Temperature-induced changes in Arabidopsis Rubisco activity and isoform expression.

In many plant species, expression of the nuclear encoded Rubisco small subunit (SSu) varies with environmental changes, but the functional role of any changes in expression remains unclear. In this study, we investigated the impact of differential expression of Rubisco SSu isoforms on carbon assimilation in Arabidopsis. Using plants grown at contrasting temperatures (10 °C and 30 °C), we confirm the previously reported temperature response of the four RbcS genes and extend this to protein expression, finding that warm-grown plants produce Rubisco containing ~65% SSu-B and cold-grown plants produce Rubisco with ~65% SSu-A as a proportion of the total pool of subunits. We find that these changes in isoform concentration are associated with kinetic changes to Rubisco in vitro: warm-grown plants produce a Rubisco having greater CO2 affinity (i.e. higher SC/O and lower KC) but lower kcatCO2 at warm measurement temperatures. Although warm-grown plants produce 38% less Rubisco than cold-grown plants on a leaf area basis, warm-grown plants can maintain similar rates of photosynthesis to cold-grown plants at ambient CO2 and 30 °C, indicating that the carboxylation capacity of warm-grown Rubisco is enhanced at warmer measurement temperatures, and is able to compensate for the lower Rubisco content in warm-grown plants. This association between SSu isoform expression and maintenance of Rubisco activity at high temperature suggests that SSu isoform expression could impact the temperature response of C3 photosynthesis.

Temperature response of Rubisco kinetics in Arabidopsis thaliana: thermal breakpoints and implications for reaction mechanisms.

Enhancement of Rubisco kinetics could improve photosynthetic efficiency, ultimately resulting in increased crop yield. However, imprecise knowledge of the reaction mechanism and the individual rate constants limits our ability to optimize the enzyme. Membrane inlet mass spectrometry (MIMS) may offer benefits over traditional methods for determining individual rate constants of the Rubisco reaction mechanism, as it can directly monitor concentration changes in CO2, O2, and their isotopologs during assays. However, a direct comparison of MIMS with the traditional radiolabel method of determining Rubisco kinetic parameters has not been made. Here, the temperature responses of Rubisco kinetic parameters from Arabidopsis thaliana were measured using radiolabel and MIMS methods. The two methods provided comparable parameters above 25 °C, but temperature responses deviated at low temperature as MIMS-derived catalytic rates of carboxylation, oxygenation, and CO2/O2 specificity showed thermal breakpoints. Here, we discuss the variability and uncertainty surrounding breakpoints in the Rubisco temperature response and the relevance of individual rate constants of the reaction mechanisms to potential breakpoints.

Temperature dependence of in vitro Rubisco kinetics in species of Flaveria with different photosynthetic mechanisms.

There is general consensus in the literature that plants with different photosynthetic mechanisms (i.e. C3 vs. C4) have Rubiscos characterised by different kinetic performances. However, potential differences in the temperature dependencies of Rubisco kinetic parameters between C3 and C4 plants are uncertain. Accordingly, six species of Flaveria with contrasting photosynthetic mechanisms (C3, C3/C4 and C4) were selected and their Rubisco Michaelis-Menten constants for CO2 and RuBP (K c and K RuBP), carboxylase catalytic turnover rate ([Formula: see text]) and CO2/O2 specificity factor (S c/o) were measured between 10 and 40 °C. The results confirmed different Rubisco characteristics between C3 and C4 plants. Rubisco from the C3 species had higher E a for K c and [Formula: see text] than that from C4 species, which were translated into differences in the temperature response of the carboxylase catalytic efficiency ([Formula: see text]/K c). However, E a did not differ for S c/o or K RuBP. Although a mechanism remains uncertain, it appears that the Asp/Glu-149-Ala and Met-309-Ile substitutions lead to differences in the temperature responses of catalysis between C3 and C4 Rubiscos in Flaveria. Therefore, the above observations are consistent with the fact that C3 species have a higher photosynthetic efficiency and ecological dominance in cool environments, with respect to C4 species in temperate environments.

Can phenotypic plasticity in Rubisco performance contribute to photosynthetic acclimation?

Photosynthetic acclimation varies among species, which likely reveals variations at the biochemical level in the pathways that constitute carbon assimilation and energy transfer. Local adaptation and phenotypic plasticity affect the environmental response of photosynthesis. Phenotypic plasticity allows for a wide array of responses from a single individual, encouraging fitness in a broad variety of environments. Rubisco catalyses the first enzymatic step of photosynthesis, and is thus central to life on Earth. The enzyme is well conserved, but there is habitat-dependent variation in kinetic parameters, indicating that local adaptation may occur. Here, we review evidence suggesting that land plants can adjust Rubisco's intrinsic biochemical characteristics during acclimation. We show that this plasticity can theoretically improve CO2 assimilation; the effect is non-trivial, but small relative to other acclimation responses. We conclude by discussing possible mechanisms that could account for biochemical plasticity in land plant Rubisco.

Field analysis of photoprotection in co-occurring cool climate C(3) and C(4) grasses.

C(4) 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 C(4) species to tolerate cool climates, by focusing on two questions: (1) Do chlorophyll fluorescence responses differ seasonally between co-occurring C(3) and C(4) grasses in the field? (2) Does xanthophyll-mediated photoprotection differ between the two pathways? Spartina pectinata (C(4) ) and Calamogrostis canadensis (C(3) ) 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 C(3) and C(4) 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 C(4) species is a potential limiting factor to C(4) success at high latitudes, but our findings suggest that it is unlikely via a photoinhibitory feedback mechanism.

Quantifying the amount and activity of Rubisco in leaves.

The CO2-fixing enzyme Rubisco plays a crucial biological role as a primary determinant of both plant yield and the response of the biosphere to global change. Here, we describe techniques for measuring the amount and activity of Rubisco in higher plants. To accommodate a range of experimental capabilities, we describe basic radioisotopic methods as well as non-radioactive techniques. The required calculations are included. We discuss problems that commonly arise during the extraction and assay of Rubisco.

Changes in Rubisco kinetics during the evolution of C4 photosynthesis in Flaveria (Asteraceae) are associated with positive selection on genes encoding the enzyme.

Rubisco, the primary photosynthetic carboxylase, evolved 3-4 billion years ago in an anaerobic, high CO(2) atmosphere. The combined effect of low CO(2) and high O(2) levels in the modern atmosphere, and the inability of Rubisco to distinguish completely between CO(2) and O(2), leads to the occurrence of an oxygenation reaction that reduces the efficiency of photosynthesis. Among land plants, C(4) photosynthesis largely solves this problem by facilitating a high CO(2)/O(2) ratio at the site of Rubisco that resembles the atmosphere in which the ancestral enzyme evolved. The prediction that such conditions favor Rubiscos with higher kcat(CO2) and lower CO(2)/O(2) specificity (S(C/O)) is well supported, but the structural basis for the differences between C(3) and C(4) Rubiscos is not clear. Flaveria (Asteraceae) includes C(3), C(3)-C(4) intermediate, and C(4) species with kinetically distinct Rubiscos, providing a powerful system in which to study the biochemical transition of Rubisco during the evolution from C(3) to C(4) photosynthesis. We analyzed the molecular evolution of chloroplast rbcL and nuclear rbcS genes encoding the large subunit (LSu) and small subunit (SSu) of Rubisco from 15 Flaveria species. We demonstrate positive selection on both subunits, although selection is much stronger on the LSu. In Flaveria, two positively selected LSu amino acid substitutions, M309I and D149A, distinguish C(4) Rubiscos from the ancestral C(3) species and statistically account for much of the kinetic difference between the two groups. However, although Flaveria lacks a characteristic "C(4)" SSu, our data suggest that specific residue substitutions in the SSu are correlated with the kinetic properties of Rubisco in this genus.

Vegetative phase change and photosynthesis in Eucalyptus occidentalis: architectural simplification prolongs juvenile traits.

To understand the effect of shoot architecture on vegetative and reproductive phase changes, seedlings of Eucalyptus occidentalis (Myrtaceae) were grown as free-branching or as single-stem plants, the latter treatment resulting from the continual removal of axillary vegetative buds. In E. occidentalis, vegetative phase change was characterized by increasing leaf length/width ratios. In contrast to the behaviour of other woody species subjected to architectural manipulation of this kind, vegetative phase change was faster in branched plants than in single-stem plants, which continued to exhibit juvenile leaf morphology throughout the duration of this study. However, the first appearance of flowers occurred approximately synchronously in both treatments after 9 months, demonstrating that vegetative phase change and floral transition are developmentally uncoupled in this species. Leaf morphological changes that characterized phase change lagged behind changes in leaf anatomy and gas exchange. In single-stem plants with juvenile leaves, leaf intercellular airspace (20.9%) was almost double that in branched plants with adult foliage (11.2%). Photosynthetic gas exchange analyses indicated that the juvenile leaves of single-stem plants had greater Rubisco and electron transport capacities than those of free-branching plants. Higher leaf N concentrations were recorded in single-stem plants than in branched plants. These observations support the hypothesis that the complexity of shoot architecture impacts the rate of vegetative phase change, but does not affect reproductive phase change in this species.

Temperature responses of photosynthesis and respiration in Populus balsamifera L.: acclimation versus adaptation.

To examine the role of acclimation versus adaptation on the temperature responses of CO(2) assimilation, we measured dark respiration (R(n)) and the CO(2) response of net photosynthesis (A) in Populus balsamifera collected from warm and cool habitats and grown at warm and cool temperatures. R(n) and the rate of photosynthetic electron transport (J) are significantly higher in plants grown at 19 versus 27 degrees C; R(n) is not affected by the native thermal habitat. By contrast, both the maximum capacity of rubisco (V(cmax)) and A are relatively insensitive to growth temperature, but both parameters are slightly higher in plants from cool habitats. A is limited by rubisco capacity from 17-37 degrees C regardless of growth temperature, and there is little evidence for an electron-transport limitation. Stomatal conductance (g(s)) is higher in warm-grown plants, but declines with increasing measurement temperature from 17 to 37 degrees C, regardless of growth temperature. The mesophyll conductance (g(m)) is relatively temperature insensitive below 25 degrees C, but g(m) declines at 37 degrees C in cool-grown plants. Plants acclimated to cool temperatures have increased R(n)/A, but this response does not differ between warm- and cool-adapted populations. Primary carbon metabolism clearly acclimates to growth temperature in P. balsamifera, but the ecotypic differences in A suggest that global warming scenarios might affect populations at the northern and southern edges of the boreal forest in different ways.

Rubisco, Rubisco activase, and global climate change.

Global warming and the rise in atmospheric CO(2) will increase the operating temperature of leaves in coming decades, often well above the thermal optimum for photosynthesis. Presently, there is controversy over the limiting processes controlling photosynthesis at elevated temperature. Leading models propose that the reduction in photosynthesis at elevated temperature is a function of either declining capacity of electron transport to regenerate RuBP, or reductions in the capacity of Rubisco activase to maintain Rubisco in an active configuration. Identifying which of these processes is the principal limitation at elevated temperature is complicated because each may be regulated in response to a limitation in the other. Biochemical and gas exchange assessments can disentangle these photosynthetic limitations; however, comprehensive assessments are often difficult and, for many species, virtually impossible. It is proposed that measurement of the initial slope of the CO(2) response of photosynthesis (the A/C(i) response) can be a useful means to screen for Rubisco activase limitations. This is because a reduction in the Rubisco activation state should be most apparent at low CO(2) when Rubisco capacity is generally limiting. In sweet potato, spinach, and tobacco, the initial slope of the A/C(i) response shows no evidence of activase limitations at high temperature, as the slope can be accurately modelled using the kinetic parameters of fully activated Rubisco. In black spruce (Picea mariana), a reduction in the initial slope above 30 degrees C cannot be explained by the known kinetics of fully activated Rubisco, indicating that activase may be limiting at high temperatures. Because black spruce is the dominant species in the boreal forest of North America, Rubisco activase may be an unusually important factor determining the response of the boreal biome to climate change.

The biochemistry of Rubisco in Flaveria.

C(4) plants have been reported to have Rubiscos with higher maximum carboxylation rates (kcat(CO(2))) and Michaelis-Menten constants (K(m)) for CO(2) (K(c)) than the enzyme from C(3) species, but variation in other kinetic parameters between the two photosynthetic pathways has not been extensively examined. The CO(2)/O(2) specificity (S(C/O)), kcat(CO(2)), K(c), and the K(m) for O(2) (K(o)) and RuBP (K(m-RuBP)), were measured at 25 degrees C, in Rubisco purified from 16 species of Flaveria (Asteraceae). Our analysis included two C(3) species of Flaveria, four C(4) species, and ten C(3)-C(4) or C(4)-like species, in addition to other C(4) (Zea mays and Amaranthus edulis) and C(3) (Spinacea oleracea and Chenopodium album) plants. The S(C/O) of the C(4) Flaveria species was about 77 mol mol(-1), which was approximately 5% lower than the corresponding value in the C(3) species. For Rubisco from the C(4) Flaverias kcat(CO(2)) and K(c) were 23% and 45% higher, respectively, than for Rubisco from the C(3) plants. Interestingly, it was found that the K(o) for Rubisco from the C(4) species F. bidentis and F. trinervia were similar to the C(3) Flaveria Rubiscos (approximately 650 microM) while the K(o) for Rubisco in the C(4) species F. kochiana, F. australasica, Z. mays, and A. edulis was reduced more than 2-fold. There were no pathway-related differences in K(m-RuBP). In the C(3)-C(4) species kcat(CO(2)) and K(c) were generally similar to the C(3) Rubiscos, but the K(o) values were more variable. The typical negative relationships were observed between S(C/O) and both kcat(CO(2)) and K(c), and a strongly positive relationship was observed between kcat(CO(2)) and Kc. However, the statistical significance of these relationships was influenced by the phylogenetic relatedness of the species.

The temperature response of photosynthesis in tobacco with reduced amounts of Rubisco.

The reasons for the decline in net CO2 assimilation (A) above its thermal optimum are controversial. We tested the hypothesis that increasing the ratio of Rubisco activase to Rubisco catalytic site concentration would increase the activation state of Rubisco at high temperatures. We measured photosynthetic gas exchange, in vivo electron transport (J) and the activation state of Rubisco between 15 and 45 degrees C, at 38 and 76 Pa ambient CO2, in wild-type (WT) and anti-rbcS tobacco. The Rubisco content of the anti-rbcS lines was 30% (S7-1) or 6% (S7-2) of WT, but activase levels were the same in the three genotypes. Anti-rbcS plants had lower A than WT at all temperatures, but had a similar thermal optimum for photosynthesis as WT at both CO2 levels. In WT plants, Rubisco was fully activated at 32 degrees C, but the activation state declined to 64% at 42 degrees C. By contrast, the activation state of Rubisco was above 90% in the S7-1 line, between 15 and 42 degrees C. Both A and J declined about 20% from T(opt) to the highest measurement temperatures in WT and the S7-1 line, but this was fully reversed after a 20 min recovery at 35 degrees C. At 76 Pa CO2, predicted rates of RuBP regeneration-limited photosynthesis corresponded with measured A in WT tobacco at all temperatures, and in S7-1 tobacco above 40 degrees C. Our observations are consistent with the hypothesis that the high temperature decline in A in the WT is because of an RuBP regeneration limitation, rather than the capacity of Rubisco activase to maintain high Rubisco activation state.

The temperature response of C(3) and C(4) photosynthesis.

We review the current understanding of the temperature responses of C(3) and C(4) photosynthesis across thermal ranges that do not harm the photosynthetic apparatus. In C(3) species, photosynthesis is classically considered to be limited by the capacities of ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco), ribulose bisphosphate (RuBP) regeneration or P(i) regeneration. Using both theoretical and empirical evidence, we describe the temperature response of instantaneous net CO(2) assimilation rate (A) in terms of these limitations, and evaluate possible limitations on A at elevated temperatures arising from heat-induced lability of Rubisco activase. In C(3) plants, Rubisco capacity is the predominant limitation on A across a wide range of temperatures at low CO(2) (<300 microbar), while at elevated CO(2), the limitation shifts to P(i) regeneration capacity at suboptimal temperatures, and either electron transport capacity or Rubisco activase capacity at supraoptimal temperatures. In C(4) plants, Rubisco capacity limits A below 20 degrees C in chilling-tolerant species, but the control over A at elevated temperature remains uncertain. Acclimation of C(3) photosynthesis to suboptimal growth temperature is commonly associated with a disproportional enhancement of the P(i) regeneration capacity. Above the thermal optimum, acclimation of A to increasing growth temperature is associated with increased electron transport capacity and/or greater heat stability of Rubisco activase. In many C(4) species from warm habitats, acclimation to cooler growth conditions increases levels of Rubisco and C(4) cycle enzymes which then enhance A below the thermal optimum. By contrast, few C(4) species adapted to cooler habitats increase Rubisco content during acclimation to reduced growth temperature; as a result, A changes little at suboptimal temperatures. Global change is likely to cause a widespread shift in patterns of photosynthetic limitation in higher plants. Limitations in electron transport and Rubisco activase capacity should be more common in the warmer, high CO(2) conditions expected by the end of the century.

C4 grasses in boreal fens: their occurrence in relation to microsite characteristics.

C(4) plants are rare in cool climates, an ecological pattern attributable to their poor photosynthetic performance at low temperatures relative to C(3) species. However, some C(4) species are able to persist at high latitudes and high elevations, possibly due to the characteristics of the particular microsites they inhabit in these otherwise unfavourable environments. One such species is Muhlenbergia glomerata, which occurs above 60 degrees N in Canada and is found in the atypical C(4) habitat of boreal fens. In this study, we evaluate how microsite features affect the success of M. glomerata in boreal fens. We surveyed 19 populations across northern Ontario during the summers of 1999 and 2000. The ground coverage by woody vegetation was the most important parameter affecting the presence or absence of M. glomerata. Woody plants covered over 50% of the ground area in plots where M. glomerata is absent, but less than 20% where it is present. The minimum light intensity threshold for the presence of the C(4) species was about 32% of full-sunlight at plant height. Surprisingly, in boreal fens M. glomerata was largely restricted to the wetter moss hollows, rather than occurring on the dry hummocks where its greater water use efficiency might have been advantageous. Woody species dominated the hummocks, but were uncommon in the hollows. In these cool northern climates M. glomerata apparently persists because sufficient periods of temperatures favourable to C(4) photosynthesis occur, but this persistence likely requires some factor that suppresses the woody vegetation.

C4 photosynthesis at low temperature. A study using transgenic plants with reduced amounts of Rubisco.

C(4) plants are rare in the cool climates characteristic of high latitudes and elevations, but the reasons for this are unclear. We tested the hypothesis that CO(2) fixation by Rubisco is the rate-limiting step during C(4) photosynthesis at cool temperatures. We measured photosynthesis and chlorophyll fluorescence from 6 degrees C to 40 degrees C, and in vitro Rubisco and phosphoenolpyruvate carboxylase activity from 0 degrees C to 42 degrees C, in Flaveria bidentis modified by an antisense construct (targeted to the nuclear-encoded small subunit of Rubisco, anti-RbcS) to have 49% and 32% of the wild-type Rubisco content. Photosynthesis was reduced at all temperatures in the anti-Rbcs plants, but the thermal optimum for photosynthesis (35 degrees C) did not differ. The in vitro turnover rate (kcat) of fully carbamylated Rubisco was 3.8 mol mol(-)(1) s(-)(1) at 24 degrees C, regardless of genotype. The in vitro kcat (Rubisco Vcmax per catalytic site) and in vivo kcat (gross photosynthesis per Rubisco catalytic site) were the same below 20 degrees C, but at warmer temperatures, the in vitro capacity of the enzyme exceeded the realized rate of photosynthesis. The quantum requirement of CO(2) assimilation increased below 25 degrees C in all genotypes, suggesting greater leakage of CO(2) from the bundle sheath. The Rubisco flux control coefficient was 0.68 at the thermal optimum and increased to 0.99 at 6 degrees C. Our results thus demonstrate that Rubisco capacity is a principle control over the rate of C(4) photosynthesis at low temperatures. On the basis of these results, we propose that the lack of C(4) success in cool climates reflects a constraint imposed by having less Rubisco than their C(3) competitors.

Quo vadis C(4)? An ecophysiological perspective on global change and the future of C(4) plants.

C(4) plants are directly affected by all major global change parameters, often in a manner that is distinct from that of C(3) plants. Rising CO(2) generally stimulates C(3) photosynthesis more than C(4), but C(4) species still exhibit positive responses, particularly at elevated temperature and arid conditions where they are currently common. Acclimation of photosynthesis to high CO(2) occurs in both C(3) and C(4) plants, most notably in nutrient-limited situations. High CO(2) aggravates nitrogen limitations and in doing so may favor C(4) species, which have greater photosynthetic nitrogen use efficiency. C(4) photosynthesis is favored by high temperature, but global warming will not necessarily favor C(4) over C(3) plants because the timing of warming could be more critical than the warming itself. C(3) species will likely be favored where harsh winter climates are moderated, particularly where hot summers also become drier and less favorable to C(4) plant growth. Eutrophication of soils by nitrogen deposition generally favors C(3) species by offsetting the superior nitrogen use efficiency of C(4) species; this should allow C(3) species to expand at the expense of C(4) plants. Land-use change and biotic invasions are also important global change factors that affect the future of C(4) plants. Human exploitation of forested landscapes favors C(4) species at low latitude by removing woody competitors and opening gaps in which C(4) grasses can establish. Invasive C(4) grasses are causing widespread forest loss in Asia, the Americas and Oceania by accelerating fire cycles and reducing soil nutrient status. Once established, weedy C(4) grasses can prevent woodland establishment, and thus arrest ecological succession. In sum, in the future, certain C(4) plants will prosper at the expense of C(3) species, and should be able to adjust to the changes the future brings.