New insights into plant glycoside hydrolase family 32 in Agave species

Abstract

In order to optimize the use of agaves for commercial applications, an understanding of fructan metabolism in these species at the molecular and genetic level is essential. Based on transcriptome data, this report describes the identification and molecular characterization of cDNAs and deduced amino acid sequences for genes encoding fructosyltransferases, invertases and fructan exohydrolases (FEH) (enzymes belonging to plant glycoside hydrolase family 32) from four different agave species (A. tequilana, A. deserti, A. victoriae-reginae, and A. striata). Conserved amino acid sequences and a hypervariable domain allowed classification of distinct isoforms for each enzyme type. Notably however neither 1-FFT nor 6-SFT encoding cDNAs were identified. In silico analysis revealed that distinct isoforms for certain enzymes found in a single species, showed different levels and tissue specific patterns of expression whereas in other cases expression patterns were conserved both within the species and between different species. Relatively high levels of in silico expression for specific isoforms of both invertases and fructosyltransferases were observed in floral tissues in comparison to vegetative tissues such as leaves and stems and this pattern was confirmed by Quantitative Real Time PCR using RNA obtained from floral and leaf tissue of A. tequilana. Thin layer chromatography confirmed the presence of fructans with degree of polymerization (DP) greater than DP three in both immature buds and fully opened flowers also obtained from A. tequilana.

Keywords: agave; amino acid motifs; floral tissue; fructans; in silico expression; plant glycoside hydrolase family 32; transcriptome analysis.

Figure 1

Dendrogram showing the relationship between amino acid sequences of PGHF32 members from four agave species. Letters indicate different clades, sequences with activity confirmed by in vitro analysis are indicated by ^*. Atq-A. tequilana, Ad-A. deserti, Avr- A. victoriae-reginae, Ast-A. striata, Ao-A. officinalis, Ac-A. cepa. Numbers indicate Bootstrap values.

Figure 2

Comparison of conserved amino acid motifs specific to PGHF32. Motifs are indicated above each column. Dark green squares indicate residues conserved in all sequences.

Figure 3

Comparison of amino acid sequences within the 18 amino acid hypervariable region. (A) Sequences from fructosyltransferases (a–d) and vacuolar invertases (e). The red box indicates a putatively new invertase isoform. (B) Sequences from (a) fructan exohydrolases (AtCwinv3 has been re classified as an FEH) and Cell wall invertases (b). Dark green squares indicate residues conserved in all sequences.

Figure 4

In silico expression analysis of PGHF32 isoforms from three Agave species. (A) Isoforms of A. tequilana, (B) Isoforms of A. striata, (C) Isoforms of A. victoriae-reginae. TPM-Transcripts per million reads, Enzyme names defined in abreviations.

Figure 5

qRT-PCR analysis of transcripts encoding 1-SST, 6G-FFT, and Cwinv type enzymes in different floral tissues of A. tequilana. (A) Example of flower buds and floral tissues used for qRT-PCR analysis a, complete umbel; b, whole buds; c, dissected tepals; d, anthers; e, pistils and ovaries. (B) qRT-PCR expression patterns of PGHF32 genes in floral tissues.

Figure 6

TLC analysis of floral tissue from A. tequilana. (A) Example of immature floral buds, (B) Example of a fully developed A. tequilana flower. (C) TLC of fructans extracted from: IFB-immature floral buds and FL fully developed A. tequilana flowers. G-glucose, F-fructose, S-sucrose, 1-K-1kestose, DP-degree of polymerization. St-standards.