David S. Kubien

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.

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.

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 CO2 assimilation, we measured dark respiration (Rn) and the CO2 response of net photosynthesis (A) in Populus balsamifera collected from warm and cool habitats and grown at warm and cool temperatures. Rn and the rate of photosynthetic electron transport (J) are significantly higher in plants grown at 19 versus 27°C; Rn is not affected by the native thermal habitat. By contrast, both the maximum capacity of rubisco (Vcmax) 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°C regardless of growth temperature, and there is little evidence for an electron-transport limitation. Stomatal conductance (gs) is higher in warm-grown plants, but declines with increasing measurement temperature from 17 to 37°C, regardless of growth temperature. The mesophyll conductance (gm) is relatively temperature insensitive below 25°C, but gm declines at 37°C in cool-grown plants. Plants acclimated to cool temperatures have increased Rn/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.

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.

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.

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.

Differences in the Structure and Gas Exchange Physiology of Juvenile and Adult Leaves in Metrosideros excelsa

In many plant species, ontogeny is characterized by the production of different leaf forms, but the functional significance of this phenomenon is unclear. In Metrosideros excelsa (Myrtaceae), vegetative phase change is characterized by a transition from glabrous juvenile foliage to adult leaves that possess a dense pubescence on their abaxial surface. We examined the changes to anatomical and physiological leaf characteristics that accompany phase change in this species. There was no consistent change to δ13C during ontogeny in M. excelsa, indicating that the ratio of intercellular to ambient CO2 remained relatively constant. Rates of photosynthesis were lower in adult versus juvenile foliage, apparently because of a reduction in resources invested toward carbon gain. The stomatal conductance to water vapor tended to decline as the development of pubescence progressed but increased in fully pubescent adult leaves. The stomatal limitation to photosynthetic carbon gain (Ls) exceeded 40% in juvenile and transition leaves but was less than 30% in adult leaves. During vegetative phase change in M. excelsa, the control of water loss appears to shift from a physiological to a physical basis, possibly associated with a reallocation of leaf resources away from photosynthesis.

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 (Kc and KRuBP), carboxylase catalytic turnover rate (