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New insights into plant glycoside hydrolase family 32 in Agave species.

Avila de Dios E, Gomez Vargas AD, Damián Santos ML, Simpson J - Front Plant Sci (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Engineering, Centro de Investigación y Estudios Avanzados Irapuato, Mexico.

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.

No MeSH data available.


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.
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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.

Mentions: Based only on the 18 amino acid hypervariable loop region a reasonably good correlation is obtained between the groups formed in the dendrogram and enzyme activity. Only 5 of the 106 sequences indicated with red boxes in Supplementary Figure 3 show no strong correlation between activity and the groups defined by sequence analysis. AtCwinv3 indicated by a stippled red box was initially classified as a cell wall invertase but later shown to be an FEH and is correctly placed in the corresponding clade. When the 18 amino acid loop sequences were aligned by putative or confirmed enzyme activity, distinct patterns of conserved amino acids could be determined for each of the enzyme types (Figures 3A,B). As indicated, three amino acids: lysine, tyrosine, and glycine at positions 1, 12, and 15 respectively within the 18 amino acid motif are conserved in all sequences. This minimal pattern distinguishes the FEH group from the other enzyme types. In contrast the closely related Cwinv group, in addition to the minimal 3, has 6 additional conserved amino acids, the vacuolar invertases 5 and the 1-SST, 6G-FFT, and 1FFT fructosyltransferases 6, 9, and 5 additional conserved amino acids respectively (Table 2). The conserved amino acids within the 18-residue motif for the 6SFT type enzyme is based on closely related reported sequences whose identities have not yet been confirmed by activity and shows strong conservation with only 2 of the 18 residues found to vary. Although Cwinvs and FEHs could clearly be distinguished based on the hypervariable loop region, different forms of FEH type enzymes could not be accurately determined. The newly identified Vinv, Cwinv, FEH, and FFT genes from the different agave species show good correlations with the conserved amino acid patterns and putative enzyme types, supporting the initial classification based on the dendrogram in Figure 1.


New insights into plant glycoside hydrolase family 32 in Agave species.

Avila de Dios E, Gomez Vargas AD, Damián Santos ML, Simpson J - Front Plant Sci (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4524927&req=5

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.
Mentions: Based only on the 18 amino acid hypervariable loop region a reasonably good correlation is obtained between the groups formed in the dendrogram and enzyme activity. Only 5 of the 106 sequences indicated with red boxes in Supplementary Figure 3 show no strong correlation between activity and the groups defined by sequence analysis. AtCwinv3 indicated by a stippled red box was initially classified as a cell wall invertase but later shown to be an FEH and is correctly placed in the corresponding clade. When the 18 amino acid loop sequences were aligned by putative or confirmed enzyme activity, distinct patterns of conserved amino acids could be determined for each of the enzyme types (Figures 3A,B). As indicated, three amino acids: lysine, tyrosine, and glycine at positions 1, 12, and 15 respectively within the 18 amino acid motif are conserved in all sequences. This minimal pattern distinguishes the FEH group from the other enzyme types. In contrast the closely related Cwinv group, in addition to the minimal 3, has 6 additional conserved amino acids, the vacuolar invertases 5 and the 1-SST, 6G-FFT, and 1FFT fructosyltransferases 6, 9, and 5 additional conserved amino acids respectively (Table 2). The conserved amino acids within the 18-residue motif for the 6SFT type enzyme is based on closely related reported sequences whose identities have not yet been confirmed by activity and shows strong conservation with only 2 of the 18 residues found to vary. Although Cwinvs and FEHs could clearly be distinguished based on the hypervariable loop region, different forms of FEH type enzymes could not be accurately determined. The newly identified Vinv, Cwinv, FEH, and FFT genes from the different agave species show good correlations with the conserved amino acid patterns and putative enzyme types, supporting the initial classification based on the dendrogram in Figure 1.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Engineering, Centro de Investigación y Estudios Avanzados Irapuato, Mexico.

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.

No MeSH data available.