UDP-glucose 4-epimerase isoforms UGE2 and UGE4 cooperate in providing UDP-galactose for cell wall biosynthesis and growth of Arabidopsis thaliana

Abstract

Five Arabidopsis thaliana genes that encode UDP-glucose 4-epimerase (UGE) and represent two ancient plant UGE clades might be involved in the regulation of cell wall carbohydrate biosynthesis. We tested this hypothesis in a genome-wide reverse genetic study. Despite significant contributions of each gene to total UGE activity, none was essential for normal growth on soil. uge2 uge4 displayed dramatic general growth defects, while other mutant combinations were partially aberrant. UGE2 together with UGE3 influenced pollen development. UGE2 and UGE4 synergistically influenced cell wall galactose content, which was correlated with shoot growth. UGE2 strongly and UGE1 and UGE5 lightly supported UGE4 in influencing root growth and cell wall galactose content by affecting galactan content. By contrast, only UGE4 influenced xyloglucan galactosylation in roots. Secondary hypocotyl thickening and arabinogalactan protein carbohydrate structure in xylem parenchyma depended on the combination of UGE2 and UGE4. As opposed to cell wall galactose content, tolerance to external galactose strictly paralleled total UGE activity. We suggest a gradual recruitment of individual UGE isoforms into specific roles. UGE2 and UGE4 influence growth and cell wall carbohydrate biosynthesis throughout the plant, UGE3 is specialized for pollen development, and UGE1 and UGE5 might act in stress situations.

Figure 1.

Phylogeny of Plant UGE Isoforms. Phylogeny of UGE proteins from Chlamydomonas reinhardtii (Cr), Physcomitrella patens (Pp), Populus trichocarpa (Pt), Arabidopsis thaliana (At), and Oryza sativa (Os) excluding N- and C-terminal sequences. The tree was rooted with the Chlamydomonas sequence. Percentages are bootstrap values obtained from 100 bootstrap replicates.

Figure 2.

Spatial Expression Patterns of UGE:GUS Constructs. (A) Top, UGE1:GUS to UGE5:GUS expression in 8-d-old light-grown seedlings (left) and 4-d-old dark-grown seedlings (right). Bottom, details of expression in root tips of 5-d-old light-grown seedlings. (B) UGE1:GUS to UGE5:GUS expression in sections of secondary thickened hypocotyls of 6-week-old plants.

Figure 3.

Rosette and Root Phenotypes of uge Mutants. (A) Rosettes of 22-d-old wild-type Columbia (Col-0) and selected uge mutant plants. (B) Top, roots of 4-d-old wild-type Col-0 and selected uge mutant plants. Bottom, root tips with division and elongation zones magnified 2.5 times. Bars = 1 mm (top) and 0.4 mm (bottom).

Figure 4.

Floral Phenotypes of uge Multiple Mutants. (A) General growth phenotypes in uge2,4 and uge1,2,4. Bar = 500 μm. (B) Floral organs are formed but expand abnormally in uge1,2,4. Bar = 200 μm. (C) Pollen phenotype in uge2,3. Bar = 50 μm.

Figure 5.

Correlation of Biochemical and Growth Parameters of Shoots of uge Mutants. The dot plots show the comparison between UGE activity and galactose content of cell walls (A), rosette diameter (B), and the amount of galactose-containing xyloglucan oligosaccharides (XGO) (C) and pectic galactan (D). All values are represented in percentages of the wild-type level. The plots also show linear regressions including all genotypes ([B] to [D]; r² = 0.855, 0.627, and 0.583 respectively) or excluding uge2,4 and uge1,2,4 ([A]; r² = 0.391). For clarity, only some data points are labeled. All numerical values can be found in Supplemental Tables 2 to 5 online.

Figure 6.

Correlation of Biochemical and Growth Parameters of Roots of uge Mutants. The dot plots show the comparison between UGE activity and galactose content of cell walls (A), root length ([B] and [C]), and the amount of galactose-containing xyloglucan oligosaccharides (XGO) (D) and pectic galactan (E). All values are represented in percentages of the wild-type level. The plots also show linear regressions including all genotypes tested ([E]; r² = 0.617), lines with the UGE4 wild-type background ([A], black line; r² = 0.414), the uge4 mutant allele ([B]; r² = 0.871), or with a UGE2 wild-type allele and a uge4 mutant allele ([A] and [C], gray lines; r² = 0.674 and 0.894, respectively). For clarity, only some data points are labeled. All numerical values can be found in Supplemental Tables 2 to 5 online.

Figure 7.

Distribution of Cell Wall Epitopes in Secondary Thickened Hypocotyls of uge Mutants. Transverse sections through secondary thickened hypocotyls of 8-week-old wild-type (top), uge2,4 (middle), and uge1,2,4 (bottom) plants. LM10 recognizes an unsubstituted xylan in the secondary walls of xylem (McCartney et al., 2005); CCRC-M1 binds to α-l-fucosyl (1→2)-β-d-galactosyl side chains of xyloglucan (Puhlmann et al., 1994); LM5 recognizes (1→4)-β-d-galactan side chains of pectic rhamnogalacturonan I (Jones et al., 1997); LM2 recognizes a β-d-glucuronosyl residue on AGP (Yates et al., 1996); CCRC-M7 recognizes an arabinosylated (1→6)-β-d-galactan of arabinogalactan II (Steffan et al., 1995); and JIM13 binds to AGPs (Yates et al., 1996). Arrowheads indicate xylem vessels. Antibody labeling is shown in green, and autofluorescence counterstain is shown in purple. Bar = 100 μm.

Figure 8.

Effects of d-Galactose on uge Mutants. Seedlings were germinated on plates containing standard medium supplemented with different concentrations of d-galactose and documented 12 d after germination. Signs of galactose toxicity are reduced growth, anthocyanin production, and, in the most severe cases, chlorosis and necrosis. Toxicity is moderate in the wild type but most strongly enhanced in multiple uge mutants that display the strongest reduction of UGE activity. The complete lack of pigmentation in uge1,2,5 und uge2,5 seedlings caused by galactose is currently unexplained. However, it might indicate that one mode of action of galactose toxicity in plants is the inhibition of chlorophyll biosynthesis or chloroplast development.