Relative nutritional quality of C3 and C4 grasses for a graminivorous lepidopteran,Paratrytone melane (Hesperiidae)

Summary We tested the hypothesis that C₄ grasses are inferior to C₃ grasses as host plants for herbivorous insects by measuring the relative performance of larvae of a graminivorous lepidopteran, Paratrytone melane (Hesperiidae), fed C₃ and C₄ grasses. Relative growth rates and final weights were higher in larvae fed a C₃ grass in Experiment I. However, in two additional experiments, relative growth rates and final weights were not significantly different in larvae fed C₃ and C₄ grasses. We examined two factors which are believed to cause C₄ grasses to be of lower nutritional value than C₃ grasses: foliar nutrient levels and nutrient digestibility. In general, foliar nutrient levels were higher in C₃ grasses. In Experiment I, protein and soluble carbohydrates were digested from a C₃ and a C₄ grass with equivalent efficiencies. Therefore, differences in larval performance are best explained by higher nutrient levels in the C₃ grass in this experiment. In Experiment II, soluble carbohydrates were digested with similar efficiencies from C₃ and C₄ grasses but protein was digested with greater efficiency from the C₃ grasses. We conclude (1) that the bundle sheath anatomy of C₄ grasses is not a barrier to soluble carbohydrate digestion and does not have a nutritionally significant effect on protein digestion and (2) that P. melane may consume C₄ grasses at compensatory rates.     

Preference for C4 shade grasses increases hatchling performance in the butterfly,Bicyclus safitza

The Miocene radiation of C₄ grasses under high‐temperature and low ambient CO₂ levels occurred alongside the transformation of a largely forested landscape into savanna. This inevitably changed the host plant regime of herbivores, and the simultaneous diversification of many consumer lineages, including Bicyclus butterflies in Africa, suggests that the radiations of grasses and grazers may be evolutionary linked. We examined mechanisms for this plant–herbivore interaction with the grass‐feeding Bicyclus safitza in South Africa. In a controlled environment, we tested oviposition preference and hatchling performance on local grasses with C₃ or C₄ photosynthetic pathways that grow either in open or shaded habitats. We predicted preference for C₃ plants due to a hypothesized lower processing cost and higher palatability to herbivores. In contrast, we found that females preferred C₄ shade grasses rather than either C₄ grasses from open habitats or C₃ grasses. The oviposition preference broadly followed hatchling performance, although hatchling survival was equally good on C₄ or C₃ shade grasses. This finding was explained by leaf toughness; shade grasses were softer than grasses from open habitats. Field monitoring revealed a preference of adults for shaded habitats, and stable isotope analysis of field‐sampled individuals confirmed their preference for C₄ grasses as host plants. Our findings suggest that plant–herbivore interactions can influence the direction of selection in a grass‐feeding butterfly. Based on this work, we postulate future research to test whether these interactions more generally contribute to radiations in herbivorous insects via expansions into new, unexploited ecological niches.     

Performance of a generalist grasshopper on a C<Subscript>3</Subscript> and a C<Subscript>4</Subscript> grass: compensation for the effects of elevated CO<Subscript>2</Subscript> on plant nutritional quality

The increasing CO₂ concentration in Earths atmosphere is expected to cause a greater decline in the nutritional quality of C₃ than C₄ plants. As a compensatory response, herbivorous insects may increase their feeding disproportionately on C₃ plants. These hypotheses were tested by growing the grasses Lolium multiflorum C₃) and Bouteloua curtipendula C₄) at ambient (370 ppm) and elevated (740 ppm) CO₂ levels in open top chambers in the field, and comparing the growth and digestive efficiencies of the generalist grasshopper Melanoplus sanguinipes on each of the four plant × CO₂ treatment combinations. As expected, the nutritional quality of the C₃ grass declined to a greater extent than did that of the C₄ grass at elevated CO₂; protein levels declined in the C₃ grass, while levels of carbohydrates (sugar, fructan and starch) increased. However, M. sanguinipes did not significantly increase its consumption rate to compensate for the lower nutritional quality of the C₃ grass grown under elevated CO₂. Instead, these grasshoppers appear to use post-ingestive mechanisms to maintain their growth rates on the C₃ grass under elevated CO₂. Consumption rates of the C₃ and C₄ grasses were also similar, demonstrating a lack of compensatory feeding on the C₄ grass. We also examined the relative efficiencies of nutrient utilization from a C₃ and C₄ grass by M. sanguinipes to test the basis for the C₄ plant avoidance hypothesis. Contrary to this hypothesis, neither protein nor sugar was digested with a lower efficiency from the C₄ grass than from the C₃ grass. A novel finding of this study is that fructan, a potentially large carbohydrate source in C₃ grasses, is utilized by grasshoppers. Based on the higher nutrient levels in the C₃ grass and the better growth performance of M. sanguinipes on this grass at both CO₂ levels, we conclude that C₃ grasses are likely to remain better host plants than C₄ grasses in future CO₂ conditions.     

Insect herbivory on C3 and C4 grasses

Summary This study tested the hypothesis that grasses with the C₄ photosynthetic pathway are avoided as a food source by insect herbivores in natural communities. Insects were sampled from ten pairs of C₃–C₄ grasses and their distributions analyzed by paired comparisons tests. Results showed no statistically significant differences in herbivore utilization of C₃–C₄ species. However, there was a trend towards heavier utilization of C₃ species when means for both plant groups were compared. In particular, Homoptera and Diptera showed heavier usage of C₃ plants. Significant correlations between insect abundances and plant protein levels suggest that herbivores respond to the higher protein content of C₃ grasses. ¹³C values for six of the most common grasshopper species in the study area indicated that three species fed on C₃ plants, two species fed on C₄ plants, and one species consumed a mixture of C₃ and C₄ tissue.Welder Wildlife Refuge Contribution Number 213     

Changes in western wheatgrass foliage quality following defoliation: consequences for a graminivorous grasshopper

We determined the effects of defoliation by a graminivorous grasshopper on the foliage quality of the C3 plant, western wheatgrass (Pascopyrum smithii [Rydb] A. Love). Additionally, we determined the effects of this defoliation upon the subsequent feeding of the graminivorous grasshopper Phoetaliotes nebrascensis Thomas (Orthoptera: Acrididae). In field and greenhouse studies, graminivorous grasshopper herbivory altered the quality of remaining western wheatgrass foliage. In the greenhouse, severe (50% foliage removal) grasshopper grazing (638 grasshoppers/m² for 72h) resulted in decreased foliar nitrogen (–12%), carbohydrate (–11%) and water (–2.5%) concentrations, and increased phenolic concentrations (+43%). These changes were associated with decreased adult female grasshopper mass gain, consumption rate, approximate digestibility, and food conversion efficiencies. In the field, moderate (14% foliage removal) grasshopper grazing (20 grasshoppers/m² for 20 days) led to a 10% reduction in foliar nitrogen concentrations. Foliage quality changes in the field were not associated with any reductions in grasshopper mass gain, consumption rates, food digestibility, or conversion efficiencies. The results presented here are consistent with the hypothesis that defoliation leads to a reallocation of carbon and nitrogen compounds within the plant such that foliage quality for P. nebrascensis is reduced.     

Grasshoppers efficiently process C4 grass leaf tissues: implications for patterns of host-plant utilization

Leaf‐chewing insects are commonly believed to be unable to crush the nutrient‐rich bundle sheath cells (BSC) of C₄ grasses. This physical constraint on digestion is thought to reduce the nutritional quality of these grasses substantially. However, recent evidence suggests that BSC are digested by grasshoppers. To directly assess the ability of grasshoppers to digest C₄ grass BSC, leaf particles of Bouteloua curtipendula (Poaceae) were examined from the digestive tracts of two grasshopper species: Camnula pellucida (Scudder) (primarily a grass feeder) and Melanoplus sanguinipes (Fabricius) (a forb and grass generalist) (Orthoptera: Acrididae). Transmission electron microscopy was used to make the first observations of BSC crushing by herbivorous insects. Camnula pellucida and M. sanguinipes crushed over 58% and 24%, respectively, of the BSC in ingested leaf tissues. In addition, chloroplast and cell membranes were commonly disrupted in uncrushed BSC, permitting soluble nutrients to be extracted, even when BSC walls remain intact. The greater efficiency with which C. pellucida crushes BSC is consistent with the idea that grass‐feeding species are better adapted for handling grass leaf tissues than are generalist species. By demonstrating the effectiveness with which the BSC of B. curtipendula can be crushed and extracted by both species of grasshoppers, this study suggests one reason why C₄ grasses are not generally avoided by grasshoppers: at least some C₄ grasses can be more easily digested than has been hypothesized.     

Interspecific competition among grasshoppers and their effect on plant abundance in experimental field environments

Summary We tested whether grasshoppers in experimental field environments, i.e. cages (40×40 cm) placed on existing old field vegetation, (1) were limited in density by plant abundance and/or nitrogen content, (2) exhibited interspecific competition, and (3) altered the relative abundance of different plant species. We examined interactions among a pair of early season grasshopper species (May–June; Arphia conspersa and Pardalophora apiculata) and a late season pair (July–August; Melanoplus femur-rubrum and Melanoplus bivittatus). Each grasshopper species was placed in cages by itself and with another grasshopper species. Grasshoppers generally survived at higher density in fertilized cages and they reduced plant abundance relative to empty cages, suggesting that grasshoppers may be food limited at these densities. In unfertilized plots, early season grasshoppers preferred grasses (Schizachyrium scoparium and Poa pratensis) and favored the growth of forbs (especially Solidago spp.). However, late in summer, Melanoplus spp. preferred Solidago spp. and favored the growth of grasses.The pattern of grasshopper survivorship and plant reduction within these experimental environments provide preliminary support for some of the predictions of resource competition theory. Grasshoppers exhibited interspecific competition only if they significantly reduced plant biomass. If two species competed, a grasshopper species was eliminated only if the superior competitor, when living by itself, could reduce plant biomass to a significantly lower level than the inferior competitor. Competitors persisted only if they did not differ in their ability to reduce plant biomass or reduced the abundance of different plant species.     

Distribution of C3 and C4 grasses at different altitudes in a temperate arid region of Argentina

Summary The distribution of native C₃ and C₄ grasses in a temperate arid region of Mendoza, Argentina, was studied in six areas at different altitudes. C₄ species predominate at low elevations in both relative species abundance and plant cover. At high elevations C₃ species are dominant in cover and composition. At medium altitudes (1100–1600 m) grass species composition is balanced but plant cover of C₃ species is greater. Of 31 genera in the whole area, 19 were C₄. Only the genera Stipa (C₃) and Aristida (C₄) were present in all the six areas surveyed. The pattern of grass distribution shows high correlation with evapotranspiration and temperature parameters, but low correlation with rainfall. The relation between grass distribution and different climatic parameters is discussed.     

The effects of mandible morphology and photosynthetic pathway on selective herbivory in grasshoppers

Summary In the present study the following hypotheses were tested: 1) Selective herbivory is influenced by the photosynthetic pathway of plants, and 2) selective herbivory is influenced by the mandible morphology of herbivores. To test these hypotheses, grasshoppers with different mandibular patterns were offered C₃ and C₄ plants in controlled feeding experiments.Mandible morphology was found to be significantly associated with energy assimilation and feeding frequency. Grasshoppers with herbivorous mandibles had greater assimilation efficiencies and fed significantly less often than grasshoppers with forbivorous mandibles. These results indicate grasshoppers (with comparable caloric requirements) feed with a frequency determined, in part, by the functional morphology of their mandibles.Photosynthetic pathway also influenced the foraging behavior of grasshoppers tested. As predicted by the C₄ avoidance hypothesis (Caswell et al. 1973), C₄ plants were fed upon significantly less often than C₃ plants. These results suggest grasshoppers may discriminate between C₃ and C₄ plants, but apparently not on the basis of available energy.Recent studies of insect herbivory, previously interpreted within the framework of C₄ plant avoidance, are reviewed. The results of these studies may be critically dependent on incomplete considerations of mandible morphology and photosynthetic pathway.This study is contribution 92 of the Chihuahuan Desert Research Institute     

Germination sensitivities to water potential among co‐existing C3 and C4 grasses of cool semi‐arid prairie grasslands

An untested theory states that C₄ grass seeds could germinate under lower water potentials (Ψ) than C₃ grass seeds. We used hydrotime modelling to study seed water relations of C₄ and C₃ Canadian prairie grasses to address Ψ divergent sensitivities and germination strategies along a risk‐spreading continuum of responses to limited water. C₄ grasses were Bouteloua gracilis, Calamovilfa longifolia and Schizachyrium scoparium; C₃ grasses were Bromus carinatus, Elymus trachycaulus, Festuca hallii and Koeleria macrantha. Hydrotime parameters were obtained after incubation of non‐dormant seeds under different Ψ PEG 6000 solutions. A t‐test between C₃ and C₄ grasses did not find statistical differences in population mean base Ψ (Ψ_b(50)). We found idiosyncratic responses of C₄ grasses along the risk‐spreading continuum. B. gracilis showed a risk‐taker strategy of a species able to quickly germinate in a dry soil due to its low Ψ_b(50) and hydrotime (θ_H). The high Ψ_b(50) of S. scoparium indicates it follows the risk‐averse strategy so it can only germinate in wet soils. C. longifolia showed an intermediate strategy: the lowest Ψ_b(50) yet the highest θ_H. K. macrantha, a C₃ grass which thrives in dry habitats, had the highest Ψ_b(50), suggesting a risk‐averse strategy for a C₃ species. Other C₃ species showed intermediate germination patterns in response to Ψ relative to C₄ species. Our results indicate that grasses display germination sensitivities to Ψ across the risk‐spreading continuum of responses. Thus seed water relations may be poor predictors to explain differential recruitment and distribution of C₃ and C₄ grasses in the Canadian prairies.     

Aboveground Biomass Removal by Burning and Raking Increases Diversity in a Reconstructed Prairie

Prescribed spring burning often contributes to a predominance of C₄ grasses and low forb abundance and is impractical at many sites, especially near development. We tested raking after mowing as an alternative to prescribed burning in a reconstructed Minnesota prairie. We also tested mowing without raking as a possible means of maintaining prairie communities. Frequency, flowering stem abundance, and cover were measured for all plant species and native functional groups (C₄ grasses, C₃ graminoids, forbs, legumes, and annual or biennial forbs). Mowing alone did not differ from the control in its effect on any functional groups of plants. Round‐headed bush clover (Lespedeza capitata), a legume, and Black‐eyed Susan (Rudbeckia hirta), a biennial, increased in frequency with treatments that removed biomass (i.e., fire or raking), but they did not have significantly more flowering stems. Thus, new plants established well from seed, whereas the vitality of mature plants did not change. Raking had similar effects to burning on most functional groups, although flowering stems of C₄ grasses were significantly more abundant after fire than after raking. Burning reduced some C₃ forbs and grasses and favored the dominance of C₄ grasses. Therefore, raking after mowing in the spring provides an alternative to prescribed burning that has many of the same positive aspects as fire but does not promote aggressive C₄ grasses to the same extent.     

Drought limitation of photosynthesis differs between C3 and C4 grass species in a comparative experiment

Phylogenetic analyses show that C₄ grasses typically occupy drier habitats than their C₃ relatives, but recent experiments comparing the physiology of closely related C₃ and C₄ species have shown that advantages of C₄ photosynthesis can be lost under drought. We tested the generality of these paradoxical findings in grass species representing the known evolutionary diversity of C₄ NADP‐me and C₃ photosynthetic types. Our experiment investigated the effects of drought on leaf photosynthesis, water potential, nitrogen, chlorophyll content and mortality. C₄ grasses in control treatments were characterized by higher CO₂ assimilation rates and water potential, but lower stomatal conductance and nitrogen content. Under drought, stomatal conductance declined more dramatically in C₃ than C₄ species, and photosynthetic water‐use and nitrogen‐use efficiency advantages held by C₄ species under control conditions were each diminished by 40%. Leaf mortality was slightly higher in C₄ than C₃ grasses, but leaf condition under drought otherwise showed no dependence on photosynthetic‐type. This phylogenetically controlled experiment suggested that a drought‐induced reduction in the photosynthetic performance advantages of C₄ NADP‐me relative to C₃ grasses is a general phenomenon.     

Feeding preferences of Melanoplus femurrubrum grasshoppers on native and exotic grasses: behavioral and molecular approaches

Generalist insect herbivores, such as grasshoppers, may either avoid feeding on exotic plants, potentially enabling these plants to become invasive in the introduced range, or insects may incorporate exotic plants into their diet, contributing to the biotic resistance of native communities and potentially preventing plant invasions. Accurate determination of insect diet preferences with regard to native and exotic plants can be challenging, but this information is critical for understanding the interaction between native herbivores and exotic plants, and ultimately the mechanisms underlying plant invasions. To address this, we combined behavioral and molecular approaches to accurately compare food consumption of the polyphagous red‐legged grasshopper, Melanoplus femurrubrum (De Geer) (Orthoptera: Acrididae), on native [Andropogon gerardii Vitman and Bouteloua curtipendula (Michx.) Torr.] and exotic, potentially invasive grasses [Miscanthus sinensis Andersson and Bothriochloa ischaemum (L.) Keng] (all Poaceae). We found that M. femurrubrum grasshoppers demonstrated strong feeding preferences toward exotic grasses in experiments with intact plants under both field and greenhouse conditions, but they showed no preference in experiments with clipped leaves. Additionally, we sampled the gut contents of M. femurrubrum collected in the field and identified the ingested plant species based on DNA sequences for the non‐coding region of the chloroplast trnL (UAA) gene. We found that exotic plants were prevalent in the gut contents of grasshoppers collected at study sites in Ohio and Maryland, USA. These results suggest that the generalist herbivore M. femurrubrum does not avoid feeding on exotic grasses with which they do not share coevolutionary history. In addition, by demonstrating greater food consumption of exotic plants, these grasshoppers potentially provide biotic resistance should these grasses escape cultivation and become invasive in the introduced range.     

Estimation of food composition of Hodotermes mossambicus (Isoptera: Hodotermitidae) based on observations and stable carbon isotope ratios

Abstract The diet of the harvester termite Hodotermes mossambicus was investigated at two sites with distinct dietary components: C₄ grasses (δ¹³C isotope values, ?13.8‰ to ?14.0‰) and C₃ plants (δ¹³C isotope values, ?25.6‰ to ?27.1‰). By comparing observations of food items carried into the colony by the termites and carbon isotope ratios of whole termites (that determined assimilated carbon), the relative proportion of the C₃ and C₄ plant food components of the termite diet was estimated. There was agreement between the observational data and stable carbon isotopic data, with grass representing approximately 93% of the diet of H. mossambicus at two study sites (urban and rural) on the South African highveld. However, when correcting for mass of food items, that is, C₃ and C₄, carried by termites, the proportion of grass (C₄) in the diet may be underestimated.     

Field analysis of photoprotection in co‐occurring cool climate C3 and C4 grasses

C₄ photosynthesis is particularly successful at high light intensities and high temperatures, but is relatively rare when the average growing season temperature is less than about 15°C. We tested the hypothesis that rapidly reversible photoprotection enables some C₄ species to tolerate cool climates, by focusing on two questions: (1) Do chlorophyll fluorescence responses differ seasonally between co‐occurring C₃ and C₄ grasses in the field? (2) Does xanthophyll‐mediated photoprotection differ between the two pathways? Spartina pectinata (C₄) and Calamogrostis canadensis (C₃) were sampled in a herbaceous fresh‐water meadow in New Brunswick, Canada (45°N 66°W). Non‐photochemical thermal energy dissipation (Φ_NPQ) and the epoxidation state of the xanthophyll cycle (EPS) were used as indicators of photoprotection. We observed no differential susceptibility to chronic photoinhibition (i.e. photodamage) between the C₃ and C₄ species, except potentially during spring emergence. On average, C. canadensis showed higher levels of protective dynamic photoinhibition throughout the growing season, but S. pectinata had greater Φ_NPQ and lower EPS during seasonal and daily temperature minima. The low Rubisco capacity of C₄ species is a potential limiting factor to C₄ success at high latitudes, but our findings suggest that it is unlikely via a photoinhibitory feedback mechanism.     

The small scale spatial pattern of C3 and C4 grasses depends on shrub distribution

At micro‐site scale, the spatial pattern of a plant species depends on several factors including interactions with neighbours. It has been seen that unfavourable effects generate a negative association between plants, while beneficial effects generate a positive association. In grasslands, the presence of shrubby species promotes a particular microenvironment beneath their canopy that could affect differently the spatial distribution of plants with different tolerance to abiotic conditions. We measured photosynthetic active radiation, air temperature and wind speed under shrub canopies and in adjacent open sites and analysed the spatial distribution of four grass species (two C₃ and two C₄) in relation to shrub canopy in a grazed sub‐humid natural grassland in southern Uruguay. Radiation, air temperature and wind speed were lower under shrubs than in adjacent open sites. The spatial distribution of grasses relative to the shrub canopy varied depending on the photosynthetic metabolism of grasses. C₄ grasses showed a negative association or no correlation with the shrubs, whereas C₃ grasses showed a positive association. Our results highlight the importance of the photosynthetic metabolism of the grasses in the final outcome of interactions between grasses and shrubs. Micro‐environmental conditions generated underneath shrubs create a more suitable site for the establishment of C₃ than for C₄ grasses. These results show that facilitation could be more important than previously thought in sub‐humid grasslands.     

Restoration of C4 grasses with seasonal fires in a C3/C4 grassland invaded by Prosopis glandulosa,a fire‐resistant shrub

Questions: Can prescribed fire restore C₄ perennial grasses in grassland ecosystems that have become dominated by fire‐resistant C₃ shrubs (Prosopis glandulosa) and C₃ grasses? Do fires in different seasons alter the direction of change in grass composition? Location: Texas, USA. Methods: We quantified short‐ and long‐term (12 yr post‐fire) herbaceous functional group cover and diversity responses to replicated seasonal fire treatments: (1) repeated‐winter fires (three in 5 yr), (2) repeated‐summer fires (two in 3 yr), and (3) alternate‐season fires (two winter and one summer in 4 yr), compared with a no‐fire control. Results: Summer fires were more intense than winter fires, but all fire treatments temporarily decreased Prosopis and C₃ annual grass cover. The alternate‐season fire treatment caused a long‐term increase in C₄ mid‐grass cover and functional group diversity. The repeated‐summer fire treatment increased C₄ short‐grass cover but also caused a long‐term increase in bare ground. The repeated winter fire treatment had no long‐term effects on perennial grass cover. Mesquite post‐fire regrowth had increasingly negative impacts on herbaceous cover in all fire treatments. Conclusions: Summer fire was necessary to shift herbaceous composition toward C₄ mid‐grasses. However, the repeated‐summer fire treatment may have been too extreme and caused post‐fire herbaceous composition to “over‐shift” toward less productive C₄ short‐grasses rather than C₄ mid‐grasses. This study provides some of the first long‐term data showing a possible benefit of mixing seasonal fires (i.e., the alternate‐season fire treatment) in a prescribed burning management plan to restore C₄ mid‐grass cover and enhance overall herbaceous diversity.     

Improving our understanding of environmental controls on the distribution of C3 and C4 grasses

A number of studies have demonstrated the ecological sorting of C₃ and C₄ grasses along temperature and moisture gradients. However, previous studies of C₃ and C₄ grass biogeography have often inadvertently compared species in different and relatively unrelated lineages, which are associated with different environmental settings and distinct adaptive traits. Such confounded comparisons of C₃ and C₄ grasses may bias our understanding of ecological sorting imposed strictly by photosynthetic pathway. Here, we used MaxEnt species distribution modeling in combination with satellite data to understand the functional diversity of C₃ and C₄ grasses by comparing both large clades and closely related sister taxa. Similar to previous work, we found that C₄ grasses showed a preference for regions with higher temperatures and lower precipitation compared with grasses using the C₃ pathway. However, air temperature differences were smaller (2 °C vs. 4 °C) and precipitation and % tree cover differences were larger (1783 mm vs. 755 mm, 21.3% vs. 7.7%, respectively) when comparing C₃ and C₄ grasses within the same clade vs. comparing all C₄ and all C₃ grasses (i.e., ignoring phylogenetic structure). These results were due to important differences in the environmental preferences of C₃ BEP and PACMAD clades (the two main grass clades). Winter precipitation was found to be more important for understanding the distribution and environmental niche of C₃ PACMADs in comparison with both C₃ BEPs and C₄ taxa, for which temperature was much more important. Results comparing closely related C₃–C₄ sister taxa supported the patterns derived from our modeling of the larger clade groupings. Our findings, which are novel in comparing the distribution and niches of clades, demonstrate that the evolutionary history of taxa is important for understanding the functional diversity of C₃ and C₄ grasses, and should have implications for how grasslands will respond to global change.     

A role for differential Rubisco activase isoform expression in C4 bioenergy grasses at high temperature

Rubisco activase (Rca) facilitates the release of sugar‐phosphate inhibitors at Rubisco catalytic sites during CO₂ fixation. Most plant species express two Rca isoforms, the larger Rca‐α and the shorter Rca‐β, either by alternative splicing from a single gene or expression from separate genes. The mechanism of Rubisco activation by Rca isoforms has been intensively studied in C₃ plants. However, the functional role of Rca in C₄ plants where Rubisco and Rca are located in a much higher [CO₂] compartment is less clear. In this study, we selected four C₄ bioenergy grasses and the model C₄ grass setaria (Setaria viridis) to investigate the role of Rca in C₄ photosynthesis. All five C₄ grass species contained two Rca genes, one encoding Rca‐α and the other Rca‐β, which were positioned closely together in the genomes. A variety of abiotic stress‐related motifs were identified in the Rca‐α promoter of each grass, and while the Rca‐β gene was constantly highly expressed at ambient temperature, Rca‐α isoforms were expressed only at high temperature but never surpassed 30% of Rca‐β content. The pattern of Rca‐α induction on transition to high temperature and reduction on return to ambient temperature was the same in all five C₄ grasses. In sorghum (Sorghum bicolor), sugarcane (Saccharum officinarum), and setaria, the induction rate of Rca‐α was similar to the recovery rate of photosynthesis and Rubisco activation at high temperature. This association between Rca‐α isoform expression and maintenance of Rubisco activation at high temperature suggests that Rca‐α has a functional thermo‐protective role in carbon fixation in C₄ grasses by sustaining Rubisco activation at high temperature.     

Physiological advantages of C4 grasses in the field: a comparative experiment demonstrating the importance of drought

Global climate change is expected to shift regional rainfall patterns, influencing species distributions where they depend on water availability. Comparative studies have demonstrated that C₄ grasses inhabit drier habitats than C₃ relatives, but that both C₃ and C₄ photosynthesis are susceptible to drought. However, C₄ plants may show advantages in hydraulic performance in dry environments. We investigated the effects of seasonal variation in water availability on leaf physiology, using a common garden experiment in the Eastern Cape of South Africa to compare 12 locally occurring grass species from C₄ and C₃ sister lineages. Photosynthesis was always higher in the C₄ than C₃ grasses across every month, but the difference was not statistically significant during the wettest months. Surprisingly, stomatal conductance was typically lower in the C₃ than C₄ grasses, with the peak monthly average for C₃ species being similar to that of C₄ leaves. In water‐limited, rain‐fed plots, the photosynthesis of C₄ leaves was between 2.0 and 7.4 μmol m^?2 s^?1 higher, stomatal conductance almost double, and transpiration 60% higher than for C₃ plants. Although C₄ average instantaneous water‐use efficiencies were higher (2.4–8.1 mmol mol^?1) than C₃ averages (0.7–6.8 mmol mol^?1), differences were not as great as we expected and were statistically significant only as drought became established. Photosynthesis declined earlier during drought among C₃ than C₄ species, coincident with decreases in stomatal conductance and transpiration. Eventual decreases in photosynthesis among C₄ plants were linked with declining midday leaf water potentials. However, during the same phase of drought, C₃ species showed significant decreases in hydrodynamic gradients that suggested hydraulic failure. Thus, our results indicate that stomatal and hydraulic behaviour during drought enhances the differences in photosynthesis between C₄ and C₃ species. We suggest that these drought responses are important for understanding the advantages of C₄ photosynthesis under field conditions.