Does Rubisco control the rate of photosynthesis and plant growth? An exercise in molecular ecophysiology

Experiments are described in which tobacco (Nicotiana tabacum L.) transformed with antisense rbcS to decrease expression of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) was used to evaluate the contribution of Rubisco to the control of photosynthetic rate, and the impact of a changed rate of photosynthesis on whole plant composition, allocation and growth. (1) The concept of flux control coefficients is introduced. It is discussed how, with adequate precautions, a set of wild-type and transgenic plants with varying expression of an enzyme can be used to obtain experimental values for its flux control coefficient. (2) The flux control coefficient of Rubisco for photosynthesis depends on the short-term conditions. It increases in high light, or low CO₂. (3) When plants are grown under constant irradiance, the flux control coefficient in the growth conditions is low (<0.2) at irradiances of up to 1000μmol quanta m⁻² s⁻¹. In a natural irradiance regime exceeding 1500μmol quanta m⁻² s⁻² over several hours the flux coefficient rose to 0.8–0.9. It is concluded that plants are able to adjust the balance between Rubisco and the remainder of the photosynthetic machinery, and thereby avoid a one-sided limitation of photosynthesis by Rubisco over a wide range of ambient growth irradiance regimes. (4) When the plants were grown on limiting inorganic nitrogen, Rubisco had a higher flux control coefficient (0.5). It is proposed that, in many growth conditions, part of the investment in Rubisco may be viewed as a nitrogen store, albeit bringing additional marginal advantages with respect to photosynthetic rate and water use efficiency. (5) A change in the rate of photosynthesis did not automatically translate into a change in growth rate. Several factors are identified which contribute to this buffering of growth against a changed photosynthetic rate. (6) There is an alteration in whole plant allocation, resulting in an increase in the leaf area ratio. The increase is mainly due to a higher leaf water content, and not to changes in shoot/root allocation. This increased investment in whole plant leaf area partly counteracts the decreased efficiency of photosynthesis at the biochemical level. (7) Plants with decreased Rubisco have a lower intrinsic water use efficiency and contain high levels of inorganic cations and anions. It is proposed that these are a consequence of the increased rate of transpiration, and that the resulting osmotic potential might be a contributory factor to the increased water content and expansion of the leaves. (8) Starch accumulation in source leaves is decreased when unit leaf photosynthesis is reduced, allowing a more efficient use of the fixed carbon. (9) Decreased availability of carbohydrates leads to a down-regulation of nitrate assimilation, acting via a decrease in nitrate reductase activity.     

Decrease in phosphoribulokinase activity by antisense RNA in transgenic tobacco. Relationship between photosynthesis, growth, and allocation at different nitrogen levels

To study the direct effects of photosynthesis on allocation of biomass by altering photosynthesis without altering leaf N or nitrate content, phosphoribulokinase (PRK) activity was decreased in transgenic tobacco (Nicotiana tabacum L.) with an inverted tobacco PRK cDNA and plants were grown at different N levels (0.4 and 5 mM NH4NO3). The activation state of PRK increased as the amount of enzyme was decreased genetically at both levels of N. At high N a 94% decrease in PRK activity had only a small effect (20%) on photosynthesis and growth. At low N a 94% decrease in PRK activity had a greater effect on leaf photosynthesis (decreased by up to 50%) and whole-plant photosynthesis (decreased by up to 35%) than at high N. These plants were up to 35% smaller than plants with higher PRK activities because they had less structural dry matter and less starch, which was decreased by 3- to 4-fold, but still accumulated to 24% to 31% of dry weight; young leaves contained more starch than older leaves in older plants. Leaves had a higher ion and water content, and specific leaf area was higher, but allocation between shoot and root was unaltered. In conclusion, low N in addition to a 94% decrease in PRK by antisense reduces the activity of PRK sufficient to diminish photosynthesis, which limits biomass production under conditions normally considered sink limited.     

Effects of Ambient CO2 Concentration on Growth and Nitrogen Use in Tobacco (Nicotiana tabacum) Plants Transformed with an Antisense Gene to the Small Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

Growth of the R1 progeny of a tobacco plant (Nicotiana tabacum) transformed with an antisense gene to the small subunit of ribulose-1,5-carboxylase/oxygenase (Rubisco) was analyzed under 330 and 930 [mu]bar of CO2, at an irradiance of 1000 [mu]mol quanta m-2 s-1. Rubisco activity was reduced to 30 to 50% and 13 to 18% of that in the wild type when one and two copies of the antisense gene, respectively, were present in the genome, whereas null plants and wild-type plants had similar phenotypes. At 330 [mu]bar of CO2 all antisense plants were smaller than the wild type. There was no indication that Rubisco is present in excess in the wild type with respect to growth under high light. Raising ambient CO2 pressure to 930 [mu]bar caused plants with one copy of the DNA transferred from plasmid to plant genome to achieve the same size as the wild type at 330 [mu]bar, but plants with two copies remained smaller. Differences in final size were due mostly to early differences in relative rate of leaf area expansion (m2 m-2 d-1) or of biomass accumulation (g g-1 d-1): within less than 2 weeks after germination relative growth rates reached a steady-state value similar for all plants. Plants with greater carboxylation rates were characterized by a higher ratio of leaf carbon to leaf area, and at later stages, they were characterized also by a relatively greater allocation of structural and nonstructural carbon to roots versus leaves. However, these changes per se did not appear to be causing the long-term insensitivity of relative growth rates to variations in carboxylation rate. Nor was this insensitivity due to feedback inhibition of photosynthesis in leaves grown at high partial pressure of CO2 in the air (pa) or with high Rubisco activity, even when the amount of starch approached 40% of leaf dry weight. We propose that other intrinsic rate-limiting processes that are independent of carbohydrate supply were involved. Under plentiful nitrogen supply, reduction in the amount of nitrogen invested in Rubisco was more than compensated for by an increase in leaf nitrate. Nitrogen content of organic matter, excluding Rubisco, was unaffected by the antisense gene. In contrast, it was systematically lower at elevated pa than at normal pa. Combined with the positive effects of pa on growth, this resulted in the single-dose antisense plants growing as fast at 930 [mu]bar of CO2 as the wild-type plants at 330 [mu]bar of CO2 but at a lower organic nitrogen cost.     

BSD2 is a Rubisco‐specific assembly chaperone,forms intermediary hetero‐oligomeric complexes,and is nonlimiting to growth in tobacco

The folding and assembly of Rubisco large and small subunits into L₈S₈ holoenzyme in chloroplasts involves many auxiliary factors, including the chaperone BSD2. Here we identify apparent intermediary Rubisco‐BSD2 assembly complexes in the model C₃ plant tobacco. We show BSD2 and Rubisco content decrease in tandem with leaf age with approximately half of the BSD2 in young leaves (~70 nmol BSD2 protomer.m²) stably integrated in putative intermediary Rubisco complexes that account for <0.2% of the L₈S₈ pool. RNAi‐silencing BSD2 production in transplastomic tobacco producing bacterial L₂ Rubisco had no effect on leaf photosynthesis, cell ultrastructure, or plant growth. Genetic crossing the same RNAi‐bsd2 alleles into wild‐type tobacco however impaired L₈S₈ Rubisco production and plant growth, indicating the only critical function of BSD2 is in Rubisco biogenesis. Agrobacterium mediated transient expression of tobacco, Arabidopsis, or maize BSD2 reinstated Rubisco biogenesis in BSD2‐silenced tobacco. Overexpressing BSD2 in tobacco chloroplasts however did not alter Rubisco content, activation status, leaf photosynthesis rate, or plant growth in the field or in the glasshouse at 20°C or 35°C. Our findings indicate BSD2 functions exclusively in Rubisco biogenesis, can efficiently facilitate heterologous plant Rubisco assembly, and is produced in amounts nonlimiting to tobacco growth.     

Increased cytokinin levels in transgenic PSAG12–IPT tobacco plants have large direct and indirect effects on leaf senescence, photosynthesis and N partitioning

We studied the impact of delayed leaf senescence on the functioning of plants growing under conditions of nitrogen remobilization. Interactions between cytokinin metabolism, Rubisco and protein levels, photosynthesis and plant nitrogen partitioning were studied in transgenic tobacco (Nicotiana tabacum L.) plants showing delayed leaf senescence through a novel type of enhanced cytokinin syn‐thesis, i.e. targeted to senescing leaves and negatively auto‐regulated (P_SAG12–IPT), thus preventing developmental abnormalities. Plants were grown with growth‐limiting nitrogen supply. Compared to the wild‐type, endogenous levels of free zeatin (Z)‐ and Z riboside (ZR)‐type cytokinins were increased up to 15‐fold (total ZR up to 100‐fold) in senescing leaves, and twofold in younger leaves of P_SAG12–IPT. In these plants, the senescence‐associated declines in N, protein and Rubisco levels and photosynthesis rates were delayed. Senescing leaves accumulated more (¹⁵N‐labelled) N than younger leaves, associated with reduced shoot N accumulation (–60%) and a partially inverted canopy N profile in P_SAG12–IPT plants. While root N accumulation was not affected, N translocation to non‐senescing leaves was progressively reduced. We discuss potential consequences of these modified sink–source relations, associated with delayed leaf senescence, for plant productivity and the efficiency of utilization of light and minerals.     

Decreased Rubisco activity leads to dramatic changes of nitrate metabolism,amino acid metabolism and the levels of phenylpropanoids and nicotine in tobacco antisense RBCS transformants

Tobacco transformants that express an antisense RBCS construct were used to investigate the consequences of a lesion in photosynthetic carbon metabolism for nitrogen metabolism and secondary metabolism. The results show that an inhibition of photosynthesis and decrease in sugar levels leads to a general inhibition of nitrogen metabolism, and dramatic changes in the levels of secondary metabolites. The response was particularly clear in plants that received excess nitrogen. In these conditions, a decrease of Rubisco activity led to an inhibition of nitrate reductase activity, accumulation of nitrate, a decrease of amino acid levels that was larger than the decrease of sugars, and a large decrease of chlorogenic acid and of nicotine, which are the major carbon- and nitrogen-rich secondary metabolites in tobacco leaves, respectively. Similar changes were seen when nitrogen-replete wild-type tobacco was grown in low light. The inhibition of nitrogen metabolism was partly masked when wild-type plants and antisense RBCS transformants were compared in marginal or in limiting nitrogen, because the lower growth rate of the transformants alleviated the nitrogen deficiency, leading to an increase of amino acids. In these conditions, chlorogenic acid always decreased but the decrease of nicotine was ameliorated or reversed. When the changes in internal pools are compared across all the genotypes and growth conditions, two conclusions emerge. First, decreased levels of primary metabolites lead to a dramatic decrease in the levels of secondary metabolites. Second, changes of the amino acid : sugar ratio are accompanied by changes of the nicotine:chlorogenic acid ratio.     

Growth and photosynthesis under high and low irradiance of Arabidopsis thaliana antisense mutants with reduced ribulose-1,5-bisphosphate carboxylase/oxygenase activase content.

Photosynthesis and growth to maturity of antisense ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase Arabidopsis thaliana with reduced concentrations of activase relative to wild-type (Wt) plants were measured under low (200 mumol m-2 s-1) and high (600 mumol m-2 s-1) photosynthetic photon flux density growing conditions. Both growth and photosynthesis were significantly reduced in an Arabidopsis clone (R100) with 30 to 40% Wt activase, an effect that was more pronounced in high light. The aboveground biomass of the antisense clone R100 reached 80% of Wt under low light and 65% of Wt under high light. Decreased growth in the antisense plants was attributed to reduced relative rates of growth and leaf area expansion early in development; all plants attained similar values of relative rates of growth and leaf elongation by 21 d after planting. Reductions in photosynthesis were attributed to decreased Rubisco activation in the antisense plants. Rubisco constituted about 40% of total soluble protein in both Wt and clone R100 under both light regimes. Activase content was 5% and 1.4% of total soluble protein in Wt and clone R100, respectively, and also was unaffected by growth irradiance. The stoichiometry of Rubisco to activase was estimated at 20 Rubisco active sites per activase tetramer in Wt Arabidopsis and 60 to 80 in the transgenic clone R100. We conclude that Wt Arabidopsis does not contain Rubisco activase in great excess of the amount required for optimal growth.     

Decreased ribulose-1,5-bisphosphate carboxylase/oxygenase in transgenic tobacco transformed with 'antisense'rbcS.VIII. Impact on photosynthesis and growth in tobacco growing under extreme high irradiance and high temperature

Wild-type and antisense rbcS tobacco (Nicotiana tabacum) plants were grown in a glasshouse in midsummer in Portugal with an irradiance of 1500–2000 μmol m⁻²s⁻¹ and daytime temperatures of 30–35 °C. The Rubisco content of the transformants was lower by 35, 80 and over 90% than that of the wild-type. Gas exchange was measured over three separate days. There was a near-linear relation between Rubisco content and photosynthetic rate during the period of high irradiance, allowing a flux control coefficient of 0.83–0.89 to be estimated. The relation deviated slightly from linearity, because the internal CO₂ concentration (c;) was higher in the transformants than in the wild-type (190 and 275 μmol mol⁻¹ in plants with 35 and 80% less Rubisco, respectively, compared with 175 μmol mol⁻¹ for wild-type), compensating to some extent for the decreased Rubisco content. This increase in c_i occurred because the stomatal conductance (g) remained unaltered or was even higher in plants with decreased Rubisco, despite the lower rate of CO₂ assimilation. As a consequence, water use efficiency declined. The decreased rate of photosynthesis was not accompanied by a stoichiometric decrease in apparent growth rate. These results are discussed in relation to earlier studies of the plant set in growth cabinets. It is concluded that tobacco can adjust over a wide range of growth conditions to avoid a onesided limitation by Rubisco, but that in extreme environmental conditions this capacity to adapt is exhausted.     

Complementation of the nuclear antisense rbcS-induced photosynthesis deficiency by introducing an rbcS gene into the tobacco plastid genome

The small subunit (SS) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a nuclear gene-encoded protein that is imported into chloroplasts where it assembles with the large subunit (LS) after removal of the transit peptide to form Rubisco. We have explored the possibility that the severe deficiency in photosynthesis exhibited in nuclear transgenic tobacco (line alpha5) expressing antisense rbcS coding DNA that results in low SS and Rubisco protein content [Rodermel et al. (1988) Cell 55: 673] could be complemented by introducing a copy of the rbcS gene into its plastid genome through chloroplast transformation. Two independent lines of transplastomic plants were generated, in which the tobacco rbcS coding sequence, either with or without the transit sequence, was site-specifically integrated into the plastid genome. We found that compared with the antisense plants, expression of the plastid rbcS gene in the transplastomic plants resulted in very high mRNA abundance but no increased accumulation of the SS and Rubisco protein or improvement in plant growth and photosynthesis. Therefore, there is a limitation in efficient translation of the rbcS mRNA in the plastid or an incorrect processing and modification of the plastid-synthesized SS protein that might cause its rapid degradation.     

Chloroplast downsizing under nitrate nutrition restrained mesophyll conductance and photosynthesis in rice (Oryza sativa L.) under drought conditions

The phenomenon whereby ammonium enhances the tolerance of rice seedlings (Oryza sativa L., cv. 'Shanyou 63' hybrid indica China) to water stress has been reported in previous studies. To study the intrinsic mechanism of biomass synthesis related to photosynthesis, hydroponic experiments supplying different nitrogen (N) forms were conducted; water stress was simulated by the addition of polyethylene glycol. Water stress decreased leaf water potential (Ψ(leaf)) under nitrate nutrition, while it had no negative effect under ammonium nutrition. The decreased Ψ(leaf) under nitrate nutrition resulted in chloroplast downsizing and subsequently decreased mesophyll conductance to CO(2) (g(m)). The decreased g(m) and stomatal conductance (g(s)) under nitrate nutrition with water stress restrained the CO(2) supply to the chloroplast and Rubisco. The relatively higher distribution of leaf N to Rubisco under ammonium nutrition might also be of benefit for photosynthesis under water stress. In conclusion, chloroplast downsizing induced a decline in g(m), a relatively higher decrease in g(s) under nitrate nutrition with water stress, restrained the CO(2) supply to Rubisco and finally decreased the photosynthetic rate.     

Reduction of ribulose-1,5-bisphosphate carboxylase/oxygenase content by antisense RNA reduces photosynthesis in transgenic tobacco plants

A complementary DNA for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was cloned from tobacco (Nicotiana tabacum) and fused in the antisense orientation to the cauliflower mosaic virus 35S promoter. This antisense gene was introduced into the tobacco genome, and the resulting transgenic plants were analyzed to assess the effect of the antisense RNA on Rubisco activity and photosynthesis. The mean content of extractable Rubisco activity from the leaves of 10 antisense plants was 18% of the mean level of activity of control plants. The soluble protein content of the leaves of anti-small subunit plants was reduced by the amount equivalent to the reduction in Rubisco. There was little change in phosphoribulokinase activity, electron transport, and chlorophyll content, indicating that the loss of Rubisco did not affect these other components of photosynthesis. However, there was a significant reduction in carbonic anhydrase activity. The rate of CO₂ assimilation measured at 1000 micromoles quanta per square meter per second, 350 microbars CO₂, and 25°C was reduced by 63% (mean value) in the antisense plants and was limited by Rubisco activity over a wide range of intercellular CO₂ partial pressures (p_i). In control leaves, Rubisco activity only limited the rate of CO₂ assimilation below a p_i of 400 microbars. Despite the decrease in photosynthesis, there was no reduction in stomatal conductance in the antisense plants, and the stomata still responded to changes in p_i. The unchanged conductance and lower CO₂ assimilation resulted in a higher p_i, which was reflected in greater carbon isotope discrimination in the leaves of the antisense plants. These results suggest that stomatal function is independent of total leaf Rubisco activity.     

Does Decrease in Ribulose-1,5-Bisphosphate Carboxylase by Antisense RbcS Lead to a Higher N-Use Efficiency of Photosynthesis under Conditions of Saturating CO2 and Light in Rice Plants?

Rice (Oryza sativa L.) plants with decreased ribulose-1,5-bisphosphate carboxylase (Rubisco) were obtained by transformation with the rice rbcS antisense gene under the control of the rice rbcS promoter. The primary transformants were screened for the Rubisco to leaf N ratio, and the transformant with 65% wild-type Rubisco was selected as a plant set with optimal Rubisco content at saturating CO2 partial pressures for photosynthesis under conditions of high irradiance and 25[deg]C. This optimal Rubisco content was estimated from the amounts and kinetic constants of Rubisco and the gas-exchange data. The R1 selfed progeny of the selected transformant were grown hydroponically with different N concentrations. Rubisco content in the R1 population was distributed into two groups: 56 plants had about 65% wild-type Rubisco, whereas 23 plants were very similar to the wild type. Although the plants with decreased Rubisco showed 20% lower rates of light-saturated photosynthesis in normal air (36 Pa CO2), they had 5 to 15% higher rates of photosynthesis in elevated partial pressures of CO2, (100-115 Pa CO2) than the wild-type plants for a given leaf N content. We conclude that the rice plants with 65% wild-type Rubisco show a higher N-use efficiency of photosynthesis under conditions of saturating CO2 and high irradiance.     

Effects of Nitrate Application on Amaranthus powellii Wats. : I. Changes in Photosynthesis, Growth Rates, and Leaf Area

Physiological effects of different nitrate applications were studied using the C₄ plant, Amaranthus powellii Wats. Plants were grown in a controlled environment chamber and watered daily with nutrient solutions containing 45, 10, 5, or 1 millimolar nitrate. Chloride and sulfate were used to keep the cation and phosphate concentrations constant. Total leaf nitrogen concentration, chlorophyll concentration, specific leaf mass, leaf area, relative growth rate, relative leaf growth rate, unit leaf rate (increase of dry mass per unit leaf area per day), net photosynthetic rate, and incident quantum yield decreased with decreasing nitrate concentration. The per cent decrease of unit leaf rate was similar to the decrease of light-saturated net photosynthetic rate; however, the decrease in relative growth rate was less than that of unit leaf rate because leaf area ratio (leaf area per unit dry mass) increased with decreasing nitrate concentration. Essential mineral concentrations per unit leaf area were about equal among all treatments. Leaf expansion, determined by stomatal density, decreased except for the 1 millimolar treatment which showed relatively more cell expansion but less cell division. Decreased nitrate application was correlated with higher osmotic potentials and lower pressure potentials (determined by pressure-volume curves), whereas leaf water potentials were equal among treatments. Even though total leaf area and shoot mass decreased with decreasing applied nitrate, the increase of the leaf area ratio may be related to selection for the highest possible growth rate.     

Influence of ozone on transgenic tobacco plants expressing reduced quantities of Rubisco

RbcS-antisense transformed tobacco plants (Nicotiana tabacum cv. Petit Havana) expressing reduced quantities of Rubisco protein were used to examine the role of Rubisco quantity in determining ozone (O₃) sensitivity. Transformed and wild-type plants were exposed to O₃ in the greenhouse and in the field. Stomatal conductance, net photosynthesis and Rubisco protein quantity were measured at various times. Antisense-transformed genotypes responded to O₃ by exhibiting rapid, severe foliar necrosis. The wild-type plants responded more slowly, exhibiting limited injury. Decreases in stomatal conductance, net photosynthesis or Rubisco quantity in plants exposed to O₃ were not observed in asymptomatic leaves. Total biomass was lower for the transformed genotypes and decreased in both genotypes after exposure to O₃. Shoot–root ratio and specific leaf area were higher in the transformed genotypes and increased in both genotypes with exposure to O₃. Measurements of intercellular airspace demonstrated the presence of larger intercellular spaces in the transformed plants. The indirect effects of the rbcS antisense transformation, including morphological changes in the leaf, probably rendered the transformed plants more sensitive to the oxidant. The decreased quantity of Rubisco is not thought to be directly related to increased O₃ sensitivity in the transformed plants.     

Acclimation of peanut (Arachis hypogaea L.) leaf photosynthesis to elevated growth CO2 and temperature

Peanut (Arachis hypogaea L. cv. Florunner) was grown from seed sowing to plant maturity under two daytime CO₂ concentrations ([CO₂]) of 360 μmol mol⁻¹ (ambient) and 720 μmol mol⁻¹ (elevated) and at two temperatures of 1.5 and 6.0 °C above ambient temperature. The objectives were to characterize peanut leaf photosynthesis responses to long-term elevated growth [CO₂] and temperature, and to assess whether elevated [CO₂] regulated peanut leaf photosynthetic capacity, in terms of activity and protein content of ribulose bisphosphate carboxylase-oxygenase (Rubisco), Rubisco photosynthetic efficiency, and carbohydrate metabolism. At both growth temperatures, leaves of plants grown under elevated [CO₂] had higher midday photosynthetic CO₂ exchange rate (CER), lower transpiration and stomatal conductance and higher water-use efficiency, compared to those of plants grown at ambient [CO₂]. Both activity and protein content of Rubisco, expressed on a leaf area basis, were reduced at elevated growth [CO₂]. Declines in Rubisco under elevated growth [CO₂] were 27–30% for initial activity, 5–12% for total activity, and 9–20% for protein content. Although Rubisco protein content and activity were down-regulated by elevated [CO₂], Rubisco photosynthetic efficiency, the ratio of midday light-saturated CER to Rubisco initial or total activity, of the elevated-[CO₂] plants was 1.3- to 1.9-fold greater than that of the ambient-[CO₂] plants at both growth temperatures. Leaf soluble sugars and starch of plants grown at elevated [CO₂] were 1.3- and 2-fold higher, respectively, than those of plants grown at ambient [CO₂]. Under elevated [CO₂], leaf soluble sugars and starch, however, were not affected by high growth temperature. In contrast, high temperature reduced leaf soluble sugars and starch of the ambient-[CO₂] plants. Activity of sucrose-P synthase, but not adenosine 5′-diphosphoglucose pyrophosphorylase, was up-regulated under elevated growth [CO₂]. Thus, in the absence of other environmental stresses, peanut leaf photosynthesis would perform well under rising atmospheric [CO₂] and temperature as predicted for this century.     

Genetic modification of photosynthesis with E. coli genes for trehalose synthesis

Improvement in photosynthesis per unit leaf area has been difficult to alter by breeding or genetic modification. We report large changes in photosynthesis in Nicotiana tabacum transformed with E. coli genes for the trehalose pathway. Significantly, photosynthetic capacity (CO2 assimilation at varying light and CO2, and quantum yield of PSII electron transport) per unit leaf area and per leaf dry weight were increased in lines of N. tabacum transformed with the E. coli gene otsA, which encodes trehalose phosphate synthase. In contrast, transformation with otsB, which encodes trehalose phosphate phosphatase or Trec, encoding trehalose phosphate hydrolase, produced the opposite effect. Changes in CO2 assimilation per unit leaf area were closely related to the amount and activity of Rubisco, but not to the maximum activities of other Calvin cycle enzymes. Alterations in photosynthesis were associated with trehalose 6-phosphate content rather than trehalose. When growth parameters were determined, a greater photosynthetic capacity did not translate into greater relative growth rate or biomass. This was because photosynthetic capacity was negatively related to leaf area and leaf area ratio. In contrast, relative growth rate and biomass were positively related to leaf area. These results demonstrate a novel means of modifying Rubisco content and photosynthesis, and the complexities of regulation of photosynthesis at the whole plant level, with potential benefits to biomass production through improved leaf area.