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

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.

New insights into plant glycoside hydrolase family 32 in Agave species

“…A dendrogram based on an expanded alignment including all available amino acid sequences from experimentally determined members of PGHF32 from dicotyledonous and monocotyledonous species confirmed the classification of the agave sequences (Supplementary Figure 1 ). Previously characterized sequences from A. tequilana (Cortés-Romero et al, 2012 ) are indicated in red.…”

“…RNA extraction and qRT-PCR analysis was carried out as described in Abraham Juárez et al ( 2015 ). Primers used are listed in Cortés-Romero et al ( 2012 ).…”

“…Agavin structure implies the activity of four different fructosyltransferase enzyme activities: Sucrose:sucrose 1-fructosyltransferase (1-SST), Fructan:fructan 1-fructosyl transferase (1-FFT), sucrose:fructan 6 fructosyltransferase (6-SFT), and Fructan:Fructan 6G-Fructosyltransferase (6G-FFT). Previously different isoforms of 1-SST and 6G-FFT enzymes from A. tequilana (shown in red in Supplementary Table 1 ) were characterized at the genetic and functional level in different tissues and plants of different ages (Cortés-Romero et al, 2012 ). A 1-FFT type enzyme has also been reported for A. tequilana and A. inaequidans and shown to respond differentially in terms of expression in relation to exposure to different metabolites such as hormones or sugars (Suárez-González et al, 2013 ).…”

Expression of the 1-SST and 1-FFT genes and consequent fructan accumulation in Agave tequilana and A. inaequidens is differentially induced by diverse (a)biotic-stress related elicitors

“…9 and 10), suggests that Sucinduced accumulation of FOS observed in stems of this species at 42 DAT (Figs. 2B and 6A) is the result of enzymes other than 1-FFT, such as fructan:fructan 6G-fructosyltransferase (6G-FFT) and sucrose:fructan 6-fructosyltransferase (6-SFT) (Cortés-Romero et al, 2012). This could also be said with regard to the MeJA-Hi treatment in stems.…”

The Sweet Taste of Adapting to the Desert: Fructan Metabolism in Agave Species

“…The first Agave FT to be characterized was a 1-SST from A. tequilana (Avila-Fernandez et al, 2007) and by RNAseq 15 members of PGHF32 from A. tequilana, A. deserti, and A. victoriaereginae were later identified (Avila De Dios et al, 2015). Sequence based predictions of enzyme activities have also been confirmed for some A. tequilana enzymes using the P. pastoris system (Cortes-Romero et al, 2012). cDNAs encoding 6-SFT or 1-FFT type enzymes have not yet been conclusively identified perhaps due to low or tissue specific expression.…”