Evolution Of Rice Invertase Gene Family And Its Regulation Under Drought Stress

Invertases (EC 3.2.1.26) are enzymes that catalyze the irreversible hydrolysis of sucrose to glucose and fructose. Plants contain two unrelated families of invertases: acid forms (located in the cell-wall and vacuole) and neutral/alkaline forms (thought to be located in the cytosol). Here for rice I identify the genes encoding the cell-wall group (designated OsCIN1-9), the vacuolar group (OsVIN1-2) and two ancient neutral/alkaline groups: a (OsNIN1-4) and β (OsNIN5-8). Analysis of the gene sequences allows conclusions to be drawn concerning the subcellular location of the encoded proteins and the evolution of the acid and neutral/alkaline invertase gene families as regards intron-exon structure, gene duplication, gene loss and gene expression. Compared with the relatively conserved structure of neutral/alkaline invertase gene family, the acid invertase family underwent many events of intron loss. The acid invertase genes also showed greater spatial and temporal diversity of expression than the neutral/alkaline genes.I focused on examining possible reasons for the multiplicity of the acid invertase gene family. Since enzymes of fructan synthesis are known to have evolved from vacuolar invertases, I began with OsVINl and OsVIN2 and asked whether both genes encoded active invertases (glucosyl-fructosidases) or whether one gene encoded a fructosyltransferase capable of fructan synthesis. Although rice does not accumulate fructans, in closely related cereals such as wheat and barley fructans play an important role in tolerance of drought and cold stress. I characterized OsVINl and OsVIN2 in terms of the enzymatic properties and sequence relationships of the encoded proteins, and their regulation in response to drought stress. Recombinant OsVINl and OsVIN2 proteins expressed in the yeast Pichia pastoris showed high invertase activity against 2 mM sucrose, and only low l-sucrose:sucrose fructosyltransferase activity (1-kestose synthesis) at 60-1000 mM sucrose. Furthermore, recombinant OsVINl showed significant levels of fructosyl-fructoside activity against 1-kestose, 1,1-nystose and inulin (fructan exohydrolase activity). By contrast, recombinant OsVIN2 lacked fructan exohydrolase activity and could therefore represent a major evolutionary step towards achieving a capacity for fructan synthesis. This possibility is supported by my demonstration that all known fructan fructosyltransferases of the Pooideae (in genera such as Triticum, Hordeum, Poa and Lolium) are more closely related to...