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.

Transcriptome analysis of bolting in A. tequilana reveals roles for florigen, MADS, fructans and gibberellins

“…Quantification of sucrose, glucose and fructose in the samples was carried out using the K-SUFRG kit (Megazyme) and the manufacturers instructions. Presence and estimation of DP of fructans was carried out using the kit K-FRUC kit (Megazyme) and by TLC as described in [20].…”

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

“…In silico expression patterns for genes encoding invertases and FEH across three different Agave species (A. tequilana, A. striata and A. victoriae-reginae) are consistent, whereas FT expression is highly variable (Avila De Dios et al, 2015). For example isoforms encoding 1-SST enzymes from A. tequilana and A. striata showed similar tissue specific patterns whereas those identified for A. victoriae-reginae varied widely and 6G-FFT encoding genes of A. victoriae-reginae and A. striata are strongly expressed in vegetative tissue in contrast to A. tequilana.…”

“…FT and FEH in common with vacuolar and cell wall invertases are members of Plant Glycoside Hydrolase Family 32 (PGHF32). 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).…”

“…For example isoforms encoding 1-SST enzymes from A. tequilana and A. striata showed similar tissue specific patterns whereas those identified for A. victoriae-reginae varied widely and 6G-FFT encoding genes of A. victoriae-reginae and A. striata are strongly expressed in vegetative tissue in contrast to A. tequilana. Expression patterns for all three Agave species showed high levels of expression for both FT and FEH in floral tissue (Avila De Dios et al, 2015) suggesting that fructans are not only being degraded but are also being synthesized in these organs.…”

AteqTERT expression and specific tissue activity in a 2-year-old complete plant in Agave tequilana in field conditions

“…This script allowed us to map short sequences into the transcriptomics libraries, aligning the RNA seq readings with the reference script sequence (RSEM-reference); these aligned reads were used to estimate abundance. Simultaneously, read mapped sequences are aligned with the NCBI database and homologies are identified in other species deposited in the database (Grabherr et al 2011;De Dios et al 2015;Zavala-García et al 2018).…”