Transcriptome Analysis of Air Space Type Variegation Formation in Trifolium pratense

Air space type variegation is the most diverse among species of known variegated leaf plants and is caused by conspicuous intercellular spaces between the epidermal and palisade cells and among palisade cells at non-green areas. Trifolium pratense, a species in Fabaceae with V-shaped air space type variegation, was selected to explore the application potential of variegated leaf plants and accumulate basic data on the molecular regulatory mechanism and evolutionary history of leaf variegation. We performed comparative transcriptomics on the young and adult leaflets of variegated and green plants and identified 43 candidate genes related to air space type variegation formation. Most genes were related to cell wall structure modification (CESA, CSL, EXP, FLA, PG, PGIP, PLL, PME, RGP, SKS, and XTH family genes), followed by photosynthesis (LHCB subfamily, RBCS, GOX, and AGT family genes), redox (2OG and GSH family genes), and nitrogen metabolism (NodGS family genes). Other genes were related to photooxidation, protein interaction, and protease degradation systems. The down-regulated expression of light-responsive LHCB subfamily genes and the up-regulated expression of genes related to cell wall structure modification were important conditions for air space type variegation formation in T. pratense. The up-regulated expression of ubiquitin-protein ligase enzyme (E3) related genes in the protease degradation systems were conducive to air space type variegation formation. Because these family genes are necessary for plant growth and development, the mechanism of leaf variegation formation in T. pratense might be a widely existing regulation in air space type variegation in nature.