Molecular characterization of SXD1: A novel chloroplast protein required for sucrose export from maize leaves

In recent years, many studies have implicated a role for plasmodesmata in intercellular communication between plant cells, but no mutant defective in plasmodesmal function has been fully characterized. The sucrose export defective1 (sxd1) mutant of maize demonstrates occlusion of plasmodesmata between bundle sheath and vascular parenchyma cells, and remains unique as a plant demonstrating defects in plasmodesmal structure. Along with this disruption of plasmodesmata, mature leaf blades of sxd1 plants exhibit a loss of sucrose export ability, accumulation of starch and anthocyanin, and vascular parenchyma cell death. This dissertation describes the cloning and characterization of the Sxd1 gene product, and explores the relationship between the loss of SXD1 function and the observed plasmodesmal defects. The Sxd1 gene was shown to encode a novel protein. A second null allele, sxd1-2, was identified and shown to produce a phenotype indistinguishable from that of the original allele, sxd1-1. The structure of the sxd1 locus was determined in wild-type plants and both sxd1 alleles, and the gene was mapped to chromosome 5. Expression analysis demonstrated that the gene was transcribed in all green tissues, with highest levels in maturing leaf blades. The SXD1 protein was localized to chloroplasts, in both bundle sheath and mesophyll cells. Levels of sucrose, glucose, and fructose were compared between wild-type and sxd1 plants. The mutants were fully capable of producing sucrose, and accumulated all three sugars at 5--6 times the concentrations measured in wildtype plants. Interestingly, despite the increased sugar concentrations, photosynthetic gene expression was not significantly downregulated in affected areas of sxd1 leaf blades. The Sxd1 ortholog in Arabidopsis was identified and cloned. The AtSxd1 gene product was also expressed in mature leaves and targeted to chloroplasts. Experiments altering the expression of AtSxd1 were initiated to determine the function of the protein in Arabidopsis. It was concluded that the plasmodesmal occlusion observed in sxd1 maize mutants effectively traps photosynthate within the source leaf. A model for SXD1 function is proposed, in which the protein is involved in a chloroplast-to-nucleus signaling pathway necessary for proper late-stage differentiation of maize bundle sheath cells, including the developmentally regulated modification of plasmodesmata.