C4 photosynthesis at low temperatures

Abstract. C₄ plants grown in optimum conditions are, by comparison to C₃, capable of higher maximum dry-matter yields and greater efficiencies of water and nitrogen use, yet they are rare outside the subtropics. Both latitudinal and altitudinal limits of C₄ distributions correlate most closely with a mean minimum temperature of 8-10°C during the period of active growth. The possibility that the C₄ process is inherently incapable of functioning at low temperatures is examined. The reversible effects of chilling on the quantum efficiency of C₄ photosynthesis and the functioning of the individual steps in the C₄ cycle are examined. Chilling also produces an irreversible loss of capacity to assimilate CO₂ which is directly proportional to the light received during chilling. It is suggested that the reversible reduction in capacity to assimilate CO₂ and the lack of an alternative pathway for the utilization of lightgenerated reducing power may make C₄ species more prone to chilling-dependent photoinhibition. Laboratory studies and limited field observations suggest that this damage would be most likely to occur during photosynthetic induction at the temperatures and light levels encountered on clear, cool mornings during the spring and early summer in cool climates. Even those C₄ species occurring naturally in cool climates do not appear fully capable of tolerating these conditions; indeed their growth patterns suggest that they may be adapted by avoiding 'rather than enduring' such conditions.     

On the significance of C3—C4 intermediate photosynthesis to the evolution of C4 photosynthesis

Abstract Evidence is drawn from previous studies to argue that C₃—C₄ intermediate plants are evolutionary intermediates, evolving from fully-expressed C₃ plants towards fully-expressed C₄ plants. On the basis of this conclusion, C₃—C₄ intermediates are examined to elucidate possible patterns that have been followed during the evolution of C₄ photosynthesis. An hypothesis is proposed that the initial step in C₄-evolution was the development of bundle-sheath metabolism that reduced apparent photorespiration by an efficient recycling of CO₂ using RuBP carboxylase. The CO₂-recycling mechanism appears to involve the differential compartmentation of glycine decarboxylase between mesophyll and bundle-sheath cells, such that most of the activity is in the bundlesheath cells. Subsequently, elevated phosphoenolpyruvate (PEP) carboxylase activities are proposed to have evolved as a means of enhancing the recycling of photorespired CO₂. As the activity of PEP carboxylase increased to higher values, other enzymes in the C₄-pathway are proposed to have increased in activity to facilitate the processing of the products of C₄-assimilation and provide PEP substrate to PEP carboxylase with greater efficiency. Initially, such a ‘C₄-cycle’ would not have been differentially compartmentalized between mesophyll and bundlesheath cells as is typical of fully-expressed C₄ plants. Such metabolism would have limited benefit in terms of concentrating CO₂ at RuBP carboxylase and, therefore, also be of little benefit for improving water- and nitrogen-use efficiencies. However, the development of such a limited C₄-cycle would have represented a preadaptation capable of evolving into the leaf biochemistry typical of fully-expressed C₄ plants. Thus, during the initial stages of C₄-evolution it is proposed that improvements in photorespiratory CO₂-loss and their influence on increasing the rate of net CO₂ assimilation per unit leaf area represented the evolutionary ‘driving-force’. Improved resourceuse efficiency resulting from an efficient CO₂-concentrating mechanism is proposed as the driving force during the later stages.     

Diurnal changes in phosphoenolpyruvate carboxylase and pyruvate, orthophosphate dikinase properties in the natural environment: interplay of light and temperature in a C4 thermophile

Activities of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) were measured in leaf extracts of field grown Amaranthus paniculatus L. (C₄) during a natural diurnal irradiance and temperature pattern. Enzyme assays were run at both fixed (30°C) and the corresponding leaf temperature at the time of harvest. Light activation of PEP carboxylase (PEPCase) at fixed assay temperatures was expressed as a decrease in S_0–5 (PEP) after a threshold (> 330 μmol m^–2 s^–1) photon fluence rate was surpassed at noon. Earlier in the morning, increase in apparent enzyme affinity for PEP was observed when the assay was run at leaf temperature, indicating a physiologically meaningfull effect of temperature on S^0.5 (PEP). The 3.3-fold increase in PEPCase activity at low PEP and fixed assay temperature between the minimal and maximal irradiance and temperature hours of the day, became 12.8-, 11.5- and 7.4-fold when assays were run at the corresponding leaf temperature during three diurnal cycles with respective temperature differences (max minus min) of 9.0, 8.3 and 7.4°C. The extent of malate inhibition was the same for both day and night forms of PEPCase assayed at 35°C, but increased considerably with night enzyme at 25°C. The results indicate that light increases the apparent affinity of PEPCase for PEP and that at lower temperatures malate becomes more inhibitory. Pyruvate orthophosphate dikinase activity started to increase immediately after sunrise and the 10-fold increase at fixed temperature became 14.8-, 14.2- and 13.1-fold when assays were run at the above leaf temperatures. This indicates that the light effect predominates with pyruvate, orthophosphate dikinase, while with phosphoenolpyravate carboxylase, light and temperature co-operate to increase the day enzyme activities.     

Carbon: terrestrial C4 plants

The carbon isotope composition of terrestrial C₄ plants depends on the primary carboxylation of phosphoenolpyruvate (PEP) and on the diffusion of CO₂ to the carboxylation sites, but is also influenced by the final carboxylation of ribulose-1,5-bisphosphate (RuBP). Several models have been used for reproducing this complex situation. In the present review, a particular model is applied as a means to interpret the effects of environmental and genetically determined factors on carbon isotope discrimination during C₄ photosynthesis. As a new feature, the model considers four types of limitation of the overall CO₂ assimilation rate. Both carboxylation reactions are assumed to be limited by either maximum enzyme activity or maximum substrate regeneration rate. The model is applied to experimental data on the effects of CO₂, irradiance and water stress on short-term discrimination by leaves of several C₄ species measured simultaneously with CO₂ gas exchange characteristics. In particular, different patterns of the influence of low irradiances on carbon isotope discrimination are interpreted as due to variations in that irradiance at which a transition from limitation by PEP regeneration rate and RuBP carboxylase activity to limitation by the regeneration rates of both substrates occurs. After discussing literature data on the effects of environmental conditions on carbon isotope discrimination by C₄ plants seasonal and developmental changes in carbon isotope composition, studies on the systematic and geographic distribution of C₄ plants, evolutionary and genetical aspects, and some ecological implications are reviewed.     

Photosynthetic responses to temperature of phosphoenolpyruvate carboxykinase type C4 species differing in cold sensitivity

Photosynthetic rates, the activities of key enzymes associated with the C₄ cycle and ribulose-1,5-bisphosphate carboxylase (RuBPCase), and the levels of metabolites involved in the C₄ cycle were compared between the two phosphoenolpyruvate carboxykinase (PCK) type C₄ species Spartina anglica, which is cold-tolerant, and Zoysia japonica, which is cold-sensitive, during exposure to low temperature. Plants of both species grown outside in summer were placed in a growth chamber at 27/20 °C day/night temperatures. After 1 week, plants were exposed to 20/17 °C for 1 week and then to 10/7 °C for 2 weeks. Photosynthetic rates in Z. japonica decreased progressively to about 25% during the chilling treatments. In contrast, S. anglica exhibited a 43% increase in photosynthetic rates after exposure to 20 °C for 1 week, which remained relatively constant thereafter. Consistent with these observations, most of the C₄ enzymes and RuBPCase in Z. japonica declined. Phosphoenolpyruvate carboxylase (PEPC) and PCK activities declined particularly drastically during the treatments. However, the activities of these enzymes in S. anglica showed either a slight increase or decrease upon a mild cold treatment, and remained relatively constant during further chilling treatments. There was a sharp decline in phosphoenolpyruvate in Z. japonica after exposure to 10 °C. On the other hand, metabolite levels in S. anglica were largely unaffected by the chilling treatments. These results suggest that the drastic declines of both PEPC and PCK activities may be important limiting factors responsible for cold sensitivity in C₄ photosynthesis of Z. japonica.     

Are-examination of NaCI effects on phosphoenolpyruvate carboxylase at high (physiological) enzyme concentrations

Inhibition of phosphoenolpyruvate carboxylase (EC 4.1.1 31) from the C₄-halophyte Salsola soda L. by NaCI is compeutive to phosphoenolpyruvate (PEP). Physiological (betaine, glycerol) and synthetic (polyethylene glycol) osmotica and the allosteric activator glucose-6-phosphate (G6P) increase the apparent affinity of the enzyme for PEP and also alleviate the inhibition by NaCl. Physiological osmotica that either increase the K_m(PEP) (proline) or are neutral (sorbitol), do not protect the enzyme against NaCI attack. In the absence of cosolutes and, G6P, the enzyme is self-protected when its concentration in the assay medium is increased to more physiological values. In addition, the amount of betaine needed for complete protection is inversely related to native protein concentration in the assay. Exogenous protein (bovine serum albumin or bovine skin gelatin) have no effect on either K_m(PEP), or extent of NaCl inhibition. These results can be better explained with the exclusion volume theory and the inferred assumption that both cosolutes and high protein concentration strengthen intrinsic aggregation properties of enzymes. It is suggested that the extremely high phosphoenolpyruvate carboxylase concentration in the cytoplasm and the accumulation of compatible solutes in response to water stress fully protect the enzyme in vivo against the chaotropic effects of NaCI.     

Occurrence of unstable PEP carboxylase in C4 grasses in the genus Spartina Schreb.

Abstract The aims of this work were to discover the distribution within the C₄ grass Spartina anglica of a PEP carboxylase which is very unstable during and after extraction, and to determine whether this unstable form occurs in other members of the genus. In S. anglica, only the leaf contains an unstable PEP carboxylase. Within the leaf only the major one of two isoenzymes is unstable, and this is located in the mesophyll cells. The unstable isoenzyme is inactivated during extraction and storage unless protected by bovine serum albumin or Triton X-100, and is inactivated in assay mixtures at optimum pH in the absence of PEP. Evidence is presented that inactivation is not due to degradation or inhibition during extraction and storage. The enzyme from leaves of Spartina species taxonomically closely related to S. anglica is also very unstable during and after extraction, but that from less closely related species is much more stable.     

Changes in levels of phosphoenolpyruvate carboxylase with induction of Crassulacean acid metabolism (CAM)-like behavior in the C4 plant Portulaca oleracea

Changes in levels of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31, orthophosphate: oxaloacetate carboxy-lyase, phosphorylating) were followed in leaves and stems of CAM-expressing and non-expressing Portulaca oleracea L. plants. CAM expression was induced by growing plants under an 8-h photoperiod and water stress conditions (SD-WS). Leaves and stems of these plants (designated CAM) expressed nocturnal acidification with an oscillation pattern and an amplitude characteristic of CAM plants. Generally, PEPC activity increased by ca 3-fold during the period of CAM induction. Over the day/night cycle. PEPC activity oscillated in a pattern typical of CAM plants. Treatment of the other plant group (designated as non-CAM) by growth under a 16-h photoperiod and well-watered conditions (LD-WW) did not induce expression of the tested criteria of CAM in plants. In these plants, nocturnal acidification as well as changes in the magnitude of PEPC, activity and fluctuation pattern were undetectable. SDS-PAGE of leaf extracts of the CAM-expressing plants and the corresponding densitometric scans show progressive increase in the amount of PEPC subunit protein (ca 95 kDa) during the period of CAM induction. These results show that induction of CAM-like characteristics in the C₄ plant Portulaca oleracea is also accompanied by increased PEPC activity, which may be partly due to an increase in enzyme synthesis.     

Comparative ecophysiology of C3 and C4 plants

Abstract. In this review we relate the physiological significance of C₄ photosynthesis to plant performance in nature. We begin with an examination of the physiological consequences of the C₄ pathway on photosynthesis, then discuss the ecophysiological performance of C₄ plants in contrasting environments. We then compare the performance of C₃ and C₄ plants when they occur together in similar habitats, and finally discuss the distribution of C₄ photosynthesis with respect to the physical environment, phylogeny, and life form.     

Evidence for the expression of morphological and biochemical characteristics of C3-photosynthesis in chlorohyllous callus cultures of Zea mays

A Zea mays callus culture containing chlorophyll was established and grown photomixotrophically. Cell chloroplast structure, and pigment and soluble protein contents were examined. Expression of some key enzymes of C₄ carbon metabolism was compared with that of etiolated (heterotrophic) and green photoautotrophic leaves. Chlorophyll content of the callus was 15–20% that of green leaves. Soluble protein content of callus was half that of leaf cells. Electron microscopic observations showed that green callus cells contained only typical granal chloroplasts. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.38) activities in green callus were ca 30% those of green leaves but 2–3 times higher than in etiolated leaves. Quantitative enzyme protein determination, using antibodies specific to maize leaf Rubisco showed that the chloroplastic carboxylase represented about 7% of total soluble protein in green callus, in parallel to its low chlorophyll content. The specific activity of Rubisco in callus and leaves was unchanged. Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) activity in green callus was about 20% that of green leaves and similar to that measured in etiolated leaves. Apparent K_m (PEP) values (0.08 mM) for PEPC isolated from green callus and etiolated leaves were very different from values (0.5 mM) obtained with PEPC from green leaves. These kinetic characteristics together with the absence of inhibition by malate and activation by glucose-6-phosphate suggest that the properties of PEPC isolated from green callus and etiolated maize leaves are very similar to those of PEPPC from C₃ plants. Using PEPC antibodies specific to green maize leaf enzyme, immunotitration of PEPC preparations containing identical enzyme units allowed complete precipitation of the green leaf enzyme with increasing antibody volumes. In contrast, 60–70% of the activity of PEPC from etiolated and green callus was inhibited, suggesting low affinity for the maize green leaf PEPC antiserum (typical C₄ form). Ouchterlony double diffusion tests revealed only partial recognition of PEPC in green callus and etiolated leaves. NAD-malate dehydrogenase (NAD-MDH, EC 1.1.1.37) activity in callus was 2 and 3 times higher, respectively, than in etiolated and green leaves. NADP-malic enzyme (NADP-ME, EC 1.1.1.40) activity in callus cultures was much lower than in green leaves. All our data support the hypothesis that cultures of fully dedifferentiated chlorophyllous tissues of Zea mays possess a C₃-like metabolism.     

Effects of stabilizing solutes on salt activation of phosphoenolpyruvate carboxylase from various plant sources

Phosphoenolpyruvate carboxylase (PEP carboxylase EC 4.1.1.31) was extracted from various halophytic, semi-halophytic and glycophytic plant species. When the enzyme of those extracts was substrate protected, and in the presence of 1.6 m M PEP in the reaction mixture, the activity of PEP carboxylase was increased by 100 m M NaCl, and the activity range in the presence of NaCl was expanded. No correlation could be established between the response of the enzyme to ions and various plant characteristics, such as taxonomic status, salt tolerance or carbon fixation pathways. Salt activation of PEP carboxylase was substrate (PEP) dependent, but the minimal substrate concentration varied in different species.
Effects of the stabilizing solutes PEP, betaine, proline and glycerol on the kinetic properties of PEP carboxylase from Zea mays (L.) cv. Hazera were analyzed. In the absence of NaCl the slope of the Hill plot (n_It) tended to rise in the presence of these solutes. Stabilization of the enzyme with betaine or glycerol caused a decrease in K'. while K' and VTO increased in the presence of PEP. NaCl (100 mM) caused an increase in both K' and V^max in the protected as well as in the unprotected enzyme, except for PEP protection, where K' decreased somewhat. In the presence of the protectants, glycerol and PEP, the effect of NaCl on V^max, was 2–4 times higher than its effect on the non-protected enzyme.     

Evolution of C4 phosphoenolpyruvate carboxylase

C4 plants are known to be of polyphyletic origin and to have evolved independently several times during the evolution of angiosperms. This implies that the C4 isoform of phosphoenolpyruvate carboxylase (PEPC) originated from a nonphotosynthetic PEPC gene that was already present in the C3 ancestral species. To meet the special requirements of the C4 photosynthetic pathway the expression program of the C4 PEPC gene had to be changed to achieve a strong and selective expression in leaf mesophyll cells. In addition, the altered metabolite concentrations around C4 PEPC in the mesophyll cytoplasm necessitated changes in the enzyme's kinetic and regulatory properties. To obtain insight into the evolutionary steps involved in these altered enzyme characteristics, and even the order of these steps, the dicot genus Flaveria (Asteraceae) appears to be the experimental system of choice. Flaveria contains closely related C3, C3-C4, and C4 species that can be ordered by their gradual increase in C4 photosynthetic traits. The C4 PEPC of F. trinervia, which is encoded by the ppcA gene class, possesses typical kinetic and regulatory features of a C4-type PEPC. Its nearest neighbor is the orthologous ppcA gene of the C3 species F. pringlei. This latter gene encodes a typical nonphotosynthetic C3-type PEPC which is believed to be similar to the C3 ancestral PEPC. This pair of orthologous PEPCs has been used to map C4-specific molecular determinants for the kinetic and regulatory characteristics of C4 PEPCs. The most notable finding from these investigations was the identification of a C4 PEPC invariant site-specific mutation from alanine (C3) to serine (C4) at position 774 that was a necessary and late step in the evolution of C3 to C4 PEPC. The C3-C4 intermediate ppcA PEPCs are used to identify the sequence of events leading from a C3- to a C4-type PEPC.     

The light induction of maize phosphoenolpyruvate carboxylase kinase translatable mRNA requires transcription but not translation

We have previously demonstrated that the level of translatable mRNA for phosphoenolpyruvate carboxylase kinase in maize leaves is increased in response to light ( Hartwell et al. 1996 ; Plant Journal 10 , 1071–1078). To identify the steps required for this increase, we have examined the effects of protein and RNA synthesis inhibitors. The RNA synthesis inhibitors actinomycin D and cordycepin (500 μ M ) strongly inhibited the light-induced increases in kinase translatable mRNA and the apparent phosphorylation state of phosphoenolpyruvate carboxylase, as judged by its sensitivity to inhibition by L -malate. The protein synthesis inhibitors cycloheximide and puromycin blocked the light-induced increase in the apparent phosphorylation state of phosphoenolpyruvate carboxylase but not the increase in kinase translatable mRNA. Indeed, the amount of phosphoenolpyruvate carboxylase kinase translatable mRNA after 3 h of illumination of leaves treated with either 1 m M puromycin or 100 μ M cycloheximide was double that in illuminated control leaves. Each inhibitor reduced the light-induction of two control genes, malic enzyme and pyruvate, phosphate dikinase. Thus the light induction of phosphoenolpyruvate carboxylase kinase translatable mRNA requires RNA synthesis, but not protein synthesis.     

A study of photorespiratory ammonia production in the C4 plant Amaranthus edulis, using mutants with altered photosynthetic capacities

Previous studies have indicated that the rate of photorespiration in C₄ plants is low or negligible. In this study, wild-type and mutant leaves of the C₄ plant Amaranthus edulis were treated with the glutamine synthetase inhibitor, phosphinothricin and the glycine decarboxylase inhibitor, aminoacetonitrile, at different concentrations of CO₂. The time course of ammonia accumulation in leaves of the wild type was compared with a mutant lacking phosphoenolpyruvate carboxylase activity (EC 4.1.1.31), and with three different mutants that accumulated glycine. The increase in the concentration of ammonia in the leaves, stimulated by the treatments was used as a measurement of the rate of photorespiration in C₄ plants. The application of glutamine and glycine maintained the rate of photorespiratory ammonia production for a longer period in the wild type, and increased the rate in a mutant lacking phosphoenolpyruvate carboxylase suggesting that there was a lack of amino donors in these plants. The calculated rate of photorespiration in Amaranthus edulis wild-type leaves when the supply of amino donors was enough to maintain the photorespiratory nitrogen flow, accounted for approximately 6% of the total net photosynthetic CO₂ assimilation rate. In a mutant lacking phosphoenolpyruvate carboxylase, however, this rate increased to 48%, when glutamine was fed to the leaf, a value higher than that found in some C₃ plants. In mutants of Amaranthus edulis that accumulated glycine, the rate of photorespiration was reduced to 3% of the total net CO₂ assimilation rate. The rate of ammonia produced during photorespiration was 60% of the total produced by all metabolic reactions in the leaves. The data suggests that photorespiration is an active process in C₄ plants, which can play an important role in photosynthetic metabolism in these plants.     

Comparative studies of coupled assays for phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase (PEPC) from higher plants is usually assayed by using malate dehydrogenase (MDH) as a coupling enzyme. To avoid erroneous readings caused by metal ions, which convert oxaloacetate (OAA) to pyruvate, lactic dehydrogenase can be included. Reporting the total NADH used by both coupling enzymes gives the total OAA production. Microbial PEPC has been assayed by employing citrate synthase (CS) as a coupling enzyme which detects the reaction of CoA with Ellman's reagent. Comparable K_m values for MgPEP are found with the two assays. When MDH alone is used as the coupling system, the V_max value is about 60% larger than the one found with the CS assay. However, when MDH is added to the CS assay without the NADH cofactor, V_max is brought back to the same level as that with the NADH-coupled enzyme. Malate inhibition of PEPC assayed with the CS coupling system is blocked by low concentrations of citrate in the range produced in the assay. High concentrations of citrate inhibit PEPC. Glucose-6-phosphate in concentrations higher than 1 m M blocks the response of PEPC to added MDH in the CS assay.     

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.     

Distribution of C3 and C4 grasses along an altitudinal gradient in Central Argentina

The distribution pattern of C₃ and C₄ grasses was studied in eight sites located between 350 m and 2100 m along an altitudinal gradient in Central Argentina. Of 139 taxa fifty-nine are C₃ and eighty C₄. Species of the C₃ tribes (Stipeae, Poeae, Meliceae, Aveneae, Bromeae and Triticeae) and C₃ Paniceae species increase in number at higher elevations; only one C₃ species was found below 650 m. C₄ Aristideae, Pappophoreae, Eragrostideae, Cynodonteae, Andropogoneae and Paniceae increase at lower altitudes. The floristic crossover point is at about 1500 m; the ground cover cross-over point is at about 1000 m. Analysis of the relationships between % C₄ species along the gradient and nine climatic and environmental variables showed the highest correlation with July mean temperature, but all temperature variables show highly significant correlations with % C₄. Correlation with annual rainfall is lower but also significant. These results are consistent with previous research showing the relative importance of C₄ grasses as temperature increases. C₃ species make a high contribution to relative grass coverage below the C₃/C₄ floristic crossover point but are rare below 1000 m.