A high leaf-starch mutant in alfalfa has altered invertase activity

A mutant phenotype isolated from alfalfa (Medicago sativa L) cv. Excalibur appeared to have dramatic changes in source-sink relations and the partitioning of carbohydrates. Leaves emerged normally, but starch accumulated in the chloroplasts of the palisade mesophyll cells with maturity. Subsequently, the cells of the palisade layer lost chlorophyll, exhibited ultrastructural symptoms of senescence, and necrotic spotting appeared on the adaxial surface of fully expanded leaves. Segregation analysis of this phenotype, designated as hls (high-leaf starch), with F1, BC1, and F2 progenies revealed that two independent dominant genes condition the trait. The hls phenotype had five-fold more starch in mature leaves and less in the taproot than in the normal leaf phenotypes, suggesting a blockage in transport of carbohydrate from the leaf to the root. In vitro activity assays and native polyacrylamide gel electrophoresis (PAGE) analysis revealed that most enzymes involved in carbohydrate metabolism were not altered in the hls phenotype. However, invertase activity of expanding leaves was significantly higher in plants with the hls phenotype than in those with a normal phenotype. The visual appearance of the mutation, high leaf starch content, and high invertase activity co-segregated in the progeny. Native PAGE revealed that a fast-moving invertase isozyme (F) was developmentally inactivated in normal phenotypes during leaf expansion but remained active in the hls phenotype. A biochemical model for the hls phenotype is proposed in which high invertase activity blocks phloem loading and reduces the availability of cytosolic inorganic orthophosphate for exchange with triose-phosphate across the chloroplast envelope, thereby promoting starch accumulation