Evidence for Light and Tissue Specific Regulation of Genes Involved in Fructan Metabolism in <i>Agave tequilana</i>

Plant Glycoside Hydrolase Family 32 (PGHF32) contains the fructosyltransferases and fructan exohydrolase enzymes responsible for fructan metabolism, in addition to closely related vacuolar and cell wall acid invertases. <i>Agave</i> species produce complex and dynamic fructan molecules (agavins) requiring 4 different fructosyltransferase activities (1-SST, 1-FFT, 6G-FFT and 6-SFT) for their synthesis. Combined analysis of RNAseq and genome data for <i>A. tequilana</i> led to the characterization of the genes encoding 3 fructosyltransferases for this species and support the hypothesis that no separate 6-SFT type enzyme exists in <i>A. tequilana,</i> suggesting that at least one of the fructosyltransferases identified may have multiple enzymatic activities. Structures for PGHF32 genes varied for <i>A. tequilana</i> and between other plant species but were conserved for different enzyme types within a species. The observed patterns are consistent with the formation of distinct gene structures by intron loss. Promoter analysis of the PGHF32 genes identified abundant putative regulatory motifs for light regulation and tissue-specific expression, and these regulatory mechanisms were confirmed experimentally for leaf tissue. Motifs for phytohormone response, carbohydrate metabolism and dehydration responses were also uncovered. Based on the regulatory motifs, full-length cDNAs for MYB, GATA, DOF and GBF transcription factors were identified and their phylogenetic distribution determined by comparison with other plant species. In silico expression analysis for the selected transcription factors revealed both tissue-specific and developmental patterns of expression, allowing candidates to be identified for detailed analysis of the regulation of fructan metabolism in <i>A. tequilana</i> at the molecular level.