 to 650 nanomoles per milligram Chl under 350 microliters per liter CO₂ with increasing light intensity. The calculated RuBP concentration of 6 millimolar (corresponded to 60 nanomoles per milligram Chl) was about two times above the estimated RuBP binding-site concentration on ribulose bisphosphate carboxylase-oxygenase (Rubisco) of ~2.6 millimolar in maize bundle sheath chloroplasts in the light. The ratio of RuBP/PGA increased with decreasing light intensity under 350 microliters per liter CO₂. These results suggest that RuBP carboxylation is under control of light intensity possibly due to a limited supply of CO₂ to Rubisco through the C₄ cycle whose activity is highly dependent on light intensity. Pyruvate level increased with increasing light intensity as long as photosynthesis rate increased. A positive relationship between levels of PGA and those of pyruvate during steady-state photosynthesis under various conditions suggests that an elevated concentration of PGA increases the carbon input into the C₄ cycle through the conversion of PGA to PEP and consequently the level of total intermediates of the C₄ cycle can be raised to mediate higher photosynthesis rate.     

Quantification of Apoplastic Potassium Content by Elution Analysis of Leaf Lamina Tissue from Pea (Pisum sativum L. cv Argenteum)

K⁺ content and concentration within the apoplast of mesophyll tissue of pea (Pisum sativum L., cv Argenteum) leaflets were determined using an elution procedure. Following removal of the epidermis, a 1 centimeter (inside diameter) glass cylinder was attached to the exposed mesophyll tissue and filled with 5 millimolar CaCl₂ solution (1°C). From time-course curves of cumulative K⁺ diffusion from the tissue, the amount of K⁺ of extracellular origin was estimated. Apoplastic K⁺ contents for leaves from plants cultured in nutrient solution containing 2 or 10 millimolar K⁺ were found to range from 1 to 4.5 micromoles per gram fresh weight, comprising less than 3% of the total K⁺ content within the lamina tissue. Assuming an apoplastic solution volume of 0.04 to 0.1 milliliters per gram fresh weight and a Donnan cation exchange capacity of 2.63 micromoles per gram fresh weight (experimentally determined), the K⁺ concentration within apoplastic solution was estimated at 2.4 to 11.8 millimolar. Net movement of Rb⁺ label from the extracellular compartment within mesophyll tissue into the symplast was demonstrated by pulse-chase experiments. It was concluded that the mesophyll apoplast in pea has a relatively low capacitance as an ion reservoir. Apoplastic K⁺ content was found to be highly sensitive to changes in xylem solution concentration.     

A Comparison of Dark Respiration between C(3) and C(4) Plants

Lower respiratory costs were hypothesized as providing an additional benefit in C₄ plants compared to C₃ plants due to less investment in proteins in C₄ leaves. Therefore, photosynthesis and dark respiration of mature leaves were compared between a number of C₄ and C₃ species. Although photosynthetic rates were generally greater in C₄ when compared to C₃ species, no differences were found in dark respiration rates of individual leaves at either the beginning or after 16 h of the dark period. The effects of nitrogen on photosynthesis and respiration of individual leaves and whole plants were also investigated in two species that occupy similar habitats, Amaranthus retroflexus (C₄) and Chenopodium album (C₃). For mature leaves of both species, there was no relationship between leaf nitrogen and leaf respiration, with leaves of both species exhibiting a similar rate of decline after 16 h of darkness. In contrast, leaf photosynthesis increased with increasing leaf nitrogen in both species, with the C₄ species displaying a greater photosynthetic response to leaf nitrogen. For whole plants of both species grown at different nitrogen levels, there was a clear linear relationship between net CO₂ uptake and CO₂ efflux in the dark. The dependence of nightly CO₂ efflux on CO₂ uptake was similar for both species, although the response of CO₂ uptake to leaf nitrogen was much steeper in the C₄ species, Amaranthus retroflexus. Rates of growth and maintenance respiration by whole plants of both species were similar, with both species displaying higher rates at higher leaf nitrogen. There were no significant differences in leaf or whole plant maintenance respiration between species at any temperature between 18 and 42°C. The data suggest no obvious differences in respiratory costs in C₄ and C₃ plants.     

Effect of the Long-Term Elevation of CO(2) Concentration in the Field on the Quantum Yield of Photosynthesis of the C(3) Sedge, Scirpus olneyi

CO₂ concentration was elevated throughout 3 years around stands of the C₃ sedge Scirpus olneyi on a tidal marsh of the Chesapeake Bay. The hypothesis that tissues developed in an elevated CO₂ atmosphere will show an acclimatory decrease in photosynthetic capacity under light-limiting conditions was examined. The absorbed light quantum yield of CO₂ uptake (ø_abs and the efficiency of photosystem II photochemistry were determined for plants which had developed in open top chambers with CO₂ concentrations in air of 680 micromoles per mole, and of 351 micromoles per mole as controls. An Ulbricht sphere cuvette incorporated into an open gas exchange system was used to determine ø_abs and a portable chlorophyll fluorimeter was used to estimate the photochemical efficiency of photosystem II. When measured in an atmosphere with 10 millimoles per mole O₂ to suppress photorespiration, shoots showed a ø_abs of 0.093 ± 0.003, with no statistically significant difference between shoots grown in elevated or control CO₂ concentrations. Efficiency of photosystem II photochemistry was also unchanged by development in an elevated CO₂ atmosphere. Shoots grown and measured in 680 micromoles per mole of CO₂ in air showed a ø_abs of 0.078 ± 0.004 compared with 0.065 ± 0.003 for leaves grown and measured in 351 micromoles per mole CO₂ in air; a highly significant increase. In accordance with the change in ø_abs, the light compensation point of photosynthesis decreased from 51 ± 3 to 31 ± 3 micro-moles per square meter per second for stems grown and measured in 351 and 680 micromoles per mole of CO₂ in air, respectively. The results suggest that even after 3 years of growth in elevated CO₂, there is no evidence of acclimation in capacity for photosynthesis under light-limited conditions which would counteract the stimulation of photosynthetic CO₂ uptake otherwise expected through decreased photorespiration.     

Photosynthesis by isolated protoplasts, protoplast extracts, and chloroplasts of wheat: influence of orthophosphate, pyrophosphate, and adenylates

Protoplasts, protoplast extracts (intact chloroplasts plus extrachloroplastic material), and chloroplasts isolated from protoplasts of wheat (Triticum aestivum) have rates of photosynthesis as measured by light-dependent O₂ evolution of about 100 to 150 micromoles of O₂ per milligram of chlorophyll per hour at 20 C and saturating bicarbonate. The assay conditions sufficient for this activity were 0.4 molar sorbitol, 50 millimolar N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid KOH (pH 7.6), and 10 millimolar NaHCO₃ with protoplast, plus a requirement of 1 to 10 millimolar ethylenediaminetetraacetate (EDTA) and 0.2 to 0.5 millimolar inorganic orthophosphate (Pi) with protoplast extracts and chloroplasts. Protoplast extracts evolved approximately 6 micromoles of O₂ per milligram of chlorophyll before photosynthesis became largely dependent on exogenous Pi while photosynthesis by chloroplasts had a much stronger dependence on exogenous Pi from the outset.

Photosynthesis by chloroplasts from 6-day-old wheat plants under optimum levels of Pi was similar to that with the addition of 5 millimolar inorganic pyrophosphate (PPi) plus 0.2 millimolar adenosine-5′-diphosphate (ADP). Either PPi or ADP added separately inhibited photosynthesis. When chloroplasts were incubated in the dark for 2 to 6 minutes, photosynthesis was strongly inhibited by 5 millimolar PPi and this inhibiting was relieved by including adenosine-5′-triphosphate (ATP) or ADP (0.2 to 0.6 millimolar). Chloroplasts from 9-day-old wheat leaves were slightly less sensitive to inhibition by PPi and showed little or no inhibition by ADP.

Chloroplasts isolated from protoplasts and assayed with 0.3 millimolar Pi added before illumination have an induction time from less than 1 minute up to 16 minutes depending on the time of the assay after isolation and the components of the medium. In order to obtain maximum rates of photosynthesis and minimum induction time, NaHCO₃ and chelating agents, EDTA or PPi (+ATP), are required in the chloroplast isolation, resuspension and assay medium. With these inclusions in the isolation and resuspension medium the induction time decreased rapidly during the first 20 to 30 minutes storage of chloroplasts on ice. Requirements for isolating intact and photosynthetically functional chloroplasts from wheat protoplasts are discussed.

     

Characterization of Growth, Water Relations, and Proline Accumulation in Sodium Sulfate Tolerant Callus of Brassica napus L. cv Westar (Canola)

Unselected and sodium sulfate tolerant callus cultures of Brassica napus L. cv Westar were grown on media supplemented with mannitol, NaCl, or Na₂SO₄. In all cases, growth of tolerant callus, measured on a fresh weight or dry weight basis, was greater than that of unselected callus, which was also subject to necrosis on high levels of salt. Tissue water potential became more negative in both unselected and tolerant callus grown in the presence of mannitol or Na₂SO₄. Water potentials in unselected callus were more negative than those of the tolerant tissues; but over a range of Na₂SO₄ concentrations both cultures displayed osmotic adjustment, maintaining relatively constant turgor. Proline accumulation in both unselected and tolerant callus was low (15 to 20 micromoles per gram dry weight) in the absence of stress, but increased on media supplemented with mannitol, NaCl, or Na₂SO₄. Increases in proline concentration were approximately linear in tolerant callus, reaching a maximum of 130 to 175 micromoles per gram dry weight. In unselected callus, concentrations were higher, reaching 390 to 520 micromoles per gram dry weight. Proline accumulation was correlated with inhibition of growth, and there was a negative correlation between proline concentration and culture age for tolerant callus.