Water deficits affect caffeate O-methyltransferase, lignification, and related enzymes in maize leaves. A proteomic investigation

Abstract

Drought is a major abiotic stress affecting all levels of plant organization and, in particular, leaf elongation. Several experiments were designed to study the effect of water deficits on maize (Zea mays) leaves at the protein level by taking into account the reduction of leaf elongation. Proteomic analyses of growing maize leaves allowed us to show that two isoforms of caffeic acid/5-hydroxyferulic 3-O-methyltransferase (COMT) accumulated mostly at 10 to 20 cm from the leaf point of insertion and that drought resulted in a shift of this region of maximal accumulation toward basal regions. We showed that this shift was due to the combined effect of reductions in growth and in total amounts of COMT. Several other enzymes involved in lignin and/or flavonoid synthesis (caffeoyl-CoA 3-O-methyltransferase, phenylalanine ammonia lyase, methylenetetrahydrofolate reductase, and several isoforms of S-adenosyl-l-methionine synthase and methionine synthase) were highly correlated with COMT, reinforcing the hypothesis that the zone of maximal accumulation corresponds to a zone of lignification. According to the accumulation profiles of the enzymes, lignification increases in leaves of control plants when their growth decreases before reaching their final size. Lignin levels analyzed by thioacidolysis confirmed that lignin is synthesized in the region where we observed the maximal accumulation of these enzymes. Consistent with the levels of these enzymes, we found that the lignin level was lower in leaves of plants subjected to water deficit than in those of well-watered plants.

Figure 1.

Identification of two isoforms of COMT in maize leaves. Arrows indicate the proteins identified by LC-MS/MS and correlated with COMT1, CCoAOMT, and PAL on a silver-stained 2D gel of leaf proteins from the F2 inbred line of maize. The inset shows an enlargement of the framed region in the Io wild-type line (Bm3/Bm3) and in the near-isogenic line bearing the bm3 mutation (bm3/bm3). COMT1 and COMT2 are present in Bm3/Bm3 and absent in bm3/bm3.

Figure 2.

COMT1 and COMT2 profiles in response to water deficit. A and B, Relative amounts of COMT1 and COMT2 along leaf 6 of Io plants in experiment 1. C and D, Relative amounts of COMT1 and COMT2 along leaf 6 of Io plants in experiment 2 at day 3. The inset in C shows the elongation rate of leaf 6 in experiment 2. Vertical bars show se (n = 3 in experiment 1; n = 2 in experiment 2). RA, Relative amount.

Figure 3.

Relative amounts of COMT isoforms in function of tissue age in Io leaves of experiment 2. Relative amounts of COMT1 in leaves harvested at day 3 (A), day 4 (B), and day 5 (C) are shown. Relative amounts of COMT2 in leaves harvested at day 3 (D), day 4 (E), and day 5 (F) also are shown. Vertical bars show se (n = 4 to 11). RA, Relative amount.

Figure 4.

Elongation rate of leaf 6 of F2 plants in experiment 3. Average days of harvesting are indicated for controls (C1 to C3), and plants subjected to mild (M) and severe (S1 and S2) water deficit. Vertical bars show se (n = 3).

Figure 5.

Variation of relative amounts of COMT with leaf development. A, Silver-stained 2D gel of leaf proteins from the F2 inbred line in experiment 3. Arrowheads indicate COMT1 (left) and COMT2 (right). B, Relative amount of COMT1 along the leaves of F2 plants in experiment 3. C, Relative amount of COMT1 in function of tissue age of F2 leaves in experiment 3.

Figure 6.

Relation between COMT and lignification profiles. A to C, In vitro activity of COMT in function of relative amounts of COMT1 and COMT2 measured on 2D gels. r, Correlation coefficient. D, Measurement of uncondensed lignin units (H + G + 5OHG + S) along leaf 6 in experiment 3. E, Measurement of uncondensed lignin units in function of tissue age. F, S:G ratio along leaf 6. G, Measurement of FA along leaf 6 in experiment 3. Vertical bars show se (n = 2 in D, F, and G; n = 3–8 in E). RA, Relative amount.