Can the cold tolerance of C4 photosynthesis in Miscanthus x giganteus relative to Zea mays be explained by differences in activities and thermal properties of Rubisco?

The previous investigations show that the amount and activity of Rubisco appears the major limitation to effective C(4) photosynthesis at low temperatures. The chilling-tolerant and bioenergy feedstock species Miscanthus x giganteus (M. x giganteus) is exceptionally productive among C(4) grasses in cold climates. It is able to develop photosynthetically active leaves at temperatures 6 degrees C below the minimum for maize, and achieves a productivity even at 52 degrees N that exceeds that of the most productive C(3) crops at this latitude. This study investigates whether this unusual low temperature tolerance can be attributed to differences in the amount or kinetic properties of Rubisco relative to maize. An efficient protocol was developed to purify large amounts of functional Rubisco from C(4) leaves. The maximum carboxylation activities (V(max)), activation states, catalytic rates per active site (K(cat)) and activation energies (E(a)) of purified Rubisco and Rubisco in crude leaf extracts were determined for M. x giganteus grown at 14 degrees C and 25 degrees C, and maize grown at 25 degrees C. The sequences of M. x giganteus Rubisco small subunit mRNA are highly conserved, and 91% identical to those of maize. Although there were a few differences between the species in the translated protein sequences, there were no significant differences in the catalytic properties (V(max), K(cat), and E(a)) for purified Rubisco, nor was there any effect of growth temperature in M. x giganteus on these kinetic properties. Extracted activities were close to the observed rates of CO(2) assimilation by the leaves in vivo. On a leaf area basis the extracted activities and activation state of Rubisco did not differ significantly, either between the two species or between growth temperatures. The activation state of Rubisco in leaf extracts showed no significant difference between warm and cold-grown M. x giganteus. In total, these results suggest that the ability of M. x giganteus to be productive and maintain photosynthetically competent leaves at low temperature does not result from low temperature acclimation or adaptation of the catalytic properties of Rubisco.

Photosynthetic symmetry of sun and shade leaves of different orientations.

The photosynthetic responses to light of leaves irradiated on the adaxial or abaxial surfaces, were measured for plants with contrasting leaf orientations. For vertical-leaf species of open habitats (Eryngium yuccifolium and Silphium terebinthinaceum), photosynthetic rates were identical when irradiated on either surface. However, for horizontal-leaf species of open habitats (Ambrosia trifida and Solidago canadensis), light-saturated rates of photosynthesis for adaxial irradiation were 19 to 37% higher than rates for abaxial irradiation. Leaves of understory plants (Asarum canadense and Hydrophyllum canadense) were functionally symmetrical although they had horizontal orientation. Photosynthetic rates were measured at saturating CO2, thus differences in the response to incident irradiance presumably resulted from complex interactions of light and leaf optical properties rather than from stomatal effects. Differences in absorptance (400-700 nm) among leaf surfaces were evident for horizontal-leaf species but the primary determinant of functional symmetry was leaf anatomy. Functionally symmetrical leaves had upper and lower palisade layers of equal thickness (vertical leaves of open habitats) or were composed primarily of a single layer of photosynthetic cells (horizontal leaves of understory habitats). Photosynthetic symmetry of vertical-leaf species may be an adaptation to maximize daily integrated carbon gain and water-use efficiency, whereas asymmetry of horizontal-leaf species may be an adaptation to maximize daily integrated carbon gain and photosynthetic nutrient-use efficiency.

Forest carbon balance under elevated CO2.

Free-air CO2 enrichment (FACE) technology was used to expose a loblolly pine (Pinus taeda L.) forest to elevated atmospheric CO2 (ambient + 200 µl l-1). After 4 years, basal area of pine trees was 9.2% larger in elevated than in ambient CO2 plots. During the first 3 years the growth rate of pine was stimulated by ~26%. In the fourth year this stimulation declined to 23%. The average net ecosystem production (NEP) in the ambient plots was 428 gC m-2 year-1, indicating that the forest was a net sink for atmospheric CO2. Elevated atmospheric CO2 stimulated NEP by 41%. This increase was primarily an increase in plant biomass increment (57%), and secondarily increased accumulation of carbon in the forest floor (35%) and fine root increment (8%). Net primary production (NPP) was stimulated by 27%, driven primarily by increases in the growth rate of the pines. Total heterotrophic respiration (R h) increased by 165%, but total autotrophic respiration (R a) was unaffected. Gross primary production was increased by 18%. The largest uncertainties in the carbon budget remain in separating belowground heterotrophic (soil microbes) and autotrophic (root) respiration. If applied to temperate forests globally, the increase in NEP that we measured would fix less than 10% of the anthropogenic CO2 projected to be released into the atmosphere in the year 2050. This may represent an upper limit because rising global temperatures, land disturbance, and heterotrophic decomposition of woody tissues will ultimately cause an increased flux of carbon back to the atmosphere.

Can improvement in photosynthesis increase crop yields?

The yield potential (Yp) of a grain crop is the seed mass per unit ground area obtained under optimum growing conditions without weeds, pests and diseases. It is determined by the product of the available light energy and by the genetically determined properties: efficiency of light capture (epsilon i), the efficiency of conversion of the intercepted light into biomass (epsilon c) and the proportion of biomass partitioned into grain (eta). Plant breeding brings eta7 and epsilon i close to their theoretical maxima, leaving epsilon c, primarily determined by photosynthesis, as the only remaining major prospect for improving Yp. Leaf photosynthetic rate, however, is poorly correlated with yield when different genotypes of a crop species are compared. This led to the viewpoint that improvement of leaf photosynthesis has little value for improving Yp. By contrast, the many recent experiments that compare the growth of a genotype in current and future projected elevated [CO2] environments show that increase in leaf photosynthesis is closely associated with similar increases in yield. Are there opportunities to achieve similar increases by genetic manipulation? Six potential routes of increasing epsilon c by improving photosynthetic efficiency were explored, ranging from altered canopy architecture to improved regeneration of the acceptor molecule for CO2. Collectively, these changes could improve epsilon c and, therefore, Y p by c. 50%. Because some changes could be achieved by transgenic technology, the time of the development of commercial cultivars could be considerably less than by conventional breeding and potentially, within 10-15 years.

Photosynthesis, productivity, and yield of maize are not affected by open-air elevation of CO2 concentration in the absence of drought.

While increasing temperatures and altered soil moisture arising from climate change in the next 50 years are projected to decrease yield of food crops, elevated CO2 concentration ([CO2]) is predicted to enhance yield and offset these detrimental factors. However, C4 photosynthesis is usually saturated at current [CO2] and theoretically should not be stimulated under elevated [CO2]. Nevertheless, some controlled environment studies have reported direct stimulation of C4 photosynthesis and productivity, as well as physiological acclimation, under elevated [CO2]. To test if these effects occur in the open air and within the Corn Belt, maize (Zea mays) was grown in ambient [CO2] (376 micromol mol(-1)) and elevated [CO2] (550 micromol mol(-1)) using Free-Air Concentration Enrichment technology. The 2004 season had ideal growing conditions in which the crop did not experience water stress. In the absence of water stress, growth at elevated [CO2] did not stimulate photosynthesis, biomass, or yield. Nor was there any CO2 effect on the activity of key photosynthetic enzymes, or metabolic markers of carbon and nitrogen status. Stomatal conductance was lower (-34%) and soil moisture was higher (up to 31%), consistent with reduced crop water use. The results provide unique field evidence that photosynthesis and production of maize may be unaffected by rising [CO2] in the absence of drought. This suggests that rising [CO2] may not provide the full dividend to North American maize production anticipated in projections of future global food supply.

Potential mechanisms of low-temperature tolerance of C4 photosynthesis in Miscanthus x giganteus: an in vivo analysis.

Miscanthus x giganteus (Greef & Deuter ex Hodkinson & Renvoize) is unique among C4 species in its remarkable ability to maintain high photosynthetic productivity at low temperature, by contrast to the related C4 NADP-malic enzyme-type species Zea mays L. In order to determine the in vivo physiological basis of this difference in photosynthesis, water vapor and CO2 exchange and modulated chlorophyll fluorescence were simultaneously monitored on attached leaf segments from plants grown and measured at 25/20 degrees C or 14/11 degrees C (day/night temperature). Analysis of the response of photosynthesis to internal CO2 concentration suggested that ribulose bisphosphate carboxylase/oxygenase (Rubisco) and/or pyruvate orthophosphate dikinase (PPDK) play a more important role in determining the response to low temperature than does phosphoenolpyruvate carboxylase (PEPc). For both species at both temperatures, the linear relationship between operating efficiency of whole-chain electron transport through photosystem II (Phi(PSII)) and the efficiency of CO2 assimilation (Phi(CO2)) was unchanged and had a zero intercept, suggesting the absence of non-photosynthetic electron sinks. The major limitation at low temperature could not be solely at Rubisco or at any other point in the Calvin cycle, since this would have increased leakage of CO2 to the mesophyll and increased Phi(PSII)/Phi(CO2). This in vivo analysis suggested that maintenance of high photosynthetic rates in M. x giganteus at low temperature, in contrast to Z. mays, is most likely the result of different properties of Rubisco and/or PPDK, reduced susceptibility to photoinhibition, and the ability to maintain high levels of leaf absorptance during growth at low temperature.

Cold tolerance of C4 photosynthesis in Miscanthus x giganteus: adaptation in amounts and sequence of C4 photosynthetic enzymes.

Field-grown Miscanthus x giganteus maintains high photosynthetic quantum yields and biomass productivity in cool temperate climates. It is related to maize (Zea mays) and uses the same NADP-malic enzyme C(4) pathway. This study tests the hypothesis that M. x giganteus, in contrast to maize, forms photosynthetically competent leaves at low temperatures with altered amounts of pyruvate orthophosphate dikinase (PPDK) and Rubisco or altered properties of PPDK. Both species were grown at 25 degrees C/20 degrees C or 14 degrees C/11 degrees C (day/night), and leaf photosynthesis was measured from 5 degrees C to 38 degrees C. Protein and steady-state transcript levels for Rubisco, PPDK, and phosphoenolpyruvate carboxylase were assessed and the sequence of C(4)-PPDK from M. x giganteus was compared with other C(4) species. Low temperature growth had no effect on photosynthesis in M. x giganteus, but decreased rates by 80% at all measurement temperatures in maize. Amounts and expression of phosphoenolpyruvate carboxylase were affected little by growth temperature in either species. However, PPDK and Rubisco large subunit decreased >50% and >30%, respectively, in cold-grown maize, whereas these levels remained unaffected by temperature in M. x giganteus. Differences in protein content in maize were not explained by differences in steady-state transcript levels. Several different M. x giganteus C(4)-PPDK cDNA sequences were found, but putative translated protein sequences did not show conservation of amino acids contributing to cold stability in Flaveria brownii C(4)-PPDK. The maintenance of PPDK and Rubisco large subunit amounts in M. x giganteus is consistent with the hypothesis that these proteins are critical to maintaining high rates of C(4) photosynthesis at low temperature.