Alteration of cell wall polysaccharides through transgenic expression of UDP-Glc 4-epimerase-encoding genes in potato tubers
Introduction
Pectin consists primarily of homogalacturonan (HG) and rhamnogalacturonan I (RG-I). HG is an unbranched polymer of α-(1 → 4)-linked galacturonosyl residues, whereas the backbone of RG-I is composed of repeating α-(1 → 2)-l-rhamnose (Rha)-α-(1 → 4)-d-galacturonic acid (GalA) units with side chains connected to the rhamnose moiety (McCann & Roberts, 1991). Potato pectin is rich in galactan side chains consisting of (1 → 4)-β-d-galactose (Gal) residues connected to the O-4 position of Rha within the RG-I backbone (Sørensen et al., 2000).
The primary hemicellulose found in potato tubers is xyloglucan (Vincken et al., 2000). Xyloglucan is a polysaccharide found in primary cell walls and plays a role in the control of cell growth (Fry, 1989). Xyloglucan consists of a β-(1 → 4)-d-glucan backbone and substituted with side chains of xylose with or without additional substitution of Gal, fucose and/or arabinose (Ara). These side chains including the substituted glucose (Glc) moiety are individually annotated using a single letter code (Fry et al., 1993). In the commonly used nomenclature, G is used for an unsubstituted β-Glcp; X is an α-d-xylose (Xyl)p-(1 → 6)-β-d-Glcp; S and L are α-l-Araf-(1 → 2)-α-d-Xylp-(1 → 6)-β-d-Glcp and β-d-Galp-(1 → 2)-α-d-Xylp-(1 → 6)-β-d-Glcp, respectively; and F is an α-l-Fucp-(1 → 2)-α-l-Araf-(1 → 2)-α-d-Xylp-(1 → 6)-β-d-Glcp (Fry et al., 1993). In Solanaceae (e.g., potato), the xyloglucan consists of XXGG-type repeating units (RU), in which two out of four Glc residues in the backbone are substituted at O-6 with α-d-Xyl residues which may have additional monosaccharides such as Gal and Ara (but no Fuc) substitution attached (Vincken, Wijsman, Beldman, Niessen, & Voragen, 1996).
The composition of potato pectin can be biosynthetically modified to alter cell wall architecture. Cell wall biosynthesis requires the expression of glycosyltransferases that utilize uridine diphosphate (UDP) sugars for the assembly of cell wall polysaccharides and pectin biosynthesis is directly affected by changes in the nucleotide sugar pools (Caffall & Mohnen, 2009). They further discovered that pectin biosynthesis was directly affected by changes in the nucleotide sugar pools. Nucleotide sugars are synthesized by various types of interconverting enzymes, such as epimerases, decarboxylases and dehydrogenases, and their individual levels in the sugar pool directly affect the content of the corresponding monosaccharides in cell wall polysaccharides (Gondolf et al., 2014). The pools of UDP-sugar are directly or indirectly linked by either one-way or reversible reactions to form other UDP-sugars (Reiter & Vanzin, 2001). The Glc:Gal ratio can be influenced by UDP-glucose-4-epimerase (UGE), which catalyzes a reversible conversion of UDP-glucose (UDP-Glc) into UDP-galactose (UDP-Gal) as demonstrated in Arabidopsis mutants (Bar-Peled & O'Neill, 2011). Two potato UGE genes were isolated from a potato cDNA library using the Aradibopsis thaliana UGE1 cDNA probe (Oomen et al., 2004). UGE 51 showed a higher expression levels in tubers, flowers, stems and roots than UGE 45 (Oomen et al., 2004). Both UGEs are correlated to potato Gal content. UGE catalyze the reversible conversion of UDP-Glc to UDP-Gal (Oomen et al., 2004). In wild type potato, the epimerase activity may be limiting and could be effected by the overexpression of UGE after transgenic modification. The introduction of the gene from Arabidopsis that encodes UGE in potato increased the Gal levels in potato cell walls (Oomen et al., 2004). Oomen et al. (2004) focused primarily on the Gal levels in the cell wall materials (CWM) of UGE transgenic lines and hardly investigated the consequences of these changed levels for polysaccharide structures. Recently, we reported the appearance of shorter galactan side chains present in potato pectin after the introduction of the β-galactosidase (β-Gal) gene into wild-type potato (Huang, Kortstee et al. 2016). In contrast to the wild-type, the β-Gal transgenic lines predominantly contained XXXG-type xyloglucan in their cell walls as a side effect (Huang, Jiang et al., 2016). In UGE transgenic lines, the precise mechanism of both targeted (galactan side chain) and non-targeted modifications (other polysaccharide structures) of the cell wall polysaccharides remained unclear.
In the present study, we compared the structure of pectins found in UGE transgenic lines and wild-type potato, and identified additional changes within other polysaccharides.
Section snippets
Potato tubers
Two potato UGE genes were isolated from a potato cDNA library using the Arabidopsis thaliana UGE1 cDNA probe (Oomen et al., 2004). Both UGEs are correlated to potato Gal content. Both UGE 45 and UGE 51, were used for the preparation of a sense construct using the pBIN20 vector with wild-type potato cv. Kardal as the background to obtain UGE 45-1 and UGE 51-16 transgenic lines as described before (Oomen et al., 2004). Both UGE 45 and UGE 51 genes encoded the UGE enzyme (Oomen et al., 2004).
CWM composition of wild-type potato and transgenic lines
The starch levels in the isolated CWM from Kardal, UGE 45-1 and UGE 51-16 were 5.1%, 4.5% and 4.1% of CWM, respectively. The constituent molar monosaccharide composition of the polysaccharides in each CWM (Table 1) and the contents of constituent monosaccharides in each CWM, expressed on fresh tuber weight basis (mg/100 g tuber, Table 2) revealed distinct differences in the cell wall compositions between Kardal and UGE 45-1 and UGE 51-16 transgenic lines. The amount of carbohydrates was rather
Discussion
Previous research on CWM from UGE transgenic lines was hampered by a high content of starch (50% w/w) present in the isolated CWM (Oomen et al., 2004). Furthermore, Oomen et al. (2004) only discussed the monosaccharide content of the CWM (w/w%), and thus the overall transgenic modifications made within the UGE transgenic lines remained unidentified. In the present study, more than 99.5% of the starch was removed from the potato varieties by using an optimized procedure (Huang, Kortstee et al.
Conclusions
UGE 45-1 exhibited higher levels of Gal and longer side chains in all pectin fractions, compared to those found for the wild-type. Conversely, UGE 51-16 pectin polysaccharide fractions possessed similar or shorter side chain lengths than the wild-type. In addition, non-targeted structures had been changed in each pectin or xyloglucan polysaccharide population of UGE transgenic lines investigated. After the targeted modification on the length of galactan side chain, the other cell wall
Acknowledgments
This research is supported by the Dutch Technology Foundation STW, which is part of the Netherlands Organisation for Scientific Research (NWO) and which is partly funded by the Ministry of Economic Affairs (Project 10625).
References (31)
- et al.
The structure, function, and biosynthesis of plant cell wall pectic polysaccharides
Carbohydrate Research
(2009) - et al.
Are designer plant cell walls a realistic aspiration or will the plasticity of the plant's metabolism win out?
Current Opinion in Biotechnology
(2014) - et al.
Modification of potato cell wall pectin by introduction of rhamnogalacturonan lyase and β-galactosidase transgenes and their side effects
Carbohydrate Polymers
(2016) - et al.
Quantification of the amount of galacturonic acid residues in blocksequences in pectin homogalacturonan by enzymatic fingerprinting with exo- and endo-polygalacturonase II from Aspergillus niger
Carbohydrate Research
(2000) - et al.
Overexpression of two different potato UDP-Glc 4-epimerases can increase the galactose content of potato tuber cell walls
Plant Science
(2004) - et al.
Structural features and water holding capacities of pressed potato fibre polysaccharides
Carbohydrate Polymers
(2013) - et al.
Conformational folding of xyloglucan side chains in aqueous solution from molecular dynamics simulation
Carbohydrate Research
(2005) - et al.
Purification and characterization of endo-1,4-β-d-galactanases from Aspergillus niger and Aspergillus aculeatus: use in combination with arabinanases from Aspergillus niger in enzymic conversion of potato arabinogalactan
Carbohydrate Polymers
(1991) - et al.
Isolation and characterisation of cell wall material from olive fruit (Olea europaea cv koroneiki) at different ripening stages
Carbohydrate Polymers
(2000) - et al.
Remodelling pectin structure in potato