Genetic transformation of the C4 plant, Flaveria bidentis

An efficient and rapid transformation system for the dicotyledonous C₄ plant, Flaveria bidentis has been developed. The method involves Agrobacterium tumefaciens infection of explants followed by regeneration of shoots from kanamycin-resistant callus. Shoots first appear on callus 3–4 weeks after Agrobacterium infection, with plants in soil after another 12–15 weeks. Transformation has been verified by measuring the activity of the gusA and npt-II transgenes and by genomic Southern blots to show the stable integration of T-DNA. Most regenerated plants show normal morphology, are self-fertile and transgenes show expected Mendelian segregations in the next generation. This system allows one to test a number of gene constructions rapidly and should accelerate progress in studies of the cell-specific expression of genes coding for C₄ enzymes and the mechanisms regulating the activities of C₄ enzymes.     

Dynamic photo-inhibition and carbon gain in a C4 and a C3 grass native to high latitudes

C₄ plants are rare in the cool climates characteristic of high latitudes and altitudes, perhaps because of an enhanced susceptibility to photo‐inhibition at low temperatures relative to C₃ species. In the present study we tested the hypothesis that low‐temperature photo‐inhibition is more detrimental to carbon gain in the C₄ grass Muhlenbergia glomerata than the C₃ species Calamogrostis Canadensis. These grasses occur together in boreal fens in northern Canada. Plants were grown under cool (14/10 °C day/night) and warm (26/22 °C) temperatures before measurement of the light responses of photosynthesis and chlorophyll fluorescence at different temperatures. Cool growth temperatures led to reduced rates of photosynthesis in M. glomerata at all measurement temperatures, but had a smaller effect on the C₃ species. In both species the amount of xanthophyll cycle pigments increased when plants were grown at 14/10 °C, and in M. glomerata the xanthophyll epoxidation state was greatly reduced. The detrimental effect of low growth temperature on photosynthesis in M. glomerata was almost completely reversed by a 24‐h exposure to the warm‐temperature regime. These data indicate that reversible dynamic photo‐inhibition is a strategy by which C₄ species may tolerate cool climates and overcome the Rubisco limitation that is prevalent at low temperatures in C₄ plants.     

Low-temperature photosynthetic performance of a C4 grass and a co-occurring C3 grass native to high latitudes

The photosynthetic performance of C₄ plants is generally inferior to that of C₃ species at low temperatures, but the reasons for this are unclear. The present study investigated the hypothesis that the capacity of Rubisco, which largely reflects Rubisco content, limits C₄ photosynthesis at suboptimal temperatures. Photosynthetic gas exchange, chlorophyll a fluorescence, and the in vitro activity of Rubisco between 5 and 35 °C were measured to examine the nature of the low‐temperature photosynthetic performance of the co‐occurring high latitude grasses, Muhlenbergia glomerata (C₄) and Calamogrostis canadensis (C₃). Plants were grown under cool (14/10 °C) and warm (26/22 °C) temperature regimes to examine whether acclimation to cool temperature alters patterns of photosynthetic limitation. Low‐temperature acclimation reduced photosynthetic rates in both species. The catalytic site concentration of Rubisco was approximately 5.0 and 20 µmol m^?2 in M. glomerata and C. canadensis, respectively, regardless of growth temperature. In both species, in vivo electron transport rates below the thermal optimum exceeded what was necessary to support photosynthesis. In warm‐grown C. canadensis, the photosynthesis rate below 15 °C was unaffected by a 90% reduction in O₂ content, indicating photosynthetic capacity was limited by the capacity of P_i‐regeneration. By contrast, the rate of photosynthesis in C. canadensis plants grown at the cooler temperatures was stimulated 20–30% by O₂ reduction, indicating the P_i‐regeneration limitation was removed during low‐temperature acclimation. In M. glomerata, in vitro Rubisco activity and gross CO₂ assimilation rate were equivalent below 25 °C, indicating that the capacity of the enzyme is a major rate limiting step during C₄ photosynthesis at cool temperatures.     

Gas exchange and photosynthetic acclimation over subambient to elevated CO2 in a C3–C4 grassland

Atmospheric CO₂ (C_a) has risen dramatically since preglacial times and is projected to double in the next century. As part of a 4‐year study, we examined leaf gas exchange and photosynthetic acclimation in C₃ and C₄ plants using unique chambers that maintained a continuous C_a gradient from 200 to 550 µmol mol^?1 in a natural grassland. Our goals were to characterize linear, nonlinear and threshold responses to increasing C_a from past to future C_a levels. Photosynthesis (A), stomatal conductance (g_s), leaf water‐use efficiency (A/g_s) and leaf N content were measured in three common species: Bothriochloa ischaemum, a C₄ perennial grass, Bromus japonicus, a C₃ annual grass, and Solanum dimidiatum, a C₃ perennial forb. Assimilation responses to internal CO₂ concentrations (A/C_i curves) and photosynthetically active radiation (A/PAR curves) were also assessed, and acclimation parameters estimated from these data. Photosynthesis increased linearly with C_a in all species (P < 0.05). S. dimidiatum and B. ischaemum had greater carboxylation rates for Rubisco and PEP carboxylase, respectively, at subambient than superambient C_a (P < 0.05). To our knowledge, this is the first published evidence of A up‐regulation at subambient C_a in the field. No species showed down‐regulation at superambient C_a. Stomatal conductance generally showed curvilinear decreases with C_a in the perennial species (P < 0.05), with steeper declines over subambient C_a than superambient, suggesting that plant water relations have already changed significantly with past C_a increases. Resource‐use efficiency (A/g_s and A/leaf N) in all species increased linearly with C_a. As both C₃ and C₄ plants had significant responses in A, g_s, A/g_s and A/leaf N to C_a enrichment, future C_a increases in this grassland may not favour C₃ species as much as originally thought. Non‐linear responses and acclimation to low C_a should be incorporated into mechanistic models to better predict the effects of past and present rising C_a on grassland ecosystems.     

Stomatal responses of C3, C3-C4 and C4Flaveria species to light and intercellular CO2 concentration: implications for the evolution of stomatal behaviour

Stomatal function mediates physiological trade‐offs associated with maintaining a favourable H₂O balance in leaf tissues while acquiring CO₂ as a photosynthetic substrate. The C₃ and C₄ species appear to have different patterns of stomatal response to changing light conditions, and variation in this behaviour may have played a role in the functional diversification of the different photosynthetic pathways. In the current study, we used gain analysis theory to characterize the stomatal conductance response to light intensity in nine different C₃, C₄ and C₃‐C₄ intermediate species Flaveria species. The response of stomatal conductance (g_s) to a change in light intensity represents both a direct (related to a change in incident light intensity, I) and indirect (related to a change in intercellular CO₂ concentration, C_i) response. The slope of the line relating the change in g_s to C_i was steeper in C₄ species, compared with C₃ species, with C₃‐C₄ species having an intermediate response. This response reflects the greater relative contribution of the indirect versus direct component of the g_s versus I response in the C₄ species. The C₃‐C₄ species, Flaveria floridana, exhibited a C₄‐like response whereas the C₃‐C₄ species, Flaveria sonorensis and Flaveria chloraefolia, exhibited C₃‐like responses, similar to their hypothesized position along the evolutionary trajectory of the development of C₄ photosynthesis. There was a positive correlation between the relative contribution of the indirect component of the g_s versus I response and water use efficiency when evaluated across all species. Assuming that the C₃‐C₄ intermediate species reflect an evolutionary progression from fully expressed C₃ ancestors, the results of the current study demonstrate an increase in the contribution of the indirect component of the g_s versus I response as taxa evolve toward the C₄ extreme. The greater relative contribution of the indirect component of the stomatal response occurs through both increases in the indirect stomatal components and through decreases in the direct. Increases in the magnitude of the indirect component may be related to the maintenance of higher water use efficiencies in the intermediate evolutionary stages, before the appearance of fully integrated C₄ photosynthesis.     

Root responses along a subambient to elevated CO2 gradient in a C3–C4 grassland

Atmospheric CO₂ (C_a) concentration has increased significantly during the last 20 000 years, and is projected to double this century. Despite the importance of belowground processes in the global carbon cycle, community‐level and single species root responses to rising C_a are not well understood. We measured net community root biomass over 3 years using ingrowth cores in a natural C₃–C₄ grassland exposed to a gradient of C_a from preglacial to future levels (230–550 μmol mol^?1). Root windows and minirhizotron tubes were installed below naturally occurring stands of the C₄ perennial grass Bothriochloa ischaemum and its roots were measured for respiration, carbohydrate concentration, specific root length (SRL), production, and lifespan over 2 years. Community root biomass increased significantly (P<0.05) with C_a over initial conditions, with linear or curvilinear responses depending on sample date. In contrast, B. ischaemum produced significantly more roots at subambient than elevated C_a in minirhizotrons. The lifespan of roots with five or more neighboring roots in minirhizotron windows decreased significantly at high C_a, suggesting that after dense root growth depletes soil resource patches, plants with carbon surpluses readily shed these roots. Root respiration in B. ischaemum showed a curvilinear response to C_a under moist conditions in June 2000, with the lowest rates at C_a<300 μmol mol^?1 and peak activity at 450 μmol mol^?1 in a quadratic model. B. ischaemum roots at subambient C_a had higher SRLs and slightly higher carbohydrate concentrations than those at higher C_a, which may be related to drier soils at low C_a. Our data emphasize that belowground responses of plant communities to C_a can be quite different from those of the individual species, and suggest that complex interactions between and among roots and their immediate soil environment influence the responses of root physiology and lifespan to changing C_a.     

Salinity and sea level mediate elevated CO2 effects on C3–C4 plant interactions and tissue nitrogen in a Chesapeake Bay tidal wetland

Elevated atmospheric carbon dioxide concentrations ([CO₂]) generally increase plant photosynthesis in C₃ species, but not in C₄ species, and reduce stomatal conductance in both C₃ and C₄ plants. In addition, tissue nitrogen concentration ([N]) often fails to keep pace with enhanced carbon gain under elevated CO₂, particularly in C₃ species. While these responses are well documented in many species, implications for plant growth and nutrient cycling in native ecosystems are not clear. Here we present data on 18 years of measurement of above and belowground biomass, tissue [N] and total standing crop of N for a Scirpus olneyi‐dominated (C₃ sedge) community, a Spartina patens‐dominated (C₄ grass) community and a C₃–C₄‐mixed species community exposed to ambient and elevated (ambient +340 ppm) atmospheric [CO₂] in natural salinity and sea level conditions of a Chesapeake Bay wetland. Increased biomass production (shoots plus roots) under elevated [CO₂] in the S. olneyi‐dominated community was sustained throughout the study, averaging approximately 35%, while no significant effect of elevated [CO₂] was found for total biomass in the C₄‐dominated community. We found a significant decline in C₄ biomass (correlated with rising sea level) and a concomitant increase in C₃ biomass in the mixed community. This shift from C₄ to C₃ was accelerated by the elevated [CO₂] treatment. The elevated [CO₂] stimulation of total biomass accumulation was greatest during rainy, low salinity years: the average increase above the ambient treatment during the three wettest years (1994, 1996, 2003) was 2.9 t ha⁻¹ but in the three driest years (1995, 1999, 2002), it was 1.2 t ha⁻¹. Elevated [CO₂] depressed tissue [N] in both species, but especially in the S. olneyi where the relative depression was positively correlated with salinity and negatively related with the relative enhancement of total biomass production. Thus, the greatest amount of carbon was added to the S. olneyi‐dominated community during years when shoot [N] was reduced the most, suggesting that the availability of N was not the most or even the main limitation to elevated [CO₂] stimulation of carbon accumulation in this ecosystem.     

Photosynthesis, immediate export and carbon partitioning in source leaves of C3, C3-C4 intermediate, and C4Panicum and Flaveria species at ambient and elevated CO2 levels

Immediate export in leaves of C₃‐C₄ intermediates were compared with their C₃ and C₄ relatives within the Panicum and Flaveria genera. At 35 Pa CO₂, photosynthesis and export were highest in C₄ species in each genera. Within the Panicum, photosynthesis and export in ‘type I’ C₃‐C₄ intermediates were greater than those in C₃ species. However, ‘type I’ C₃‐C₄ intermediates exported a similar proportion of newly fixed ¹⁴C as did C₄ species. Within the Flaveria, ‘type II’ C₃‐C₄ intermediate species had the lowest export rather than the C₃ species. At ambient CO₂, immediate export was strongly correlated with photosynthesis. However, at 90 Pa CO₂, when photosynthesis and immediate export increased in all C₃ and C₃‐C₄ intermediate species, proportionally less C was exported in all photosynthetic types than that at ambient CO₂. All species accumulated starch and sugars at both CO₂ levels. There was no correlation between immediate export and the pattern of ¹⁴C‐labelling into sugars and starch among the photosynthetic types within each genus. However, during CO₂ enrichment, C₄Panicum species accumulated sugars above the level of sugars and starch normally made at ambient CO₂, whereas the C₄Flaveria species accumulated only additional starch.     

C4 photosynthesis in a single C3 cell is theoretically inefficient but may ameliorate internal CO2 diffusion limitations of C3 leaves

Attempts are being made to introduce C₄ photosynthetic characteristics into C₃ crop plants by genetic manipulation. This research has focused on engineering single‐celled C₄‐type CO₂ concentrating mechanisms into C₃ plants such as rice. Herein the pros and cons of such approaches are discussed with a focus on CO₂ diffusion, utilizing a mathematical model of single‐cell C₄ photosynthesis. It is shown that a high bundle sheath resistance to CO₂ diffusion is an essential feature of energy‐efficient C₄ photosynthesis. The large chloroplast surface area appressed to the intercellular airspace in C₃ leaves generates low internal resistance to CO₂ diffusion, thereby limiting the energy efficiency of a single‐cell C₄ concentrating mechanism, which relies on concentrating CO₂ within chloroplasts of C₃ leaves. Nevertheless the model demonstrates that the drop in CO₂ partial pressure, pCO₂, that exists between intercellular airspace and chloroplasts in C₃ leaves at high photosynthetic rates, can be reversed under high irradiance when energy is not limiting. The model shows that this is particularly effective at lower intercellular pCO₂. Such a system may therefore be of benefit in water‐limited conditions when stomata are closed and low intercellular pCO₂ increases photorespiration.     

Stomatal acclimation over a subambient to elevated CO2 gradient in a C3/C4 grassland

An investigation to determine whether stomatal acclimation to [CO₂] occurred in C₃/C₄ grassland plants grown across a range of [CO₂] (200–550 µmol mol^?1) in the field was carried out. Acclimation was assessed by measuring the response of stomatal conductance (g_s) to a range of intercellular CO₂ (a g_s–C_i curve) at each growth [CO₂] in the third and fourth growing seasons of the treatment. The g_s–C_i response curves for Solanum dimidiatum (C₃ perennial forb) differed significantly across [CO₂] treatments, suggesting that stomatal acclimation had occurred. Evidence of non–linear stomatal acclimation to [CO₂] in this species was also found as maximum g_s (g_smax; g_s measured at the lowest C_i) increased with decreasing growth [CO₂] only below 400 µmol mol^?1. The substantial increase in g_s at subambient [CO₂] for S. dimidiatum was weakly correlated with the maximum velocity of carboxylation (V_cmax; r² = 0·27) and was not associated with CO₂ saturated photosynthesis (A_max). The response of g_s to C_i did not vary with growth [CO₂] in Bromus japonicus (C₃ annual grass) or Bothriochloa ischaemum (C₄ perennial grass), suggesting that stomatal acclimation had not occurred in these species. Stomatal density, which increased with rising [CO₂] in both C₃ species, was not correlated with g_s. Larger stomatal size at subambient [CO₂], however, may be associated with stomatal acclimation in S. dimidiatum. Incorporating stomatal acclimation into modelling studies could improve the ability to predict changes in ecosystem water fluxes and water availability with rising CO₂ and to understand their magnitudes relative to the past.     

δ 13C variation of C3 and C4 plants across an Asian monsoon rainfall gradient in arid northwestern China

We have investigated carbon isotopic compositions of four plant genus/species, Bothriochloa ischaemum (C₄), Stipa bungeana (C₃), Lespedeza sp. (C₃) and Heteropappus less (C₃), along a precipitation gradient in northwest China in order to assess the impact of water availability on the carbon isotopic discrimination against ¹³C during carbon assimilation in this area. This information is necessary for reconstruction of paleovegetation, particularly paleo‐C₃/C₄ plant ratios using δ¹³C value of organic matter in loess and paleosols in the Chinese Loess Plateau. The δ¹³C of C₃ plants, as a group, exhibits a negative correlation with the annual precipitation amount with a total change and sensitivity of 5‰ and ?1.1‰/100 mm, respectively, for the precipitation range from 200 to 700 mm. The C₄ grass, B. ischaemum responds to aridity by decreasing 1.7‰ for over the precipitation range from 350 to 700 mm; the plant δ¹³C is significantly correlated with annual precipitation with a slope ?0.61‰/100 mm. This result implies that without considering the effect of water availability on the plant δ¹³C values, reconstruction of percent C₄ vegetation during the last glaciation can be overestimated by about a factor of two.     

Stomatal sensitivity to vapour pressure difference over a subambient to elevated CO2 gradient in a C3/C4 grassland

In the present study the response of stomatal conductance (g_s) to increasing leaf‐to‐air vapour pressure difference (D) in early season C₃ (Bromus japonicus) and late season C₄ (Bothriochloa ischaemum) grasses grown in the field across a range of CO₂ (200–550 µmol mol^?1) was examined. Stomatal sensitivity to D was calculated as the slope of the response of g_s to the natural log of externally manipulated D (dg_s/dlnD). Increasing D and CO₂ significantly reduced g_s in both species. Increasing CO₂ caused a significant decrease in stomatal sensitivity to D in Br. japonicus, but not in Bo. ischaemum. The decrease in stomatal sensitivity to D at high CO₂ for Br. japonicus fit theoretical expectations of a hydraulic model of stomatal regulation, in which g_s varies to maintain constant transpiration and leaf water potential. The weaker stomatal sensitivity to D in Bo. ischaemum suggested that stomatal regulation of leaf water potential was poor in this species, or that non‐hydraulic signals influenced guard cell behaviour. Photosynthesis (A) declined with increasing D in both species, but analyses of the ratio of intercellular to atmospheric CO₂ (C_i/C_a) suggested that stomatal limitation of A occurred only in Br. japonicus. Rising CO₂ had the greatest effect on g_s and A in Br. japonicus at low D. In contrast, the strength of stomatal and photosynthetic responses to CO₂ were not affected by D in Bo. ischaemum. Carbon and water dynamics in this grassland are dominated by a seasonal transition from C₃ to C₄ photosynthesis. Interspecific variation in the response of g_s to D therefore has implications for predicting seasonal ecosystem responses to CO₂.     

Above- and below-ground responses of C3–C4 species mixtures to elevated CO2 and soil water availability

We evaluated the influences of CO₂[Control, ～ 370 µ mol mol ^{? 1}; 200 µ mol mol ^{? 1} above ambient applied by free‐air CO₂ enrichment (FACE)] and soil water (Wet, Dry) on above‐ and below‐ground responses of C₃ (cotton, Gossypium hirsutum) and C₄ (sorghum, Sorghum bicolor) plants in monocultures and two density mixtures. In monocultures, CO₂ enrichment increased height, leaf area, above‐ground biomass and reproductive output of cotton, but not sorghum, and was independent of soil water treatment. In mixtures, cotton, but not sorghum, above‐ground biomass and height were generally reduced compared to monocultures, across both CO₂ and soil water treatments. Density did not affect individual plant responses of either cotton or sorghum across the other treatments. Total (cotton + sorghum) leaf area and above‐ground biomass in low‐density mixtures were similar between CO₂ treatments, but increased by 17–21% with FACE in high‐density mixtures, due to a 121% enhancement of cotton leaf area and a 276% increase in biomass under the FACE treatment. Total root biomass in the upper 1.2 m of the soil was not influenced by CO₂ or by soil water in monoculture or mixtures; however, under dry conditions we observed significantly more roots at lower soil depths ( > 45 cm). Sorghum roots comprised 81–85% of the total roots in the low‐density mixture and 58–73% in the high‐density mixture. CO₂‐enrichment partly offset negative effects of interspecific competition on cotton in both low‐ and high‐density mixtures by increasing above‐ground biomass, with a greater relative increase in the high‐density mixture. As a consequence, CO₂‐enrichment increased total above‐ground yield of the mixture at high density. Individual plant responses to CO₂ enrichment in global change models that evaluate mixed plant communities should be adjusted to incorporate feedbacks for interspecific competition. Future field studies in natural ecosystems should address the role that a CO₂‐mediated increase in C₃ growth may have on subsequent vegetation change.     

Utilization of O2 in the metabolic optimization of C4 photosynthesis

The combined effects of O₂ on net rates of photosynthesis, photosystem II activity, steady‐state pool size of key metabolites of photosynthetic metabolism in the C₄ pathway, C₃ pathway and C₂ photorespiratory cycle and on growth were evaluated in the C₄ species Amaranthus edulis and the C₃ species Flaveria pringlei. Increasing O₂ reduced net CO₂ assimilation in F. pringlei due to an increased flux of C through the photorespiratory pathway. However, in A. edulis increasing O₂ up to 5–10% stimulated photosynthesis. Analysis of the pool size of key metabolites in A. edulis suggests that while there is some O₂ dependent photorespiration, O₂ is required for maximizing C₄ cycle activity to concentrate CO₂ in bundle sheath cells. Therefore, the response of net photosynthesis to O₂ in C₄ plants may result from the balance of these two opposing effects. Under 21 versus 5% O₂, growth of A. edulis was stimulated about 30% whereas that of F. pringlei was inhibited about 40%.     

Oxygen consumption during leaf nitrate assimilation in a C3 and C4 plant: the role of mitochondrial respiration

Measurements of net fluxes of CO₂ and O₂ from leaves and chlorophyll a fluorescence were used to determine the role of mitochondrial respiration during nitrate (NO₃^–) assimilation in both a C₃ (wheat) and a C₄ (maize) plant. Changes in the assimilatory quotient (net CO₂ consumed over net O₂ evolved) when the nitrogen source was shifted from NO₃^– to NH₄⁺ (ΔAQ) provided a measure of shoot NO₃^– assimilation. According to this measure, elevated CO₂ inhibited NO₃^– assimilation in wheat but not maize. Net O₂ exchange under ambient CO₂ concentrations increased in wheat plants receiving NO₃^– instead of NH₄⁺, but gross O₂ evolution from the photosynthetic apparatus (J_O2) was insensitive to nitrogen source. Therefore, O₂ consumption within wheat photosynthetic tissue (ΔΟ₂), the difference between J_O2 and net O₂ exchange, decreased during NO₃^– assimilation. In maize, NO₃^– assimilation was insensitive to changes in intercellular CO₂ concentration (C_i); nonetheless, ΔΟ₂ at low C_i values was significantly higher in NO₃^–‐fed than in NH₄⁺‐fed plants. Changes in O₂ consumption during NO₃^– assimilation may involve one or more of the following processes: (a) Mehler ascorbate peroxidase (MAP) reactions; (b) photorespiration; or (c) mitochondrial respiration. The data presented here indicates that in wheat, the last process, mitochondrial respiration, is decreased during NO₃^– assimilation. In maize, NO₃^– assimilation appears to stimulate mitochondrial respiration when photosynthetic rates are limiting.     

Photorespiratory metabolism and immunogold localization of photorespiratory enzymes in leaves of C3 and C3-C4 intermediate species of Moricandia

Photorespiratory metabolism of the C₃-C₄ intermediate species Moricandia arvensis (L.) DC has been compared with that of the C₃ species, Moricandia moricandioides (Boiss.) Heywood. Assays of glycollate oxidase (EC 1.1.3.1), glyoxylate aminotransferases (EC 2.6.1.4, EC 2.6.1.45) and hydroxypyruvate reductase (EC 1.1.1.29) indicate that the capacity for flux through the photorespiratory cycle is similar in both species. Immunogold labelling with monospecific antibodies was used to investigate the cellular locations of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), glycollate oxidase, and glycine decarboxylase (EC 2.1.2.10) in leaves of the two species. Ribulose 1,5-bisphosphate carboxylase/oxygenase was confined to the stroma of chloroplasts and glycollate oxidase to the peroxisomes of all photosynthetic cells in leaves of both species. However, whereas glycine decarboxylase was present in the mitochondria of all photosynthetic cells in M. moricandioides, it was only found in the mitochondria of bundle-sheath cells in M. arvensis. We suggest that localized decarboxylation of glycine in the leaves of M. arvensis will lead to improved recapture of photorespired CO₂ and hence a lower rate of photorespiration.Abbreviations kDa kilodalton - RuBP ribulose-1,5-bisphosphate     

Some Morphological and Biochemical Characteristics of C3 and C4 Plants Irradiated with UV-B

Four C₃ and two C₄ plants were subjected for 350 h to an enhanced UV-B radiation (280 to 310 nm) regime simulating a 0.18 atm. cm ozone level (solar angle 55°) in growth chamber. Different degrees of response among plant species were observed. UV-B radiation reduced plant height, fresh and dry weight, protein content, total chlorophyll, inhibited net CO₂ uptake and the Hill reaction activity. Some broad-leaved species with C₃ type of carbon assimilation were more susceptible to UV-B alterations of morphological and biochemical characteristics than the narrowleaved species with C₄ type photosynthesis.     

Carbon isotope discrimination in C3-C4 intermediates

Carbon isotope discrimination in C₃–C₄ intermediates is determined by fractionations during diffusion and the biochemical fractionations occurring during CO₂ fixation. These biochemical fractionations in turn depend on the fractionation by Rubisco in the mesophyll, the amount of CO₂ fixation. These biochemical fractionations in turn depend on the fractionation by Rubisco in the mesophyll, the amount of CO₂ fixation occurring in the bundle sheath, the extent of bundle-sheath leakiness and the contribution which C₄-cycle activity makes to the CO₂ pool there. In most instances, carbon isotope discrimination in C₃–C₄ intermediates is C₃-like because only a small fraction of the total carbon fixed is fixed in the bundle sheath. In particular, this must be the case for Flaveria intermediates which initially fix substantial amounts of CO₂ into C₄-acids. In C₃–C₄ intermediates that refix photorespiratory CO₂ alone, it is possible for carbon isotope discrimination to be greater than in C₃-species, particularly at low CO₂ pressures or at high leaf temperatures. Short-term measurements of carbon isotope discrimination and gas exchange of leaves can be used to study the photosynthetic pathways of C₃-C₄ intermediates and their hybrids as has recently been done for C₃ and C₄ species.     

Green fluorescent protein as a vital elimination marker to easily screen marker-free transgenic progeny derived from plants co-transformed with a double T-DNA binary vector system

We investigated the potential of a novel double T-DNA vector for generating marker-free transgenic plants. Co-transformation methods using a double T-DNA vector or using mixture of two Agrobacterium tumefaciens strains were compared, and showed that the double T-DNA vector method could produce marker-free transgenic tobacco (Nicotiana tabacum L.) plants more efficiently. A dual marker double T-DNA vector was then constructed by assembling the green fluorescent protein (GFP) gene mgfp5 and the neomycin phosphotransferase gene nptII into the same T-DNA. The frequency of co-transformants produced by this vector was 56.3%. Co-expression of mgfp5 and nptII was found in 28 out of 29 T₁ lines, and segregation of the reporter -glucuronidase gene, gusA, from mgfp5 to nptII was found in 12 out of 29 T₁ lines. Therefore, GFP could be used as a vital marker to improve the transformation efficiency and to easily monitor the segregation of marker genes, thus facilitating screening of marker-free progeny.     

Mesophyll cell properties for some C3 and C4 species with high photosynthetic rates

Leaves of twelve C₃ species and six C₄ species were examined to understand better the relationship between mesophyll cell properties and the generally high photosynthetic rates of these plants. The CO₂ diffusion conductance expressed per unit mesophyll cell surface area (g_CO2^cell) cell was determined using measurements of the net rate of CO₂ uptake, water vapor conductance, and the ratio of mesophyll cell surface area to leaf surface area (A^mes/A). A^mes/A averaged 31 for the C₃ species and 16 for the C₄ species. For the C₃ species g_CO2^cell ranged from 0.12 to 0.32 mm s^-1, and for the C₄ species it ranged from 0.55 to 1.5 mm s^-1, exceeding a previously predicted maximum of 0.5 mm s^-1. Although the C₃ species Cammissonia claviformis did not have the highest g_CO2^cell, the combination of the highest A^mes and highest stomatal conductance resulted in this species having the greatest maximum rate of CO₂ uptake in low oxygen, 93 μmol m^-2 s^-1 (147 mg dm^-2 h^-1). The high g_CO2^cell of the C₄ species Amaranthus retroflexus (1.5 mm s^-1) was in part attributable to its thin cell wall (72 nm thick).