Photosynthetic flexibility and ecophysiological plasticity: questions and lessons from Clusia, the only CAM tree, in the neotropics

The discovery of crassulacean acid metabolism (CAM) in the trees of Clusia: arrival in the limelight of international research 8 II. Phylogeny 8 III. Photosynthetic physiotypes 10 IV. Metabolic flexibility: organic acid variations 12 V. The environmental control of photosynthetic flexibility 13 VI. Phenotypic plasticity: physiotypes and morphotypes 16 VII. Ecological amplitude and habitat impact 16 VIII. Conclusions and outlook 21 Acknowledgements 22 References 22 Summary It is the aim of this review to present a monographic survey of the neotropical genus Clusia on scaling levels from molecular phylogeny, metabolism, photosynthesis and autecological environmental responses to ecological amplitude and synecological habitat impact. Clusia is the only dicotyledonous genus with real trees performing crassulacean acid metabolism (CAM). By way of introduction, a brief historical reminiscence describes the discovery of CAM in Clusia and the consequent increase in interest in studying this particular genus of tropical shrubs and trees. The molecular phylogeny of CAM in the genus is compared with that in Kalancho? and the Bromeliaceae. At the level of metabolism and photosynthesis, the great plasticity of expression of photosynthetic physiotypes, i.e. (i) C(3) photosynthesis, (ii) CAM including CAM idling, (iii) CAM cycling and (iv) C(3)/CAM-intermediate behaviour, as well as metabolic flexibility in Clusia is illustrated. At the level of autecology, the factors water, irradiance and temperature, which control photosynthetic flexibility, are assessed. The phenotypic plasticity of physiotypes and morphotypes is described. At the level of synecology, the ecological amplitude of Clusia in the tropics and the relations to habitat are surveyed.     

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.     

Comparative ecophysiology of CAM and C3 bromeliads. III. Environmental influences on CO2 assimilation and transpiration

Abstract Field measurements of the gas exchange of epiphytic bromeliads were made during the dry season in Trinidad in order to compare carbon assimilation with water use in CAM and C₃ photosynthesis. The expression of CAM was found to be directly influenced by habitat and microclimate. The timing of nocturnal CO₂ uptake was restricted to the end of the dark period in plants found at drier habitats, and stomatal conductance in two CAM species was found to respond directly to humidity or temperature. Total night-time CO₂ uptake, when compared with malic-acid formation (measured as the dawn-dusk difference in acidity, ΔH⁺), could only account for 10–40% of the total ΔH⁺ accumulated. The remaining malic acid must have been derived from the refixation of respired CO₂ (recycling). Within the genus Aechmea (12 samples from four species), recycling was significantly correlated with night temperature at the six sample sites. Recycling was lowest in A. fendleri (54% of ΔH⁺ derived from respired CO₂), a CAM bromeliad with little water-storage parenchyma that is restricted to wetter, cooler regions of Trinidad. Gas-exchange rates of C₃ bromeliads were found to be similar to those of the CAM bromeliads, with CO₂ uptake from 1 to 3 μmol m^?2 s^?1 and stomatal conductances generally up to 100 mmol m^?2 s^?1. The midday depression of photosynthesis occurred in exposed habitats, although photosynthetically active radiation (PAR) limited photosynthesis in shaded habitats. CO₂ uptake of the C₃ bromeliad Guzmania lingulata was saturated at around 500 μmol m^?2 s^?1 PAR, suggesting that epiphytic plants found in the shaded forest understorey are shade-tolerant rather than shade-demanding. Transpiration ratios (TR) during CO₂ fixation in CAM (Phase I and IV) and C₃ bromeliads were compared at different sites in order to assess the efficiency of water utilization. For the epiphytes displaying marked uptake of CO₂, TR were found to be lower than many previously published values. In addition, the average TR values were very similar for dark CO₂ uptake in CAM (42 ± 41, n= 12), Phase IV of CAM (69 ± 36, n= 3) and for C₃ photosynthesis (99 ± 73, n= 4) in these plants. It appears that recycling of respired CO₂ by CAM bromeliads and efficient use of water in all phases of CO₂ uptake are physiological adaptations of bromeliads to arid microclimates in the humid tropics.     

Comparative ecophysiology of CAM and C3 bromeliads. I. The ecology of the Bromeliaceae in Trinidad

Abstract This article deals with the physiological ecology of the Bromeliaceae, a large neotropical family containing both terrestrial and epiphytic forms, as well as many species with crassulacean acid metabolism (CAM). The article is in two parts. In the first, we review what is known of the occurrence of CAM and C₃ species in the Bromeliaceae. The photosynthetic pathways are discussed in the context of the major taxonomic divisions within the family and the great diversity of bromeliad life-forms. Of the three subfamilies, the Pitcairnioideae contain both C₃ and CAM species and are essentially all terrestrial. In contrast, the Tillandsioideae are entirely epiphytic or saxicolous, with CAM species being restricted to the genus Tillandsia, And in the Bromelioideae all species show CAM, but terrestrial and epiphytic forms are found in about equal numbers. The evidence suggests that both CAM and the epiphytic habit arose more than once in the family's evolutionary history. In the second part we consider the photosynthetic ecology of the various bromeliad life-forms in more detail using the specific example of Trinidad (West Indies). CAM bromeliads tend to be centred on the drier regions of the island and C₃ forms on the wetter areas. However, at any one site there is a marked vertical stratification of species within the forest profile. Based on the known habitat preferences of the bromeliads, six contrasting sites were selected for field studies in Trinidad. These ranged from arid coastal scrub to montane rain forest, the vegetational and climatic characteristics of which are described here. The constancy of δ¹³C values (carbon-isotope ratios) for individual CAM species in these markedly different habitats emphasized the need for ecophysiological studies to characterize environmental effects on CO₂ assimilation and transpiration. The following papers in this series present the results of a comparative investigation of gas exchange and leaf water relations of CAM and C₃ bromeliads in situ at the various sites.     

Comparative ecophysiology of CAM and C3 bromeliads. II. Field measurements of gas exchange of CAM bromeliads in the humid tropics

Abstract The results described represent the first detailed measurements of gas exchange of epiphytic plants with crassulacean acid metabolism (CAM) in the humid tropics. A portable steady-state CO₂ and H₂O porometer was used to measure net exchange rates of CO₂ and H₂O vapour (J_CO2, J_H2O), leaf temperature (T₁), air temperature (T_A), air relative humidity (RH) and photosynthetically active radiation (PAR) for bromeliads in the field during the dry season in February and March 1983 on the tropical island of Trinidad. Different lengths of tubing (up to 25 m) were used so that the gas exchange could be measured of bromeliads in situ in their epiphytic habitats. Derived parameters such as leaf-air water-vapour-concentration difference (Δ_w), water-vapour conductance of leaves (g) and internal CO₂ partial pressure (pⁱ_CO2) could be calculated. The particular problems of making such measurements in the humid tropics due to high relative humidities and high dew-point temperatures are discussed. The long and often broad, strap-like leaves of bromeliads are well suited for measurements with the steady-state porometer. It is shown that CAM activity varies along the length of individual leaves, and variability between different leaves is also demonstrated. The major phases of CAM, i.e. nocturnal stomalal opening, CO₂ uptake and dark fixation as malic acid (Phase I), daytime stomatal closure and light-dependent assimilation of CO₂ derived from decarboxylation of the malic acid (Phase III), and late-afternoon stomatal opening with direct light-dependent assimilation of atmospheric CO₂ (Phase IV) were all clearly shown by CAM bromeliads in situ. Their expression and magnitude depended on the environmental conditions. An early-morning peak of CO₂ uptake as is characteristic of Phase II of CAM was not detected during the night-day transition. A bromeliad intermediate between C₃ and CAM, Guzmania monostachia, showed substantial net CO₂ uptake in the early morning but no net uptake integrated over the whole of the night.     

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.     

Climate controls on C3 vs. C4 productivity in North American grasslands from carbon isotope composition of soil organic matter

We analyzed the δ¹³C of soil organic matter (SOM) and fine roots from 55 native grassland sites widely distributed across the US and Canadian Great Plains to examine the relative production of C₃ vs. C₄ plants (hereafter %C₄) at the continental scale. Our climate vs. %C₄ results agreed well with North American field studies on %C₄, but showed bias with respect to %C₄ from a US vegetation database (statsgo ) and weak agreement with a physiologically based prediction that depends on crossover temperature. Although monthly average temperatures have been used in many studies to predict %C₄, our analysis shows that high temperatures are better predictors of %C₄. In particular, we found that July climate (average of daily high temperature and month's total rainfall) predicted %C₄ better than other months, seasons or annual averages, suggesting that the outcome of competition between C₃ and C₄ plants in North American grasslands was particularly sensitive to climate during this narrow window of time. Root δ¹³C increased about 1‰ between the A and B horizon, suggesting that C₄ roots become relatively more common than C₃ roots with depth. These differences in depth distribution likely contribute to the isotopic enrichment with depth in SOM where both C₃ and C₄ grasses are present.     

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.     

Mechanistic basis of differences in water-use efficiency between a CAM and a C3 species of Peperomia (Piperaceae)

Under ecologically realistic environmental conditions, the water-use efficiency (WUE) of Peperomia scandens , a CAM plant, was higher than that of the C₃ congener P. obtusifolia . This difference has been attributed to differences in stomatal activity between C₃ and CAM plants, coupled with differences in the evaporative demand of the atmosphere during which the stomata are open. This explanation has apparently not, however, been experimentally tested. Thus, WUEs were compared in these species in two experiments in which the atmospheric evaporative demand was identical (or nearly so) during the period of stomatal opening (i.e. during the night for the CAM plant and during the day for the C₃ species). In both experiments, the WUE of the CAM species was higher than that of the C₃ species. These results suggest that factors other than differences in atmospheric environmental conditions must also be responsible for the observed differences in WUE. Because CO₂ uptake rates of the CAM species were substantially lower than those of the C₃ species, the lower WUE in the CAM species resulted primarily from lower transpiration rates. Lower rates of water loss in P. scandens , relative to rates in P. obtusifolia , were ascribed, in part, to lower stomatal densities. Thus, leaf morphological differences, in addition to differences in atmospheric evaporative demand, help to explain the high WUE typically measured in CAM plants.     

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.     

Photosynthesis and the influence of CO2-enrichment on δ13C values in a C3 halophyte

Abstract Shifts in ?¹³C of the graminaceous C₃ halophyte Puccinellia nuttalliana (Schultes) Hitch. can be induced by salinization. To investigate this phenomenon, three approaches were taken: assay of carboxylases, CO₂-enrichment studies, and gas exchange analysis. Although ribulose-1,5-bisphosphate carboxylase activity decreased with salinity, phosphoenolpyruvate carboxylase activity did not increase and its levels were not atypical of C₃ plants. When plants were grown at four NaCl concentrations under atmospheres of 310 and 1300 cm³ m^?3 CO₂, the CO₂-enrichment enhanced the effects of salinity on ?¹³C. This is consistent with a biophysical explanation for salt-induced shifts in ?¹³C, whereby there is a steepening of the CO₂ diffusion gradient into the leaf. Gas exchange analysis indicated that intercellular CO₂ concentrations were depressed in the leaves of salt-affected plants. This resulted from a greatly decreased stomatal conductance coupled with only small effects on intrinsic photosynthetic capacity. Water-use efficiency was enhanced.     

RuBPCO kinetics and the mechanism of CO2 entry in C3 plants

Abstract. The CO₂ partial pressure in the chloroplasts of intact photosynthetic C₃ leaves is thought to be less than the intercellular CO₂ partial pressure. The intercellular CO₂ partial pressure can be calculated from CO₂ and H₂O gas exchange measurements, whereas the CO₂ partial pressure in the chloroplasts is unknown. The conductance of CO₂ from the intercellular space to the chloroplast stroma and the CO₂ partial pressure in the chloroplast stroma can be calculated if the properties of photosynthetic gas exchange are compared with the kinetics of the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPCO). A discrepancy between gas exchange and RuBPCO kinetics can be attributed to a deviation of CO₂ partial pressure in the chloroplast stroma from that calculated in the intercellular space. This paper is concerned with the following: estimation of the kinetic constants of RuBPCO and their comparison with the CO₂ compensation concentration; their comparison with differential uptake of ¹⁴CO₂ and ¹²CO₂; and their comparison with O₂ dependence of net CO₂ uptake of photosynthetic leaves. Discrepancy between RuBPCO kinetics and gas exchange was found at a temperature of 12.5 °C, a photosynthetic photon flux density (PPFD) of 550 μmol quanta m^?2 s^?1, and an ambient CO₂ partial pressure of 40 Pa. Consistency between RuBPCO kinetics and gas exchange was found if CO₂ partial pressure was decreased, temperature incresed and PPFD decreased. The results suggest that a discrepancy between RuBPCO kinetics and gas exchange is due to a diffusion resistance for CO₂ across the chloroplast envelope which decreases with increasing temperature. At low CO₂ partial pressure, the diffusion resistance appears to be counterbalanced by active CO₂ (or HCO₃) transport with high affinity and low maximum velocity. At low PPFD, CO₂ partial pressure in the chloroplast stroma appears to be in equilibrium with that in the intercellular space due to low CO₂ flux.     

Comparisons of photosynthesis and photoinhibition in the CAM vine Hoya australis and several C3 vines growing on the coast of eastern Australia

Abstract. The CAM vine Hoya australis and three C₃ vines, Smilax australis, Ipomoea pes-caprae and Kennedta rubicunda, were studied at a site on the coast of northeastern New South Wales, Australia. The level of CAM activity (nocturnal acid accumulation) was comparable in H. australis growing in full sunlight and in deep shade. Acclimation to shade by H. australis was indicated by thinner leaves, increased chlorophyll content, decreased chlorophyll a/b ratios, lower dark respiration rates, and lower light compensation points. When growing in full sunlight H. australis exhibited reductions in photochemical efficiency, as indicated by reduced quantum yields and F_v/F_m fluorescence from PS II as well as low rates of photosynthesis at high light. Sun leaves of H. australis experienced a massive quenching of fluorescence from PS II during normal exposure to midday irradiance which was rapidly reversible under low irradiance conditions in the late afternoon. This quenching indicated a reduction in photochemical efficiency, part of which could be accounted for by an increase in non-radiative energy dissipation, while part of it was due to one or more processes not yet identified. Changes in PS II fluorescence from shade H. australis exposed to full sunlight suggest a decrease in the rate constant for photochemistry indicative of damage to the reaction centre, as well as an increase in non-radiative energy dissipation. The C₃ vine S. australis was also shade tolerant, but exhibited little evidence of photoinhibition when growing in full sunlight. Ipomoea pescaprae and K. rubicunda, both of which were apparently shade intolerant (being found only in full sunlight), possessed high quantum yields and much higher photosynthetic capacities than either H. australis or S. australis. From this study, and several others, it appears that plants possessing CAM experience photoinhibition to a greater degree than do C₃ species in full sunlight under natural conditions, which is probably exacerbated by some degree of CAM-idling.     

Elevated CO2 enhances water relations and productivity and affects gas exchange in C3 and C4 grasses of the Colorado shortgrass steppe.

Six open‐top chambers were installed on the shortgrass steppe in north‐eastern Colorado, USA from late March until mid‐October in 1997 and 1998 to evaluate how this grassland will be affected by rising atmospheric CO₂. Three chambers were maintained at current CO₂ concentration (ambient treatment), three at twice ambient CO₂, or approximately 720 μmol mol^?1 (elevated treatment), and three nonchambered plots served as controls. Above‐ground phytomass was measured in summer and autumn during each growing season, soil water was monitored weekly, and leaf photosynthesis, conductance and water potential were measured periodically on important C₃ and C₄ grasses. Mid‐season and seasonal above‐ground productivity were enhanced from 26 to 47% at elevated CO₂, with no differences in the relative responses of C₃/C₄ grasses or forbs. Annual above‐ground phytomass accrual was greater on plots which were defoliated once in mid‐summer compared to plots which were not defoliated during the growing season, but there was no interactive effect of defoliation and CO₂ on growth. Leaf photosynthesis was often greater in Pascopyrum smithii (C₃) and Bouteloua gracilis (C₄) plants in the elevated chambers, due in large part to higher soil water contents and leaf water potentials. Persistent downward photosynthetic acclimation in P. smithii leaves prevented large photosynthetic enhancement for elevated CO₂‐grown plants. Shoot N concentrations tended to be lower in grasses under elevated CO₂, but only Stipa comata (C₃) plants exhibited significant reductions in N under elevated compared to ambient CO₂ chambers. Despite chamber warming of 2.6 °C and apparent drier chamber conditions compared to unchambered controls, above‐ground production in all chambers was always greater than in unchambered plots. Collectively, these results suggest increased productivity of the shortgrass steppe in future warmer, CO₂ enriched environments.