Adaptation of photosynthesis in marama bean Tylosema esculentum (Burchell A. Schreiber) to a high temperature, high radiation, drought-prone environment.

Marama bean, Tylosema esculentum, is a tuberous legume native to the Kalahari region of Southern Africa where it grows under high temperatures (typical daily max 37 degrees C during growing season) and radiation (frequently in excess of 2000 micromol m(-2) s(-1)) in sandy soils with low rainfall. These conditions might be expected to select for increased water-use efficiency of photosynthesis. However, marama was found to give similar leaf photosynthetic rates to other C3 plants for a given internal leaf CO2 concentration and Rubisco content. Under conditions of increasing drought, no increase in water-use efficiency of photosynthesis was observed, but stomata closed early and preceded any change in leaf water potential. The possibility of subtle adaptations of photosynthetic characteristics to its natural environment were investigated at the level of Rubisco kinetics. The specificity factor of marama Rubisco was slightly lower than that of wheat, but the apparent Km for CO2 in air (Km') was about 20% lower than that of wheat. This is consistent with better adaptation for efficient photosynthesis at high temperatures in marama compared to wheat, although the net benefit is predicted to be very small (<0.5% at 35 degrees C). The sequence of marama rbcL gene shows 27 deduced amino acid residue differences from that for wheat, and the possibility that one or more of these cause the difference in Rubisco Km' is discussed.

Physiological characterization of 'stay green' mutants in durum wheat.

Four mutants with delayed leaf senescence were selected from seed of durum wheat mutagenized with ethylmethane sulphonate. Changes in net photosynthetic rate, efficiency of photosystem II and chlorophyll concentration during the maturation and senescence of the flag leaves of both mutant and parental plants were determined under glasshouse conditions. The four mutant lines maintained photosynthetic competence for longer than the parental line and are therefore functionally 'stay green'. The mutant lines also had higher seed weights and grain yields per plant than the parental line.

Effects of water deficit and its interaction with CO(2) supply on the biochemistry and physiology of photosynthesis in sunflower.

Photosynthetic responses of sunflower plants grown for 52 d in ambient and elevated CO(2) (A=350 or E=700 micromol mol(-1), respectively) and subjected to no (control), mild or severe water deficits after 45 d were analysed to determine if E modifies responses to water deficiency. Relative water content, leaf water potential (Psi(w)) and osmotic potential decreased with water deficiency, but there were no effects of E. Growth in E decreased stomatal conductance (g(s)) and thereby transpiration, but increased net CO(2) assimilation rate (P(n), short-term measurements); therefore, water-use efficiency increased by 230% (control plants) and 380% (severe stress). Growth in E did not affect the response of P(n) to intercellular CO(2) concentration, despite a reduction of 25% in Rubisco content, because this was compensated by a 32% increase in Rubisco activity. Analysis of chlorophyll a fluorescence showed that changes in energy metabolism associated with E were small, despite the decreased Rubisco content. Water deficits decreased g(s) and P(n): metabolic limitation was greater than stomatal at mild and severe deficit and was not overcome by elevated CO(2). The decrease in P(n) with water deficiency was related to lower Rubisco activity rather than to ATP and RuBP contents. Thus, there were no important interactions between CO(2) during growth and water deficit with respect to photosynthetic metabolism. Elevated CO(2 )will benefit sunflower growing under water deficit by marginally increasing P(n), and by slowing transpiration, which will decrease the rate and severity of water deficits, with limited effects on metabolism.

Photosynthetic carbon assimilation and associated metabolism in relation to water deficits in higher plants.

Experimental studies on CO2 assimilation of mesophytic C3 plants in relation to relative water content (RWC) are discussed. Decreasing RWC slows the actual rate of photosynthetic CO2 assimilation (A) and decreases the potential rate (Apot). Generally, as RWC falls from c. 100 to c. 75%, the stomatal conductance (gs) decreases, and with it A. However, there are two general types of relation of Apot to RWC, which are called Type 1 and Type 2. Type 1 has two main phases. As RWC decreases from 100 to c. 75%, Apot is unaffected, but decreasing stomatal conductance (gs) results in smaller A, and lower CO2 concentration inside the leaf (Ci) and in the chloroplast (Cc), the latter falling possibly to the compensation point. Down-regulation of electron transport occurs by energy quenching mechanisms, and changes in carbohydrate and nitrogen metabolism are considered acclimatory, caused by low Ci and reversible by elevated CO2. Below 75% RWC, there is metabolic inhibition of Apot, inhibition of A then being partly (but progressively less) reversible by elevated CO2; gs regulates A progressively less, and Ci and CO2 compensation point, Gamma rise. It is suggested that this is the true stress phase, where the decrease in Apot is caused by decreased ATP synthesis and a consequent decreased synthesis of RuBP. In the Type 2 response, Apot decreases progressively at RWC 100 to 75%, with A being progressively less restored to the unstressed value by elevated CO2. Decreased gs leads to a lower Ci and Cc but they probably do not reach compensation point: gs becomes progressively less important and metabolic limitations more important as RWC falls. The primary effect of low RWC on Apot is most probably caused by limited RuBP synthesis, as a result of decreased ATP synthesis, either through inhibition of Coupling Factor activity or amount due to increased ion concentration. Carbohydrate synthesis and accumulation decrease. Type 2 response is considered equivalent to Type 1 at RWC below c. 75%, with Apot inhibited by limited ATP and RuBP synthesis, respiratory metabolism dominates and Ci and Gamma rise. The importance of inhibited ATP synthesis as a primary cause of decreasing Apot is discussed. Factors determining the Type 1 and Type 2 responses are unknown. Electron transport is maintained (but down-regulated) in Types 1 and 2 over a wide range of RWC, and a large reduced/oxidized adenylate ratio results. Metabolic imbalance results in amino acid accumulation and decreased and altered protein synthesis. These conditions profoundly affect cell functions and ultimately cause cell death. Type 1 and 2 responses may reflect differences in gs and in sensitivity of metabolism to decreasing RWC.

Diurnal variation of photosynthesis and photoinhibition in tea: effects of irradiance and nitrogen supply during growth in the field.

Diurnal changes in the rate of photosynthesis (A) of mature tea (Camellia sinensis (L.) O. Kuntze) bushes grown at high elevation in the field in Sri Lanka, were related to environmental conditions. Bushes were either unshaded, receiving 100% of incident photosynthetically active radiation (PAR), moderately shaded, (65% PAR) or heavily shaded (30% PAR). These treatments were combined with nitrogen fertilizer applications of 0, 360 and 720 kg ha(-1) year(-1). When recently fully expanded leaves were measured under the growing conditions on bright, clear days from dawn to dusk, A was greatest in the morning with increasing radiation between approximately 8 h and 10 h. Stomatal conductances (g(s)) and substomatal carbon dioxide concentrations (C(i)) were then large, leaf temperatures (T(L)) cool, and saturated water vapour deficits (VPD) small. However, as the irradiance, T(L) and VPD increased towards midday, A, g(s), photochemical quenching, and C(i) decreased, and non-photochemical quenching increased. In the late afternoon, irradiance, T(L) and VPD fell, but despite the relatively large increase in g(s) and C(i), A remained low; however, it recovered overnight. The zero-N treatment decreased total-N content of leaves by 50% and A by c. 20% (not significant). Leaves of unshaded plants receiving least N had significantly (P<0.05) smaller A and greater total sugar content than shaded but with abundant N, A and sugars did not differ between shade treatments. Analysis of the responses of A to environment in the morning compared to the afternoon, and of chlorophyll fluorescence, suggests that A was photoinhibited as a consequence of greatly increased PAR, whilst decreasing g(s) (related to changes in PAR, VPD and T(L)) caused C(i) to fall. End-product inhibition of A is not consistent with decreased C(i). Inhibition of A as a result of photoinhibition was minimized, but not eliminated, by abundant N. Interactions between factors regulating A in tea are discussed.

Low sink demand limits photosynthesis under P(i) deficiency.

The role of the demand for carbon assimilates (the 'sink') in regulating photosynthetic carbon assimilation (Pn: the 'source') in response to phosphate (P(i)) deficiency was examined in tobacco (Nicotiana tabacum L.). P(i) supply was maintained or withdrawn from plants, and in both treatments the source/sink ratio was decreased in some plants by darkening all but two source leaves (partially darkened plants). The remaining plants were kept fully illuminated. P(i)-sufficient plants showed little variation in rate of Pn, amounts of P(i) or phosphorylated intermediates. Withdrawal of P(i) decreased Pn by 75% under the growing conditions and at both low and high internal CO2 concentration. Concomitantly, P(i), phosphorylated intermediates and ATP contents decreased and starch increased. RuBP and activity of phosphoribulokinase closely matched the changes in Pn, but Rubisco activity remained high. Partial darkening P(i)-deficient plants delayed the loss of photosynthetic activity; Rubisco and phosphoribulokinase activities and amounts of sucrose and metabolites, particularly RuBP and G6P, were higher than in fully illuminated Pi-deficient plants. Rates of sucrose export from leaves were more than 2-fold greater than in fully illuminated P(i)-deficient plants. Greater sucrose synthesis, facilitated by increased G6P content, an activator of SPS, would recycle P(i) from the cytosol back to the chloroplast, maintaining ATP, RuBP and hence Pn. It is concluded that low sink strength imposes the primary limitation on photosynthesis in P(i)-deficient plants which restricts sucrose export and sucrose synthesis imposing an end-product synthesis limitation of photosynthesis.

Is there scope for improving balance between RuBP-regeneration and carboxylation capacities in wheat at elevated CO2?

Carboxylation and RuBP-regeneration capacities, which determine light-saturated photosynthetic rate, were analysed in leaves of spring wheat (Triticum aestivum L. cv. Minaret) grown under different atmospheric CO2 partial pressure (pCa) and N supply regimes. Capacities were estimated from a large number of gas exchange, Rubisco and ATP-synthase content measurements, and from these, the pCa at which the two capacities are equal was derived, to allow direct comparison with growth pCa. Acclimation of the balance between the two capacities to growth at elevated pCa in wheat was only partial and appears to occur mostly in older flag leaves and at low N. However, in contrast to conclusions drawn from previous analyses of these data, there was evidence of a specific effect of growth at 70 Pa pCa, where carboxylation capacity is reduced more than RuBP-regeneration capacity for a given leaf N content. A model was used to estimate the effects of fluctuations in PPFD and temperature in the growth environment on the optimal balance between these capacities. This showed that the observed balance between carboxylation and RuBP-regeneration capacities in young wheat leaves could be consistent with adaptation to the current, or even the preindustrial pCa.

Decrease of phosphoribulokinase activity by antisense RNA in transgenic tobacco: definition of the light environment under which phosphoribulokinase is not in large excess.

To test the hypothesis that the contribution of phosphoribulokinase (PRK) to the control of photosynthesis changes depending on the light environment of the plant, the response of transgenic tobacco (Nicotiana tabacum L.) transformed with antisense PRK constructs to irradiance was determined. In plants grown under low irradiance (330 micromol m(-2) s(-1)) steady-state photosynthesis was limited in plants with decreased PRK activity upon exposure to higher irradiance, with a control coefficient of PRK for CO2 assimilation of 0.25 at and above 800 micromol m(-2) s(-1). The flux control coefficient of PRK for steady-state CO2 assimilation was zero, however, at all irradiances in plant material grown at 800 micromol m(-2) s(-1) and in plants grown in a glasshouse during mid-summer (alternating shade and sun 300-1600 micromol m(-2) s(-1)). To explain these differences between plants grown under low and high irradiances, Calvin cycle enzyme activities and metabolite content were determined. Activities of PRK and other non-equilibrium Calvin cycle enzymes fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase and ribulose-1,5-bisphosphate carboxylase-oxygenase were twofold higher in plants grown at 800 micromol m(-2) s(-1) or in the glasshouse than in plants grown at 330 micromol m(-2) s(-1). Activities of equilibrium enzymes transketolase, aldolase, ribulose-5-phosphate epimerase and isomerase were very similar under all growth irradiances. The flux control coefficient of 0.25 in plants grown at 330 micromol m(-2) s(-1) can be explained because low ribulose-5-phosphate content in combination with low PRK activity limits the synthesis of ribulose-1,5-bisphosphate. This limitation is overcome in high-light-grown plants because of the large relative increase in activities of sedoheptulose-1,7-bisphosphatase and fructose-1,6-bisphosphatase under these conditions, which facilitates the synthesis of larger amounts of ribulose-5-phosphate. This potential limitation will have maintained evolutionary selection pressure for high concentrations of PRK within the chloroplast.

The regulation of component processes of photosynthesis in transgenic tobacco with decreased phosphoribulokinase activity.

Tobacco plants (Nicotiana tabacum L.) transformed with an inverted cDNA encoding ribulose 5-phosphate kinase (phosphoribulokinase,PRK; EC 2.7.1.19) were employed to study the in vivo relationship between photosynthetic electron transport and the partitioning of electron transport products to major carbon metabolism sinks under conditions of elevated ATP concentrations and limited ribulose 1,5-bisphosphate (RuBP) regeneration. Simultaneous measurements of room temperature chlorophyll fluorescence and CO2 gas exchange were conducted on intact leaves. Under ambient CO2 concentrations and light intensities above those at which the plants were grown, transformants with only 5% of PRK activity showed 'down-regulation" of PS II activity and electron transport in response to a decrease in net carbon assimilation when compared to wild-type. This was manifested as a decline in the efficiency of PS II electron transport (ΦPS II), an increase in dissipation of excess absorbed light in the antennae of PS II and a decline in: total linear electron transport (J1), electron transport dedicated to carbon assimilation (JA) and electron transport allocated to photorespiration (JL). The transformants showed no alteration in the Rubisco specificity factor measured in vitro and calculated in vivo but had a relatively smaller ratio of RuBP oxygenation to carboxylation rates (vo/vc), due to a higher CO2 concentration at the carboxylation site (Cc). The relationship between ΦPS II and ΦCO 2was similar in transformants and wild-type under photorespiratory conditions demonstrating no change in the intrinsic relationship between PS II function and carbon assimilation, however, a novel result of this study is that this similar relationship occurred at different values of quantum flux, J1, JA, JL and vo/vc in the transformant. For both wild-type and transformants, an assessment was made of the possible presence of a third major sink for electron transport products, beside RuBP oxygenation and carboxylation, the data provided no evidence for such a sink.

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 CO(2) 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 CO(2)-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.

Regulation of Photosynthetic Rate of Two Sunflower Hybrids under Water Stress.

The effect of short-term water stress on photosynthesis of two sunflower hybrids (Helianthus annuus L. cv Sungro-380 and cv SH-3622), differing in productivity under field conditions, was measured. The rate of CO(2) assimilation of young, mature leaves of SH-3622 under well-watered conditions was approximately 30% greater than that of Sungro-380 in bright light and elevated CO(2); the carboxylation efficiency was also larger. Growth at large photon flux increased assimilation rates of both hybrids. The changes in leaf composition, including cell numbers and sizes, chlorophyll content, and amounts of total soluble and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) protein, and in Rubisco activity and amount of ribulose-1,5-bisphosphate (RuBP) were determined to assess the factors regulating the differences in assimilation of the hybrids at high and low water potentials. The amounts of chlorophyll, soluble protein, Rubisco protein and the initial activity of Rubisco and its activation state did not differ significantly between hybrids. However, unstressed leaves of SH-3622 had more, smaller cells per unit area and 60% more RuBP per unit leaf area than that of Sungro-380. Water stress developing over 4 days decreased the assimilation of both hybrids similarly. Changes in the amounts of chlorophyll, soluble and Rubisco protein, and Rubisco activity and activation state were small and were not sufficient to explain the decrease in photosynthesis; neither was decreased stomatal conductance (or stomatal "patchiness"). Reduction of photosynthesis per unit leaf area from 25 to 5 micromoles CO(2) per square meter per second in both hybrids was caused by a decrease in the amount of RuBP from approximately 130 to 40 micromoles per square meter in SH-3622 and from 80 to 40 micromoles per square meter in Sungro. Differences between hybrids and their response to water stress is discussed in relation to control of RuBP regeneration.

Photosynthesis and photorespiratory CO2 evolution of water-stressed sunflower leaves.

Rates of true photosynthesis (TPS), apparent photosynthesis (APS) and photorespiration (PR) of sunflower (Helianthus annuus L., Var. Mennonite) leaves were measured in air (21% O2, 300 vpm CO2) at 25° C and 400 µEinsteins m(-2) s(-1) radiant flux density. The plants were water stressed by application of osmoticum (polyethylene glycol 4000) to the root system. TPS and APS decreased linearly from maxima at-4 bar leaf-water potential (ψ) to become very small and zero respectively at about-18 bar ψ; at smaller potential CO2 was evolved from the leaf. Statistical analysis shows that TPS and APS were more closely correlated with ψ than stomatal conductance (r s (-1)), because r s (-1) changed only in the range-4 to-13 bar but ψ exerted an effect at smaller potential. Photorespiration decreased linearly with stress and at-18 bar was 30% of the control plant rate; ψ and TPS accounted for only part of the variance in PR, both independently and in combination, and r s (-1) accounted for little of the variance. Tricarboxylic acid cycle respiration of leaves placed for 20 min in darkness, remained almost constant with changing ψ and r s (-1). It was one-third of photorespiration in control plants but increased as a proportion in severely stressed plants. The relative specific activity (RSA) of the CO2 released by PR of wellwatered plants was 90% after 20 min photosynthesis in (14)CO2 but decreased to 18% at-18 bar ψ. Therefore, under stress mpre CO2 was derived by respiration from reserve materials and less from immediate photosynthate. Elimination of CO2 production by the glycollate pathway with small oxygen concentration (1.5%), showed that the contribution of TCA cycle respiration to photorespiration was small in unstressed plants but increased at small ψ to almost the same rate as photorespiration. It is concluded that desiccation decreased photosynthesis by decreasing the stomatal conductance to CO2 diffusion and by changing the balance between CO2 assimilation and production of the leaf. As a consequence carbon flux through the glycollate pathway decreased as did the rate of CO2 produced by it. However, TCA cycle respiration in the light increased with stress, so that total photorespiration remained large. The importance of maintaining carbon flux through the glycollate pathway and TCA cycle is discussed.