Molecular and Functional Characterization of Novel Fructosyltransferases and Invertases from Agave tequilana

Celso Cortés-Romero,Aída Martínez-Hernández,Mercedes G López,Erika Mellado-Mojica,June Simpson

doi:10.1371/journal.pone.0035878

Abstract

Fructans are the main storage polysaccharides found in Agave species. The synthesis of these complex carbohydrates relies on the activities of specific fructosyltransferase enzymes closely related to the hydrolytic invertases. Analysis of Agave tequilana transcriptome data led to the identification of ESTs encoding putative fructosyltransferases and invertases. Based on sequence alignments and structure/function relationships, two different genes were predicted to encode 1-SST and 6G-FFT type fructosyltransferases, in addition, 4 genes encoding putative cell wall invertases and 4 genes encoding putative vacuolar invertases were also identified. Probable functions for each gene, were assigned based on conserved amino acid sequences and confirmed for 2 fructosyltransferases and one invertase by analyzing the enzymatic activity of recombinant Agave protein s expressed and purified from Pichia pastoris. The genome organization of the fructosyltransferase/invertase genes, for which the corresponding cDNA contained the complete open reading frame, was found to be well conserved since all genes were shown to carry a 9 bp mini-exon and all showed a similar structure of 8 exons/7 introns with the exception of a cell wall invertase gene which has 7 exons and 6 introns. Fructosyltransferase genes were strongly expressed in the storage organs of the plants, especially in vegetative stages of development and to lower levels in photosynthetic tissues, in contrast to the invertase genes where higher levels of expression were observed in leaf tissues and in mature plants.

Highlights

Agave species are carbohydrate rich plants that synthesize fructans as the main storage polysaccharides with starch found to be present only in low levels [1]
A BLAST search carried out within an A. tequilana transcriptome database obtained by the sequencing of around 29,000 clones of cDNA libraries (Martınez-Hernandez et al in preparation) revealed 33 sequences with highly significant homology to fructosyltransferase (FT) or invertase genes
Alignment of the amino acid sequences of the two A. tequilana fructosyltransferase proteins with sequences available in GenBank (1-FFT EU026119 and 1-SST DQ535031) and the complete amino acid sequences of the six putative fructosyltransferase/invertase proteins identified in this work shows the degree of conservation at the sequence level and the features characteristic of glycoside hydrolase family 32 (Figure 1)

Summary

Introduction

Agave species are carbohydrate rich plants that synthesize fructans as the main storage polysaccharides with starch found to be present only in low levels [1]. Several fructan structures have previously been characterized in other plant species and differ by their length, DP, branching, linkage between adjacent fructose molecules and the position of the glucose residue [9]. Genes encoding FTs and FEHs from several plant species have previously been isolated and functionally characterized (for a review see [22]) including a gene encoding a Sucrose:sucrose 1-fructosyltransferase (1-SST: DQ535031) from A. tequilana [23]. Invertases are responsible for the hydrolysis of sucrose to glucose and fructose and unlike the fructosyltransferases these enzymes are present in all plant species. This report describes the identification and characterization of cDNAs encoding two types of fructosyltransferases (1-SST and 6G-FFT) and two types of invertases (cell-wall and vacuolar) from Agave tequilana. Comparisons with genomic sequences were carried out for all identified genes and selected genes were chosen to analyze enzyme activity in a heterologous expression system (Pichia pastoris) and/or study gene expression patterns by qRT-PCR

Results

Discussion

Materials and Methods