The Effects of Chilling in the Light on Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activation in Tomato (Lycopersicon esculentum Mill.).

Photosynthesis rate, ribulsoe-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation state, and ribulose bisphosphate concentration were reduced after exposing tomato (Lycopersicon esculentum Mill.) plants to light at 4[deg]C for 6 h. Analysis of lysed and reconsituted chloroplasts showed that activity of the thylakoid membrane was inhibited and that Rubisco, Rubisco activase, and other soluble factors were not affected. Leaf photosynthesis rates and the ability of chilled thylakoid membranes to promote Rubisco activation recovered after 24 h at 25[deg]C. Thylakoid membranes from control tomato plants were as effective as spinach thylakoids in activating spinach Rubisco in the presence of spinach Rubisco activase. This observation is in sharp contrast to the poor ability of spinach Rubisco activase to activate tomato Rubisco (Z.-Y. Wang, G.W. Snyder, B.D. Esau, A.R. Portis, and W.L. Ogren [1992] Plant Physiol 100: 1858-1862). The ability of thylakoids from chilled tomato plants to activate Rubisco in the assay system was greatly inhibited compared to control plants. These experiments indicate that chilling tomato plants at 4[deg]C interferes with photosynthetic carbon metabolism at two sites, thioredoxin/ferredoxin reduction (G.F. Sassenrath, D.R. Ort, and A.R. Portis, Jr. [1990] Arch Biochem Biophys 282: 302-308), which limits bisphosphatase activity, and Rubisco activase, which reduces Rubisco activation state.

Leaf cavity CO2 concentrations and CO2 exchange in onion, Allium cepa L.

Onion (Allium cepa L.) plants were examined to determine the photosynthetic role of CO2 that accumulates within their leaf cavities. Leaf cavity CO2 concentrations ranged from 2250 µL L(-1) near the leaf base to below atmospheric (<350 µL L(-1)) near the leaf tip at midday. There was a daily fluctuation in the leaf cavity CO2 concentrations with minimum values near midday and maximum values at night. Conductance to CO2 from the leaf cavity ranged from 24 to 202 µmol m(-2) s(-1) and was even lower for membranes of bulb scales. The capacity for onion leaves to recycle leaf cavity CO2 was poor, only 0.2 to 2.2% of leaf photosynthesis based either on measured CO2 concentrations and conductance values or as measured directly by (14)CO2 labeling experiments. The photosynthetic responses to CO2 and O2 were measured to determine whether onion leaves exhibited a typical C3-type response. A linear increase in CO2 uptake was observed in intact leaves up to 315 µL L(-1) of external CO2 and, at this external CO2 concentration, uptake was inhibited 35.4±0.9% by 210 mL L(-1) O2 compared to 20 mL L(-1) O2. Scanning electron micrographs of the leaf cavity wall revealed degenerated tissue covered by a membrane. Onion leaf cavity membranes apparently are highly impermeable to CO2 and greatly restrict the refixation of leaf cavity CO2 by photosynthetic tissue.

Estimation of Bundle Sheath Cell Conductance in C4 Species and O2 Insensitivity of Photosynthesis.

Low conductance to CO2 of bundle sheath cells is required in C4 photosynthesis to maintain high [CO2] at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Elevated [CO2] allows high CO2 assimilation rates by this enzyme and prevents Rubisco oxygenase activity and O2 inhibition of carboxylation. Bundle sheath conductance to CO2 was estimated by chemically inhibiting phosphoenolpyruvate carboxylase and calculating the slope of the linear response of leaf CO2 uptake to [CO2]. The inhibitor 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate was supplied to detached leaves of Panicum maximum, Panicum miliaceum, and Sorghum bicolor at 4 mM. Uptake of CO2 was measured at 210 mL L-1 O2 over the CO2 concentration range of 0.34 to 28 mL L-1. Without the inhibitor, CO2 uptake increased steeply at low [CO2] and saturated at about 1 mL L-1. After inhibition, CO2 uptake was a linear function of [CO2] over much of the range tested. The slope of this CO2 response, taken as bundle sheath conductance, was 2.35, 1.96, and 1.13 mmol m-2 s-1 for P. maximum, P. miliaceum, and S. bicolor, respectively, on a leaf area basis. Conductance based on bundle sheath area was 0.76, 0.93, and 0.54 mmol m-2 s-1, respectively. Uptake of CO2 by leaves of P. maximum supplied with the inhibitor was not affected by reduction of [O2] from 210 to 20 mL L-1 over the range of [CO2] used. Because [CO2] in bundle sheath cells of inhibited leaves is likely to be much lower than ambient, the lack of O2 sensitivity of CO2 uptake cannot be ascribed to lack of O2 reaction with ribulose bisphosphate and is probably due to the low conductance of bundle sheath cells, especially at low ambient [CO2]. The likely result of reducing [O2] from 210 to 20 mL L-1 is to stimulate carboxylation of ribulose bisphosphate, thus further reducing [CO2] in bundle sheath cells and increasing CO2 diffusion to these cells from the mesophyll. However, the increase in diffusion is greatly limited by low conductance of the bundle sheath cell walls. Calculations based on estimated bundle sheath conductance show that changes in bundle sheath [CO2] of 0.085 to 0.5 mL L-1, which might be associated with reduced [O2], would have a negligible effect on CO2 uptake.

Photosynthetic Characteristics of Segregates from Hybrids between Flaveria brownii (C4 Like) and Flaveria linearis (C3-C4).

Characteristics related to C4 photosynthesis were studied in reciprocal F1 hybrids and F2 plants from Flaveria brownii (C4 like) and Flaveria linearis (C3-C4). The reciprocal F1 plants differed in 13C/12C ratios of leaves and the percentage of 14C initially incorporated into C4 acids, being more like the pollen parents in these traits. They did not differ in apparent photosynthesis or in O2 inhibition of apparent photosynthesis and differed only slightly in CO2 compensation concentration at 175 [mu]mol quanta m-2 s-1 and 400 mL L-1 O2. The 13C/12C ratios of 78 F2 progeny from the two F1 plants exhibited a normal distribution centered between those of the parents, with a few values slightly higher and lower than the parents. Apparent photosynthesis at 130 [mu]L L-1 CO2 and inhibition of photosynthesis by O2 was nearly normally distributed in the F2 population, but no values for F2 plants approached those for F. brownii (15.4 [mu]mol m-2 s-1 and 7.8%, respectively). Distribution of the CO2 compensation concentration measured at 1000 [mu]mol quanta m-2 s-1 and 400 mL L-1 of O2 in the F2 population was skewed toward F. brownii with 72% of the progeny having values <9 [mu]L of CO2 L-1 compared to 1.5 and 27.2 [mu]L L-1 for F. brownii and F. linearis, respectively. Correlations among traits of F2 plants were low (coefficients of 0.30 to -0.49), indicating that the C4- related traits are not closely linked in segregating populations. Plants in the F2 population selected for high or low apparent photosynthesis at 130 [mu]L of CO2 L-1 (six each) did not rank consistently high or low for 13C/12C ratios, O2 inhibition of apparent photosynthesis, CO2 compensation concentration, or activities of phosphoenolpyruvate carboxylase or NADP-malic enzyme. This study confirms results of earlier work that indicates independent segregation of C4 traits and also shows that the C4-like parental type can be recovered, at least for some characteristics (13C/12C ratio), in segregating populations. Recovery of fully functional C4 plants awaits further experimentation with C4 x C3 or C4 x C3-C4 hybrid plants that produce fertile progeny.

Novel characteristics of cassava, Manihot esculenta Crantz, a reputed C3-C 4 intermediate photosynthesis species.

The cassava plant, Manihot esculenta, grows exceptionally well in low fertility and drought prone environments, but the mechanisms that allow this growth are unknown. Earlier, and sometimes contradictory, work speculated about the presence of a C4-type photosynthesis in cassava leaves. In the present work we found no evidence for a C4 metabolism in mature attached cassava leaves as indicated i) by the low, 2 to 8%, incorporation of (14)CO2 into C4 organic acids in short time periods, 10 s, and the lack of (14)C transfer from C4 acids to other compounds in (12)CO2, ii) by the lack of C4 enzyme activity changes during leaf development and the inability to detect C4 acid decarboxylases, and iii) by leaf CO2 compensation values between 49 and 65 µl of CO2 1(-1) and by other infrared gas exchange photosynthetic measurements. It is concluded that the leaf biochemistry of cassava follows the C3 pathway of photosynthesis with no indication of a C3-C4 mechanism.However, cassava leaves exhibit several novel characteristics. Attached leaves have the ability to effectively partition carbon into sucrose with nearly 45% of the label in sucrose in about one min of (14)CO2 photosynthesis, contrasting with 34% in soybean (C3) and 25% in pigweed (C4). Cassava leaves displayed a strong preference for the synthesis of sucrose versus starch. Field grown cassava leaves exhibited high rates of photosynthesis and curvilinear responses to increasing sunlight irradiances with a tendency to saturate only at high irradiances, above 1500 µmol m(-2) s(-1). Morphologically, the cassava leaf has papillose epidermal cells on its lower mesophyll surface that form 'fence-like' arrangements encircling guard cells. It is proposed that the active synthesis of sugars has osmotic functions in the cassava plant and that the papillose epidermal cells function to maintain a healthy leaf water status in various environments.

Degree of C(4) Photosynthesis in C(4) and C(3)-C(4)Flaveria Species and Their Hybrids : II. Inhibition of Apparent Photosynthesis by a Phosphoenolpyruvate Carboxylase Inhibitor.

Hybrids have been made between species of Flaveria exhibiting varying levels of C(4) photosynthesis. The degree of C(4) photosynthesis expressed in four interspecific hybrids (Flaveria trinervia [C(4)] x F. linearis [C(3)-C(4)], F. brownii [C(4)-like] x F. linearis, and two three-species hybrids from F. trinervia x [F. brownii x F. linearis]) was estimated by inhibiting phosphoenolpyruvate carboxylase in vivo with 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate (DCDP). The inhibitor was fed to detached leaves at a concentration of 4 mm, and apparent photosynthesis was measured at atmospheric levels of CO(2) and at 20 and 210 mL L(-1) of O(2). Photosynthesis at 210 mL L(-1) of O(2) was inhibited 32% by DCDP in F. linearis, by 60% in F. brownii, and by 87% in F. trinervia. Inhibition in the hybrids ranged from 38 to 52%. The inhibition of photosynthesis by 210 mL L(-1) of O(2) was increased when DCDP was used, except in the C(4) species, F. trinervia, in which photosynthesis was insensitive to O(2). Except for F. trinervia, control plants with less O(2) sensitivity (more C(4)-like) exhibited a progressively greater change in O(2) inhibition of photosynthesis when treated with DCDP. This increased O(2) inhibition probably resulted from decreased CO(2) concentrations in bundle sheath cells due to inhibition of phosphoenolpyruvate carboxylase. The inhibition of photosynthesis by DCDP is concluded to underestimate the degree of C(4) photosynthesis in the interspecific hybrids because increased direct assimilation of atmospheric CO(2) by ribulose bisphosphate carboxylase may compensate for inhibition of phosphoenolpyruvate carboxylase.

Degree of C(4) Photosynthesis in C(4) and C(3)-C(4)Flaveria Species and Their Hybrids : I. CO(2) Assimilation and Metabolism and Activities of Phosphoenolpyruvate Carboxylase and NADP-Malic Enzyme.

The degree of C(4) photosynthesis was assessed in four hybrids among C(4), C(4)-like, and C(3)-C(4) species in the genus Flaveria using (14)C labeling, CO(2) exchange, (13)C discrimination, and C(4) enzyme activities. The hybrids incorporated from 57 to 88% of the (14)C assimilated in a 10-s exposure into C(4) acids compared with 26% for the C(3)-C(4) species Flaveria linearis, 91% for the C(4) species Flaveria trinervia, and 87% for the C(4)-like Flaveria brownii. Those plants with high percentages of (14)C initially fixed into C(4) acids also metabolized the C(4) acids quickly, and the percentage of (14)C in 3-phosphoglyceric acid plus sugar phosphates increased for at least a 30-s exposure to (12)CO(2). This indicated a high degree of coordination between the carbon accumulation and reduction phases of the C(4) and C(3) cycles. Synthesis and metabolism of C(4) acids by the species and their hybrids were highly and linearly correlated with discrimination against (13)C. The relationship of (13)C discrimination or (14)C metabolism to O(2) inhibition of photosynthesis was curvilinear, changing more rapidly at C(4)-like values of (14)C metabolism and (13)C discrimination. Incorporation of initial (14)C into C(4) acids showed a biphasic increase with increased activities of phosphoenolpyruvate carboxylase and NADP-malic enzyme (steep at low activities), but turnover of C(4) acids was linearly related to NADP-malic enzyme activity. Several other traits were closely related to the in vitro activity of NADP-malic enzyme but not phosphoenolpyruvate carboxylase. The data indicate that the hybrids have variable degrees of C(4) photosynthesis but that the carbon accumulation and reduction portions of the C(4) and C(3) cycles are well coordinated.

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

Lower respiratory costs were hypothesized as providing an additional benefit in C(4) plants compared to C(3) plants due to less investment in proteins in C(4) leaves. Therefore, photosynthesis and dark respiration of mature leaves were compared between a number of C(4) and C(3) species. Although photosynthetic rates were generally greater in C(4) when compared to C(3) 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 (C(4)) and Chenopodium album (C(3)). 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 C(4) 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 CO(2) uptake and CO(2) efflux in the dark. The dependence of nightly CO(2) efflux on CO(2) uptake was similar for both species, although the response of CO(2) uptake to leaf nitrogen was much steeper in the C(4) 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 degrees C. The data suggest no obvious differences in respiratory costs in C(4) and C(3) plants.

Assessing the degree of c(4) photosynthesis in c(3)-c(4) species using an inhibitor of phosphoenolpyruvate carboxylase.

An analog of phosphoenolpyruvate, 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate (DCDP), was used to inhibit phosphoenolpyruvate carboxylase and, therefore, assess the contribution of the C(4) pathway to photosynthesis in detached leaves of several C(3)-C(4) intermediate species. There was no effect of 4 millimolar DCDP on apparent photosynthesis or on inhibition of apparent photosynthesis by 210 milliliters per liter of O(2) for the C(3)-C(4) species Panicum milioides, Moricandia arvensis, and Neurachne minor or the C(3) species Flaveria pringlei. However, when leaves of Flaveria linearis (C(3)-C(4)), Flaveria brownii (C(4)-like), and Flaveria trinervia (C(4)) were fed 4 millimolar DCDP, photosynthesis was reduced 32, 60, and 90%, respectively. Photosynthetic inhibition by 210 milliliter per liter of O(2) was also significantly increased in 4 millimolar DCDP-fed leaves of F. linearis and F. brownii but not in F. trinervia when compared with control values. These results with DCDP clearly demarcate C(3)-C(4) species into species including Panicum, Moricandia, and Neurachne whose reduced photorespiration occurs without any C(4) photosynthetic involvement and species of Flaveria in which C(4) photosynthesis contributes to CO(2) assimilation.

Comparative effects of growth irradiance on photosynthesis and leaf anatomy of Flaveria brownii (C4-like), Flaveria linearis (C 3-C 4) and their F 1 hybrid.

Photosynthetic rates and related anatomical characteristics of leaves developed at three levels of irradiance (1200, 300 and 80 umol · m(-2) · s(-1)) were determined in the C4-like species Flaveria brownii A.M. Powell, the C3-C4-intermediate species F. linearis Lag., and the F1 hybrid between them (F. brownii × F. linearis). In the C3-C4 and F1 plants, increases in photosynthetic capacity per unit leaf area were strongly correlated with changes in mesophyll area per unit leaf area. The C4-like plant F. brownii, however, showed a much lower correlation between photosynthetic capacity and mesophyll area per unit leaf area. Plants of F. brownii developed at high irradiance showed photosynthetic rates per unit of mesophyll cell area 50% higher than those plants developed at medium irradiance. These results along with an increase in water-use efficiency are consistent with an increase of C4 photosynthesis in high-irradiance-grown F. brownii plants, whereas in the other two genotypes such plasticity seems to be absent. Photosynthetic discrimination against (13)C in the three genotypes was less at high than at low irradiance, with the greatest change occurring in F. brownii. Discrimination against (13)C expressed as δ (13)C was linearly correlated (r (2) = 0.81; P<0.001) with the ratio of bundle-sheath volume to mesophyll cell area when all samples from the three genotypes were combined. This tissue ratio increased for F. brownii and the F1 hybrid as growth irradiance increased, indicating a greater tendency towards Kranz anatomy. The results indicated that F. brownii had plasticity in its C4-related anatomical and physiological characteristics as a function of growth irradiance, whereas plasticity was less evident in the F1 hybrid and absent in F. linearis.