Effects of leaf age during drought and recovery on photosynthesis, mesophyll conductance and leaf anatomy in wheat leaves.

statement: Mesophyll conductance (g m) was negatively correlated with wheat leaf age but was positively correlated with the surface area of chloroplasts exposed to intercellular airspaces (S c). The rate of decline in photosynthetic rate and g m as leaves aged was slower for water-stressed than well-watered plants. Upon rewatering, the degree of recovery from water-stress depended on the age of the leaves, with the strongest recovery for mature leaves, rather than young or old leaves. Diffusion of CO2 from the intercellular airspaces to the site of Rubisco within C3 plant chloroplasts (gm) governs photosynthetic CO2 assimilation (A). However, variation in g m in response to environmental stress during leaf development remains poorly understood. Age-dependent changes in leaf ultrastructure and potential impacts on g m, A, and stomatal conductance to CO2 (g sc) were investigated for wheat (Triticum aestivum L.) in well-watered and water-stressed plants, and after recovery by re-watering of droughted plants. Significant reductions in A and g m were found as leaves aged. The oldest plants (15 days and 22 days) in water-stressed conditions showed higher A and gm compared to irrigated plants. The rate of decline in A and g m as leaves aged was slower for water-stressed compared to well-watered plants. When droughted plants were rewatered, the degree of recovery depended on the age of the leaves, but only for g m. The surface area of chloroplasts exposed to intercellular airspaces (S c) and the size of individual chloroplasts declined as leaves aged, resulting in a positive correlation between g m and S c. Leaf age significantly affected cell wall thickness (t cw), which was higher in old leaves compared to mature/young leaves. Greater knowledge of leaf anatomical traits associated with g m partially explained changes in physiology with leaf age and plant water status, which in turn should create more possibilities for improving photosynthesis using breeding/biotechnological strategies.

Rubisco oligomers composed of linked small and large subunits assemble in tobacco plastids and have higher affinities for CO2 and O2.

Manipulation of Rubisco within higher plants is complicated by the different genomic locations of the large (L; rbcL) and small (S; RbcS) subunit genes. Although rbcL can be accurately modified by plastome transformation, directed genetic manipulation of the multiple nuclear-encoded RbcS genes is more challenging. Here we demonstrate the viability of linking the S and L subunits of tobacco (Nicotiana tabacum) Rubisco using a flexible 40-amino acid tether. By replacing the rbcL in tobacco plastids with an artificial gene coding for a S40L fusion peptide, we found that the fusions readily assemble into catalytic (S40L)8 and (S40L)16 oligomers that are devoid of unlinked S subunits. While there was little or no change in CO2/O2 specificity or carboxylation rate of the Rubisco oligomers, their Kms for CO2 and O2 were reduced 10% to 20% and 45%, respectively. In young maturing leaves of the plastome transformants (called ANtS40L), the S40L-Rubisco levels were approximately 20% that of wild-type controls despite turnover of the S40L-Rubisco oligomers being only slightly enhanced relative to wild type. The reduced Rubisco content in ANtS40L leaves is partly attributed to problems with folding and assembly of the S40L peptides in tobacco plastids that relegate approximately 30% to 50% of the S40L pool to the insoluble protein fraction. Leaf CO2-assimilation rates in ANtS40L at varying pCO2 corresponded with the kinetics and reduced content of the Rubisco oligomers. This fusion strategy provides a novel platform to begin simultaneously engineering Rubisco L and S subunits in tobacco plastids.

The catalytic properties of hybrid Rubisco comprising tobacco small and sunflower large subunits mirror the kinetically equivalent source Rubiscos and can support tobacco growth.

Plastomic replacement of the tobacco (Nicotiana tabacum) Rubisco large subunit gene (rbcL) with that from sunflower (Helianthus annuus; rbcL(S)) produced tobacco(Rst) transformants that produced a hybrid Rubisco consisting of sunflower large and tobacco small subunits (L(s)S(t)). The tobacco(Rst) plants required CO(2) (0.5% v/v) supplementation to grow autotrophically from seed despite the substrate saturated carboxylation rate, K(m), for CO(2) and CO(2)/O(2) selectivity of the L(s)S(t) enzyme mirroring the kinetically equivalent tobacco and sunflower Rubiscos. Consequently, at the onset of exponential growth when the source strength and leaf L(s)S(t) content were sufficient, tobacco(Rst) plants grew to maturity without CO(2) supplementation. When grown under a high pCO(2), the tobacco(Rst) seedlings grew slower than tobacco and exhibited unique growth phenotypes: Juvenile plants formed clusters of 10 to 20 structurally simple oblanceolate leaves, developed multiple apical meristems, and the mature leaves displayed marginal curling and dimpling. Depending on developmental stage, the L(s)S(t) content in tobacco(Rst) leaves was 4- to 7-fold less than tobacco, and gas exchange coupled with chlorophyll fluorescence showed that at 2 mbar pCO(2) and growth illumination CO(2) assimilation in mature tobacco(Rst) leaves remained limited by Rubisco activity and its rate (approximately 11 micromol m(-2) s(-1)) was half that of tobacco controls. (35)S-methionine labeling showed the stability of assembled L(s)S(t) was similar to tobacco Rubisco and measurements of light transient CO(2) assimilation rates showed L(s)S(t) was adequately regulated by tobacco Rubisco activase. We conclude limitations to tobacco(Rst) growth primarily stem from reduced rbcL(S) mRNA levels and the translation and/or assembly of sunflower large with the tobacco small subunits that restricted L(s)S(t) synthesis.