Increased chloroplast area in the rice bundle sheath through cell specific perturbation of brassinosteroid signalling

In the leaves of C ₃ species such as rice, mesophyll cells contain the largest compartment of photosynthetically active chloroplasts. In contrast, plants that use the derived and more efficient C ₄ photosynthetic pathway have a significant chloroplast compartment in both bundle sheath and mesophyll cells. Accordingly, the evolution of C ₄ photosynthesis from the ancestral C ₃ state requires an increase in the chloroplast compartment of the bundle sheath. Here we investigated the potential to increase chloroplast compartment size in rice bundle sheath cells by manipulating brassinosteroid signalling. Treatment with brassinazole, a brassinosteroid biosynthesis inhibitor, increased leaf chlorophyll content and increased the number but decreased the area of chloroplasts in bundle sheath cells. Constitutive overexpression of the BRASSINAZOLE RESISTANT 1 ( OsBZR1 ) transcription factor increased bundle sheath chloroplast area by up to 45% but plants became chlorotic. However, when OsBZR1 was placed under the control of a bundle sheath specific promoter, the negative effects on growth and viability were removed whilst chloroplast area still increased. In summary, we report a role for brassinosteroids in controlling chloroplast area and number in rice and conclude that cell-specific manipulation of brassinosteroid signalling can be used to manipulate the chloroplast compartment in rice bundle sheath cells.