UDP-glucose dehydrogenases of maize: a role in cell wall pentose biosynthesis

Abstract

UDPGDH (UDP-D-glucose dehydrogenase) oxidizes UDP-Glc (UDP-D-glucose) to UDP-GlcA (UDP-D-glucuronate), the precursor of UDP-D-xylose and UDP-L-arabinose, major cell wall polysaccharide precursors. Maize (Zea mays L.) has at least two putative UDPGDH genes (A and B), according to sequence similarity to a soya bean UDPGDH gene. The predicted maize amino acid sequences have 95% similarity to that of soya bean. Maize mutants with a Mu-element insertion in UDPGDH-A or UDPGDH-B were isolated (udpgdh-A1 and udpgdh-B1 respectively) and studied for changes in wall polysaccharide biosynthesis. The udpgdh-A1 and udpgdh-B1 homozygotes showed no visible phenotype but exhibited 90 and 60-70% less UDPGDH activity respectively than wild-types in a radiochemical assay with 30 microM UDP-glucose. Ethanol dehydrogenase (ADH) activity varied independently of UDPGDH activity, supporting the hypothesis that ADH and UDPGDH activities are due to different enzymes in maize. When extracts from wild-types and udpgdh-A1 homozygotes were assayed with increasing concentrations of UDP-Glc, at least two isoforms of UDPGDH were detected, having K(m) values of approx. 380 and 950 microM for UDP-Glc. Leaf and stem non-cellulosic polysaccharides had lower Ara/Gal and Xyl/Gal ratios in udpgdh-A1 homozygotes than in wild-types, whereas udpgdh-B1 homozygotes exhibited more variability among individual plants, suggesting that UDPGDH-A activity has a more important role than UDPGDH-B in UDP-GlcA synthesis. The fact that mutation of a UDPGDH gene interferes with polysaccharide synthesis suggests a greater importance for the sugar nucleotide oxidation pathway than for the myo-inositol pathway in UDP-GlcA biosynthesis during post-germinative growth of maize.

Figure 1. Alignment of predicted UDPGDH protein from soya bean and maize

The predicted soya bean protein (id AAB58398) is compared with the proteins predicted for the two maize contigs of EST (ids 658123 and 658125 from the GENOPLANTE GnpSeq database). Proteins predicted from 658123 and 658125 are referred to here as UDPGDH-A and UDPGDH-B respectively. Underline indicates a deviation from the other two sequences; where all three sequences differ at the same site, the three residues are all underlined.

Figure 2. Alignment of contigs 658123 and 658125 (cf. Figure 1)

The ↑ specifies the position of the udpgdh-B1 insertion in the 658125 sequence; the ↓ indicates the location of the udpgdh-A1 insertion in the 658123 sequence. Emboldening indicates the start and stop codons of the predicted protein and the sequence corresponding (or approximately corresponding) to the UDPGDH-specific primer used, CCAATCCTCGTGTCCTTGCCC.

Figure 3. UDPGDH activities in developing maize leaves: radiochemical assay

(A) UDPGDH activities in two wild-types (wt) and two homozygous udpgdh-A1 mutant individuals (mut). (B) UDPGDH activities in two wild-types (wt) and two homozygous udpgdh-B1 mutant individuals (mut). Activities in ‘pkat’ are reported for the particular assay conditions used (30 μM UDP-[¹⁴C]Glc), no account being taken of the K_m value(s) for this substrate. FW, fresh weight.

Figure 4. UDPGDH and ADH activities in developing maize leaves: spectrophotometric assay

The assay mixture contained 5 mM UDP-Glc or ethanol as substrate. (A) Enzymic activities in three wild-types (wt) and three homozygous udpgdh-A1 mutant individuals (mut). (B) Enzymic activities in two wild-types (wt) and two homozygous udpgdh-B1 mutant individuals (mut). FW, fresh weight.

Figure 5. Effect of UDP-Glc concentration on UDPGDH activity in extracts from wild-type and homozygous udpgdh-A1 mutants

UDPGDH activity in extracts from developing leaves of the wild-type and homozygous udpgdh-A1 mutants was assayed by the radiochemical method with increasing concentrations of UDP-Glc. By subtraction of the observed mutant activity (isoenzyme B only; dotted line) from that of the wild-type (isoenzymes A and B; solid line), the activity due to isoenzyme A is estimated (broken line). FW, fresh weight.

Figure 6. Deduced Hanes plots for isoenzymes UDPGDH-A (▼) and UDPGDH-B (○)

Figure 7. Sugar residue composition of cell wall polysaccharides from wild-type and udpgdh mutants

The graphs show pentose/hexose ratios of non-cellulosic cell wall polysaccharides in the first and the third mature leaves (counting from the soil) and in the second-lowest stem internode of wild-type maize plants and homozygotes of udpgdh-A1 (graphs A and B) or udpgdh-B1 (graphs C and D). Black=wild-type; grey=mutant. Error bars indicate the standard errors of the ratios measured on (n=5 for wt-A and udpgdh-A1, n=4 for wt-B and n=3 for udpgdh-B1) individuals of each genotype. Means of the mutant ratios that differ statistically significantly from those of the corresponding wild-types are indicated (Student's t test, *P<0.05).