Deactivation Of Rubisco Research Articles

Rubisco deactivation and chloroplast electron transport rates co-limit photosynthesis above optimal leaf temperature in terrestrial plants

Net photosynthetic CO2 assimilation rate (An) decreases at leaf temperatures above a relatively mild optimum (Topt) in most higher plants. This decline is often attributed to reduced CO2 conductance, increased CO2 loss from photorespiration and respiration, reduced chloroplast electron transport rate (J), or deactivation of Ribulose-1,5-bisphosphate Carboxylase Oxygenase (Rubisco). However, it is unclear which of these factors can best predict species independent declines in An at high temperature. We show that independent of species, and on a global scale, the observed decline in An with rising temperatures can be effectively accounted for by Rubisco deactivation and declines in J. Our finding that An declines with Rubisco deactivation and J supports a coordinated down-regulation of Rubisco and chloroplast electron transport rates to heat stress. We provide a model that, in the absence of CO2 supply limitations, can predict the response of photosynthesis to short-term increases in leaf temperature.

Faster than expected Rubisco deactivation in shade reduces cowpea photosynthetic potential in variable light conditions

Cowpea is the major source of vegetable protein for rural populations in sub-Saharan Africa and average yields are not keeping pace with population growth. Each day, crop leaves experience many shade events and the speed of photosynthetic adjustment to this dynamic environment strongly affects daily carbon gain. Rubisco activity is particularly important because it depends on the speed and extent of deactivation in shade and recovers slowly on return to sun. Here, direct biochemical measurements showed a much faster rate of Rubisco deactivation in cowpea than prior estimates inferred from dynamics of leaf gas exchange in other species1–3. Shade-induced deactivation was driven by decarbamylation, and half-times for both deactivation in shade and activation in saturating light were shorter than estimates from gas exchange (≤53% and 79%, respectively). Incorporating these half-times into a model of diurnal canopy photosynthesis predicted a 21% diurnal loss of productivity and suggests slowing Rubisco deactivation during shade is an unexploited opportunity for improving crop productivity.

Carboxylation Capacity Can Limit C3 Photosynthesis at Elevated CO2 throughout Diurnal Cycles.

The response of carbon fixation in C3 plants to elevated CO2 is relatively larger when photosynthesis is limited by carboxylation capacity (VC) than when limited by electron transport (J). Recent experiments under controlled, steady-state conditions have shown that photosynthesis at elevated CO2 may be limited by VC even at limiting PPFD. These experiments were designed to test whether this also occurs in dynamic field environments. Leaf gas exchange was recorded every 5 min using two identical instruments both attached to the same leaf. The CO2 concentration in one instrument was controlled at 400 μmol mol−1 and one at 600 μmol mol−1. Leaves were exposed to ambient sunlight outdoors, and cuvette air temperatures tracked ambient outside air temperature. The water content of air in the leaf cuvettes was kept close to that of the ambient air. These measurements were conducted on multiple, mostly clear days for each of three species, Glycine max, Lablab purpureus, and Hemerocallis fulva. The results indicated that in all species, photosynthesis was limited by VC rather than J at both ambient and elevated CO2 both at high midday PPFDs and also at limiting PPFDs in the early morning and late afternoon. During brief reductions in PPFD due to midday clouds, photosynthesis became limited by J. The net result of the apparent deactivation of Rubisco at low PPFD was that the relative stimulation of diurnal carbon fixation at elevated CO2 was larger than would be predicted when assuming limitation of photosynthesis by J at low PPFD.

Overexpression of both Rubisco and Rubisco activase rescues rice photosynthesis and biomass under heat stress.

Global warming threatens food security by decreasing crop yields through damage to photosynthetic systems, especially Rubisco activation. We examined whether co-overexpression of Rubisco and Rubisco activase improves the photosynthetic and growth performance of rice under high temperatures. We grew three rice lines-the wild-type (WT), a Rubisco activase-overexpressing line (oxRCA) and a Rubisco- and Rubisco activase-co-overexpressing line (oxRCA-RBCS)-and analysed photosynthesis and biomass at 25 and 40°C. Compared with the WT, the Rubisco activase content was 153% higher in oxRCA and 138% higher in oxRCA-RBCS, and the Rubisco content was 27% lower in oxRCA and similar in oxRCA-RBCS. The CO2 assimilation rate (A) of WT was lower at 40°C than at 25°C, attributable to Rubisco deactivation by heat. On the other hand, that of oxRCA and oxRCA-RBCS was maintained at 40°C, resulting in higher A than WT. Notably, the dry weight of oxRCA-RBCS was 26% higher than that of WT at 40°C. These results show that increasing the Rubisco activase content without the reduction of Rubisco content could improve yield and sustainability in rice at high temperature.

Variation in Responses of Photosynthesis and Apparent Rubisco Kinetics to Temperature in Three Soybean Cultivars.

Recent in vivo assays of the responses of Rubisco to temperature in C3 plants have revealed substantial diversity. Three cultivars of soybean (Glycine max L. Merr.), Holt, Fiskeby V, and Spencer, were grown in indoor chambers at 15, 20, and 25 °C. Leaf photosynthesis was measured over the range of 15 to 30 °C, deliberately avoiding higher temperatures which may cause deactivation of Rubisco, in order to test for differences in temperature responses of photosynthesis, and to investigate in vivo Rubisco kinetic characteristics responsible for any differences observed. The three cultivars differed in the optimum temperature for photosynthesis (from 15 to 30 °C) at 400 μmol mol−1 external CO2 concentration when grown at 15 °C, and in the shapes of the response curves when grown at 25 °C. The apparent activation energy of the maximum carboxylation rate of Rubisco differed substantially between cultivars at all growth temperatures, as well as changing with growth temperature in two of the cultivars. The activation energy ranged from 58 to 84 kJ mol−1, compared with the value of 64 kJ mol−1 used in many photosynthesis models. Much less variation in temperature responses occurred in photosynthesis measured at nearly saturating CO2 levels, suggesting more diversity in Rubisco than in electron transport thermal properties among these soybean cultivars.

Activation state of Rubisco decreases with the nitrogen accumulation during the reproductive stage in soybean [Glycine max (L.) Merr.

The CO2 assimilation rate, N content, Rubisco activation state, and protein distribution to Rubisco and Rubisco activase in the leaf were analyzed for four soybean genotypes at two growth stages under field conditions. The CO2 assimilation rate was largely constant except for one genotype during the reproductive stage. Rubisco activation state significantly decreased with the increase of N content during this period in three of four genotypes. When standardized by total soluble protein amount, Rubisco activase amount was constant during this period in three genotypes. The results in the present study showed the deactivation of Rubisco with the N accumulation, which can result in the constant rate of CO2 assimilation during the reproductive stage of soybean. The deactivation of Rubisco was attributed to an unknown mechanism other than to the deficiency of Rubisco activase.

Effects of genetic manipulation of the activity of photorespiration on the redox state of photosystem I and its robustness against excess light stress under CO2-limited conditions in rice.

Under CO2-limited conditions such as during stomatal closure, photorespiration is suggested to act as a sink for excess light energy and protect photosystem I (PSI) by oxidizing its reaction center chlorophyll P700. In this study, this issue was directly examined with rice (Oryza sativa L.) plants via genetic manipulation of the amount of Rubisco, which can be a limiting factor for photorespiration. At low [CO2] of 5Pa that mimicked stomatal closure condition, the activity of photorespiration in transgenic plants with decreased Rubisco content (RBCS-antisense plants) markedly decreased, whereas the activity in transgenic plants with overproduction of Rubisco (RBCS-sense plants) was similar to that in wild-type plants. Oxidation of P700 was enhanced at [CO2] of 5Pa in wild-type and RBCS-sense plants. PSI was not damaged by excess light stress induced by repetitive saturated pulse-light (rSP) in the presence of strong steady-state light. On the other hand, P700 was strongly reduced in RBCS-antisense plants at [CO2] of 5Pa. PSI was also damaged by rSP illumination. These results indicate that oxidation of P700 and the robustness of PSI against excess light stress are hampered by the decreased activity of photorespiration as a result of genetic manipulation of Rubisco content. It is also suggested that overproduction of Rubisco does not enhance photorespiration as well as CO2 assimilation probably due to partial deactivation of Rubisco.

Shade compromises the photosynthetic efficiency of NADP-ME less than that of PEP-CK and NAD-ME C4 grasses.

The high energy cost and apparently low plasticity of C4 photosynthesis compared with C3 photosynthesis may limit the productivity and distribution of C4 plants in low light (LL) environments. C4 photosynthesis evolved numerous times, but it remains unclear how different biochemical subtypes perform under LL. We grew eight C4 grasses belonging to three biochemical subtypes [NADP-malic enzyme (NADP-ME), NAD-malic enzyme (NAD-ME), and phosphoenolpyruvate carboxykinase (PEP-CK)] under shade (16% sunlight) or control (full sunlight) conditions and measured their photosynthetic characteristics at both low and high light. We show for the first time that LL (during measurement or growth) compromised the CO2-concentrating mechanism (CCM) to a greater extent in NAD-ME than in PEP-CK or NADP-ME C4 grasses by virtue of a greater increase in carbon isotope discrimination (∆P) and bundle sheath CO2 leakiness (ϕ), and a greater reduction in photosynthetic quantum yield (Φmax). These responses were partly explained by changes in the ratios of phosphoenolpyruvate carboxylase (PEPC)/initial Rubisco activity and dark respiration/photosynthesis (Rd/A). Shade induced a greater photosynthetic acclimation in NAD-ME than in NADP-ME and PEP-CK species due to a greater Rubisco deactivation. Shade also reduced plant dry mass to a greater extent in NAD-ME and PEP-CK relative to NADP-ME grasses. In conclusion, LL compromised the co-ordination of the C4 and C3 cycles and, hence, the efficiency of the CCM to a greater extent in NAD-ME than in PEP-CK species, while CCM efficiency was less impacted by LL in NADP-ME species. Consequently, NADP-ME species are more efficient at LL, which could explain their agronomic and ecological dominance relative to other C4 grasses.

Relationship between Rubisco activase and Rubisco contents in transgenic rice plants with overproduced or decreased Rubisco content

ABSTRACTOverproduction of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; E.C. 4.1.1.39) in rice did not necessarily improve photosynthesis. The reason for this was that a partial deactivation of Rubisco occurred in RBCS-overexpressed rice plants. Since a negative correlation between the amounts of Rubisco activase (RCA) and Rubisco has been reported for plants with overproduced or decreased RCA, the possibility that RCA content declines in RBCS-overexpressed rice plants was considered. The relationship between RCA and Rubisco contents was examined in RBCS-overexpressed and RBCS-antisense rice plants. Whereas the ratio of RCA to Rubisco contents in RBCS-antisense plants increased three- to fourfold as compared with that of the wild-type levels, this ratio decreased 60–70% of the wild-type levels in RBCS-overexpressed rice plants. Thus, an apparent trade-off between the amounts of RCA and Rubisco was observed in RBCS-transgenic rice plants. However, the amounts of several Calvin–Benson cycle enzymes changed in a similar manner to that of RCA in both types of RBCS-transgenic rice plants. When the relationships between the amounts of these enzymes, including RCA, and those of total leaf-N minus Rubisco-N and trichloroacetic acid (TCA)-soluble N were examined, there were no differences between them irrespective of genotypes. These results indicate that the negative correlation between the amounts of RCA and Rubisco in RBCS-transgenic rice plants is the result of a change in N allocation to Rubisco in transgenic rice plants rather than a trade-off. Such a negative correlation was also found for other Calvin–Benson cycle enzymes. In addition, since the amounts of these Calvin–Benson cycle enzymes and RCA were highly correlated with their mRNA levels irrespective of genotype, it is suggested that changes in the amounts of these proteins are regulated at their transcript levels by a change in N allocation to Rubisco.

In Vitro Characterization of Thermostable CAM Rubisco Activase Reveals a Rubisco Interacting Surface Loop.

To maintain metabolic flux through the Calvin-Benson-Bassham cycle in higher plants, dead-end inhibited complexes of Rubisco must constantly be engaged and remodeled by the molecular chaperone Rubisco activase (Rca). In C3 plants, the thermolability of Rca is responsible for the deactivation of Rubisco and reduction of photosynthesis at moderately elevated temperatures. We reasoned that crassulacean acid metabolism (CAM) plants must possess thermostable Rca to support Calvin-Benson-Bassham cycle flux during the day when stomata are closed. A comparative biochemical characterization of rice (Oryza sativa) and Agave tequilana Rca isoforms demonstrated that the CAM Rca isoforms are approximately10°C more thermostable than the C3 isoforms. Agave Rca also possessed a much higher in vitro biochemical activity, even at low assay temperatures. Mixtures of rice and agave Rca form functional hetero-oligomers in vitro, but only the rice isoforms denature at nonpermissive temperatures. The high thermostability and activity of agave Rca mapped to the N-terminal 244 residues. A Glu-217-Gln amino acid substitution was found to confer high Rca activity to rice Rca Further mutational analysis suggested that Glu-217 restricts the flexibility of the α4-β4 surface loop that interacts with Rubisco via Lys-216. CAM plants thus promise to be a source of highly functional, thermostable Rca candidates for thermal fortification of crop photosynthesis. Careful characterization of their properties will likely reveal further protein-protein interaction motifs to enrich our mechanistic model of Rca function.

The Plastid Casein Kinase 2 Phosphorylates Rubisco Activase at the Thr-78 Site but Is Not Essential for Regulation of Rubisco Activation State.

Rubisco activase (RCA) is essential for the activation of Rubisco, the carboxylating enzyme of photosynthesis. In Arabidopsis, RCA is composed of a large RCAα and small RCAβ isoform that are formed by alternative splicing of a single gene (At2g39730). The activity of Rubisco is controlled in response to changes in irradiance by regulation of RCA activity, which is known to involve a redox-sensitive disulfide bond located in the carboxy-terminal extension of the RCAα subunit. Additionally, phosphorylation of RCA threonine-78 (Thr-78) has been reported to occur in the dark suggesting that phosphorylation may also be associated with dark-inactivation of RCA and deactivation of Rubisco. In the present study, we developed site-specific antibodies to monitor phosphorylation of RCA at the Thr-78 site and used non-reducing SDS-PAGE to monitor the redox status of the RCAα subunit. By immunoblotting, phosphorylation of both RCA isoforms occurred at low light and in the dark and feeding peroxide or DTT to leaf segments indicated that redox status of the chloroplast stroma was a critical factor controlling RCA phosphorylation. Use of a knockout mutant identified the plastid-targeted casein kinase 2 (cpCK2α) as the major protein kinase involved in RCA phosphorylation. Studies with recombinant cpCK2α and synthetic peptide substrates identified acidic residues at the –1, +2, and +3 positions surrounding Thr-78 as strong positive recognition elements. The cpck2 knockout mutant had strongly reduced phosphorylation at the Thr-78 site but was similar to wild type plants in terms of induction kinetics of photosynthesis following transfer from darkness or low light to high light, suggesting that if phosphorylation of RCA Thr-78 plays a direct role it would be redundant to redox regulation for control of Rubisco activation state under normal conditions.

Temperature response of CO2 exchange in three tropical tree species.

Tropical forests play a critical role in the global carbon cycle, but our limited understanding of the physiological sensitivity of tropical forest trees to environmental factors complicates predictions of tropical carbon fluxes in a changing climate. We determined the short-term temperature response of leaf photosynthesis and respiration of seedlings of three tropical tree species from Panama. For one of the species net CO2 exchange was also measured in situ. Dark respiration of all species increased linearly - not exponentially - over a ~30°C temperature range. The early-successional species Ficus insipida Willd. and Ochroma pyramidale (Cav. ex Lam.) Urb. had higher temperature optima for photosynthesis (Topt) and higher photosynthesis rates at Topt than the late-successional species Calophyllum longifolium Willd. The decrease in photosynthesis above Topt could be assigned, in part, to observed temperature-stimulated photorespiration and decreasing stomatal conductance (gS), with unmeasured processes such as respiration in the light, Rubisco deactivation, and changing membrane properties probably playing important additional roles, particularly at very high temperatures. As temperature increased above Topt, gS of laboratory-measured leaves first decreased, followed by an increase at temperatures >40-45°C. In contrast, gS of canopy leaves of F. insipida in the field continued to decrease with increasing temperature, causing complete suppression of photosynthesis at ~45°C, whereas photosynthesis in the laboratory did not reach zero until leaf temperature was ~50°C. Models parameterised with laboratory-derived data should be validated against field observations when they are used to predict tropical forest carbon fluxes.

Decreased CO2 availability and inactivation of Rubisco limit photosynthesis in cotton plants under heat and drought stress in the field

Heat and drought stresses are often coincident and constitute major factors limiting global crop yields. A better understanding of plant responses to the combination of these stresses under production environments will facilitate efforts to improve yield and water use efficiencies in a climatically changing world. To evaluate photosynthetic performance under dry-hot conditions, four cotton (Gossypium barbadense L.) cultivars, Monseratt Sea Island (MS), Pima 32 (P32), Pima S-6 (S6) and Pima S-7 (S7), were studied under well-watered (WW) and water-limited (WL) conditions at a field site in central Arizona. Differences in canopy temperature and leaf relative water content under WL conditions indicated that, of the four cultivars, MS was the most drought-sensitive and S6 the most drought-tolerant. Net CO2 assimilation rates (A) and stomatal conductances (gs) decreased and leaf temperatures increased in WL compared to WW plants of all cultivars, but MS exhibited the greatest changes. The response of A to the intercellular CO2 concentration (A–Ci) showed that, along with stomatal closure, non-stomatal factors associated with heat stress also limited A under WL conditions, especially in MS. The activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) decreased in WL compared to WW plants, consistent with thermal inhibition of Rubisco activase activity. The extent of Rubisco deactivation could account for the metabolic limitation to photosynthesis in MS. Taken together, these data reveal the complex relationship between water availability and heat stress for field-grown cotton plants in a semi-arid environment. Both diffusive (drought-stress-induced) and biochemical (heat-stress-induced) limitations contributed to decreased photosynthetic performance under dry-hot conditions.

Changes at the 3′-untranslated region stabilize Rubisco activase transcript levels during heat stress in Arabidopsis

Inhibition of photosynthesis by heat stress is accompanied by functional impairment of Rubisco's chaperone, activase (RCA), resulting in deactivation of Rubisco. Since activase is extremely sensitive to thermal denaturation, changes in expression of RCA at the transcript or protein level could provide a mechanism for acclimation of photosynthesis to prolonged heat stress. Using quantitative real-time PCR (qPCR) we show steady-state RCA transcript levels in Arabidopsis thaliana are stabilized during prolonged exposure to moderate heat (35°C). A survey of RCA transcripts indicates heat stress did not alter the relative abundance of transcripts encoding α and β-isoforms of activase that are produced by alternative splicing of the pre-mRNA. Instead, mRNA stabilization in heat-stressed plants coincided with a significant reduction in the average length of activase 3'-untranslated regions, and was associated with enrichment of an uncharacterized activase mRNA splice variant, AtRCAβ2. Transcript-specific qPCR revealed AtRCAβ2 mRNA was more stable than AtRCAα and AtRCAβ mRNA in heat-stressed plants. Using an inducible transgenic system, we found that RCA transcripts lacking their native 3'-untranslated region were significantly more stable than their full-length counterparts in vivo. Using this system, stability of the RCA protein was examined over 24h in vivo, in the absence of RCA transcription. At both optimal and elevated temperatures, RCA protein levels remained stable in plants lacking RCA mRNA, but increased when RCA mRNA was present, particularly in heat-stressed plants. This study reveals a possible mechanism, involving post-transcriptional regulation of an important photosynthesis regulatory gene, for acclimation of photosynthesis to heat stress.

Oxidative stress and leaf senescence

BackgroundSenescence is an important developmental process that leads to the cell death through highly regulated genetically controlled processes in plants. Biotic and abiotic Oxidative stresses can also artificially induce senescence and increase the production of reactive oxygen species (ROS) specifically in chloroplast. One of the important oxidative stresses is paraquat that induces deviation of electron from photosynthesis electron chain and lead to the production of more ROS in chloroplast. Plants have evolved special adoptive mechanism to reallocate nutrient to reproductive and juvenile organs in senescence and different oxidative stresses. Rubisco seems to be the most abundant protein in plants and is involved in many changes during senescence.ResultsIn the present study, the effects of ROS on Rubisco during senescence and oxidative stresses were evaluated by measuring photosynthesis factors such as net photosynthesis rate (Pn), stomatal conductance (G), evaporation rate (E), intra cellular CO2 concentration (Ci), fluorescence and total protein during three stages of development. Our results showed that in paraquat treated plants, CO2 assimilation is the most effective factor that refers to Rubisco damages. The highest correlation and regression coefficient belonged to Ci, while correlation coefficient between photosynthesis rate and total protein was much smaller.ConclusionIt appears in the early stage of oxidative stresses such as exposing to paraquat, ROS has the most effect on Rubisco activity that induces more susceptibility to Rubisco specific protease. Moreover, Rubisco deactivation acts as an initiative signal for Rubisco degradation.

Rubisco activity in Mediterranean species is regulated by the chloroplastic CO2 concentration under water stress.

Water stress decreases the availability of the gaseous substrate for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) by decreasing leaf conductance to CO2. In spite of limiting photosynthetic carbon assimilation, especially in those environments where drought is the predominant factor affecting plant growth and yield, the effects of water deprivation on the mechanisms that control Rubisco activity are unclear. In the present study, 11 Mediterranean species, representing different growth forms, were subject to increasing levels of drought stress, the most severe one followed by rewatering. The results confirmed species-specific patterns in the decrease in the initial activity and activation state of Rubisco as drought stress and leaf dehydration intensified. Nevertheless, all species followed roughly the same trend when Rubisco activity was related to stomatal conductance (gs) and chloroplastic CO2 concentration (Cc), suggesting that deactivation of Rubisco sites could be induced by low Cc, as a result of water stress. The threshold level of Cc that triggered Rubisco deactivation was dependent on leaf characteristics and was related to the maximum attained for each species under non-stressing conditions. Those species adapted to low Cc were more capable of maintaining active Rubisco as drought stress intensified.

Mild experimental climate warming induces metabolic impairment and massive mortalities in southern African quartz field succulents

Current thermal regimes for many southern African succulent species of the subfamily Ruschioideae, which rapidly diversified during the cooler Pleistocene period, may be close to their tolerable extremes, and likely exceeded with anticipated future climate warming. This hypothesis was tested by exposing succulent species of different size and architecture to differently elevated temperatures approximating future African climate scenarios (2.5–3.8 °C increases in mean annual daily temperature maxima) using transparent hexagonal open-top chambers of different heights. Air temperatures, soil water potentials and amounts of fog and dew precipitation were monitored hourly in the differently heated open-top chambers and ambient environment, and changes in species leaf densities and canopy covers precisely determined in these chambers and ambient environment from high resolution digital images taken at 3-monthly intervals spanning a 12-month monitoring period. Photochemical efficiencies and activities of the photosynthetic enzyme Rubisco were also measured in one widespread dwarf succulent species following 2-h exposures of its populations in a forced draft oven to eight different heat intensities (range: 40–54 °C), the highest closely matching the temperature extreme of 54.8 °C recorded in the most intensively heated open-top chambers. After 12-months warming, all succulent species displayed massively (up to 90.2%) reduced leaf densities and canopy covers in the differently heated open-top chambers, with small sparsely branched species comprising single leaf pairs per axis exhibiting much greater reductions than large, shrubby or creeping species with multiple leaves. Noteworthy, was that fog and dew precipitation levels and soil water potentials at the centres of the least intensively heated chambers did not differ significantly from those in the ambient environment, even during the critical dry summer and early autumn seasons. However, leaf density and canopy reductions in these chambers were of similar magnitude to those in the most intensively heated chambers where fog and dew precipitation levels and soil water potentials were significantly reduced. These findings identified elevated temperatures as the principal cause of the observed massive reductions in leaf density and canopy cover and supported the hypothesis that mild anthropogenic warming could exceed the thermal thresholds of many southern African quartz field succulents leading to metabolic impairment. This impairment explained by an observed loss in the catalytic efficiency of Rubisco at daytime temperature extremes exceeding 54 °C, preceded by a decrease in PSII electron transport commencing at temperatures much lower than the threshold for Rubisco de-activation.

Association of Rubisco activase with chaperonin-60 : a possible mechanism for protecting photosynthesis during heat stress

Previous studies have shown that inhibition of photosynthesis by moderate heat stress is a consequence of Rubisco deactivation, caused in part by the thermal instability of Rubisco activase. This involvement of Rubisco activase was confirmed in heat stress and recovery experiments using transgenic Arabidopsis plants. Compared with wild-type plants, photosynthesis, the effective quantum yield of photosystem II, and Rubisco activation were less thermotolerant and recovered more slowly in transgenic Arabidopsis plants with reduced levels of Rubisco activase. Immunoblots showed that 65% of the Rubisco activase was recovered in the insoluble fraction after heat stress in leaf extracts of transgenic but not wild-type plants, evidence that deactivation of Rubisco was a consequence of thermal denaturation of Rubisco activase. The transgenic Arabidopsis plants used in this study contained a modified form of Rubisco activase that facilitated affinity purification of Rubisco activase and proteins that potentially interact with Rubisco activase during heat stress. Sequence analysis and immunoblotting identified the beta-subunit of chaperonin-60 (cpn60beta), the chloroplast GroEL homologue, as a protein that was bound to Rubisco activase from leaf extracts prepared from heat-stressed, but not control plants. Analysis of the proteins by non-denaturing gel electrophoresis showed that cpn60beta was associated with Rubisco activase in a high molecular mass complex. Immunoblot analysis established that the apparent association of cpn60beta with Rubisco activase was dynamic, increasing with the duration and intensity of the heat stress and decreasing following recovery. Taken together, these data suggest that cpn60beta plays a role in acclimating photosynthesis to heat stress, possibly by protecting Rubisco activase from thermal denaturation.

Low stomatal and internal conductance to CO2 versus Rubisco deactivation as determinants of the photosynthetic decline of ageing evergreen leaves

A novel A-Ci curve (net CO2 assimilation rate of a leaf -An- as a function of its intercellular CO2 concentration -Ci) analysis method (Plant, Cell & Environment 27, 137-153, 2004) was used to estimate the CO2 transfer conductance (gi) and the maximal carboxylation (Vcmax) and electron transport (Jmax) potentials of ageing, non-senescing Pseudotsuga menziesii leaves in relation to their nitrogen (N) content and protein and pigment composition. Both gi and the stomatal conductance (gsc) of leaves were closely coupled to Vcmax, Jmax and An with all variables decreasing with increasing leaf age. Consequently, both Ci and Cc (chloroplastic CO2 concentration) remained largely conserved through successive growing seasons. The N content of leaves, as well as the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and other sodium dodecyl sulfate-soluble proteins, increased during the first three growing seasons, then stabilized or decreased only slightly afterwards. Thus, the age-related photosynthetic nitrogen use efficiency (PNUE) decline of leaves was not a consequence of decreased allocation of N towards Rubisco and other proteins involved in bioenergetics and light harvesting. Rather, loss of photosynthetic capacity was the result of the decreased activation state of Rubisco and proportional down-regulation of electron transport towards the photosynthetic carbon reduction (PCR) and photorespiratory (PCO) cycles in response to a reduction of CO2 supply to the chloroplasts' stroma. This study emphasizes the regulatory potential and homeostaticity of Cc- rather than photosynthetic metabolites or Ci- in relation to the commonly observed correlation between photosynthesis and gsc.

Modulation of Rubisco activase gene expression during heat stress in cotton ( Gossypium hirsutum L.) involves post-transcriptional mechanisms

Inhibition of photosynthesis by heat stress involves deactivation of Rubisco and is exacerbated by the low thermal stability of Rubisco's chaperone, activase. Activase structure, activity and protein expression have been the focus of previous work examining the effect of heat stress on this enzyme. Relatively little is known about the response of activase gene expression to moderate heat stress or the extent to which transcriptional mechanisms control acclimation of photosynthesis to heat stress. In the current study, the effect of moderate heat stress on the diurnal expression of three activase genes in cotton ( Gossypium hirsutum L.), including a newly identified member of the gene family, was examined. All three activase genes displayed a diurnal pattern of expression under optimal growth conditions, as determined by real-time RT-PCR. Although steady-state mRNA levels were reduced in the short-term by heat stress, transcript levels appeared to recover by the next diurnal cycle. Nuclear run-on assays utilizing real-time RT-PCR suggested mRNA stability, not synthesis, was responsible for the observed acclimatization. Identification of activase transcripts with alternative 3′-untranslated regions suggests a possible means for increasing transcript stability and hastening recovery of steady-state mRNA levels during heat stress. Together the results indicate activase gene expression is influenced by post-transcriptional mechanisms that may contribute to acclimation of photosynthesis during extended periods of heat stress.