Drought and exogenous abscisic acid alter hydrogen peroxide accumulation and differentially regulate the expression of two maize RD22-like genes

Abstract

Increased biosynthesis of abscisic acid (ABA) occurs in plants in response to water deficit, which is mediated by changes in the levels of reactive oxygen species such as H₂O₂. Water deficit and ABA induce expression of some RD22-like proteins. This study aimed to evaluate the effect of water deficit and exogenous ABA (50 µM ABA applied every 24 hours for a total of 72 hours) on H₂O₂ content in Zea mays (maize) and to characterise genes encoding two putative maize RD22-like proteins (designated ZmRD22A and ZmRD22B). The expression profiles of the two putative maize RD22-like genes in response to water deficit and treatment with ABA were examined in leaves. In silico analyses showed that the maize RD22-like proteins share domain organisation with previously characterized RD22-like proteins. Both water deficit and exogenous ABA resulted in increased H₂O₂ content in leaves but the increase was more pronounced in response to water deficit than to exogenous ABA. Lignin content was not affected by exogenous ABA, whereas it was decreased by water deficit. Expression of both RD22-like genes was up-regulated by drought but the ZmRD22A gene was not influenced by exogenous ABA, whereas ZmRD22B was highly responsive to exogenous ABA.

Figure 1

Sequence alignment and domain organization of five BURP domain-containing proteins. At5G25610 (AtRD22), AAQ57584 (BnBDC1), Glyma06g08540 (GmRD22), GRMZM2G446170 (ZmRD22A) and GRMZM2G800586 (ZmRD22B) were aligned, resulting in 136 identical sites (29% identity) and a pairwise identity of 52.4%. Similar domains and structural components were identified and are indicated as follows: the hydrophobic signal peptides are encircled in blue, the conserved amino acids in the conserved region are shown in green, The TxV (x is any amino acid) repeats are shown in red, the BURP-domains are shown in yellow and the conserved motif of the BURP-domain is underlined with yellow.

Figure 2

Effect of exogenous abscisic acid and water deficit on relative water content, H₂O₂ content, lipid peroxidation and lignin content in maize leaves. (A) Relative water content of maize leaves in response to ABA or water deficit. (B) Levels of H₂O₂ in maize leaves upon application of ABA or exposure to water deficit. (C) Changes in lipid peroxidation based on measuring MDA content in leaves. (D) Cell wall lignin content of maize leaves subjected to ABA treatment or water deficit. Data represent means of three independent experiments. The error bars signify standard errors, where bars with the same letters are statistically similar at p < 0.05.

Figure 3

Semi-qRT-PCR analysis of the effect of water deficit or exogenous ABA on the expression of ZmRD22A and ZmRD22B in maize leaves. (A) Expression levels of ZmRD22A in response to water deficit. (B) Influence of water deficit on the expression of ZmRD22B. In both (A and B), controls are indicated by the first bar (water control). (C) Expression levels of ZmRD22A in response to exogenous ABA. (D) Response of ZmRD22B expression to exogenous ABA. In both (C and D), the controls are shown by the first and second bars (water and methanol controls, respectively) and the ABA treatment is shown by the third bar. In all cases, expression levels were measured relative to Zm18S rRNA expression. The error bars signify standard error in data from three independent experiments. Bars with the same letters are statistically similar where p < 0.05.

Figure 4

Quantitative RT-PCR analysis of ZmRD22A and ZmRD22B expression in response to exogenous ABA in maize leaves. (A) ZmRD22A transcript accumulation relative to maize β-tubulin in the 2^nd youngest leaves of maize seedlings treated with 50 µM ABA. (B) Representation of ZmRD22B transcript accumulation in the 2^nd youngest leaves, calculated in relation to Zmβ-tubulin, in response to exogenous treatment of maize seedlings with 50 µM ABA. In all the graphs, the water control is indicated by the first bar, the methanol control by the second bar and the ABA treatment by the third bar. Bars are means of three independent experiments and expression levels are signified as arbitrary values. The error bars signify standard deviation, bars with the same letters are statistically similar where p < 0.05.

Figure 5

Expression of ZmRD22B in various regions of leaves in response to ABA treatments. Transcript accumulation of ZmRD22B, relative to Zmβ-tubulin, in various leaf regions of the 2^nd youngest leaves of ABA-treated maize seedlings were measured by qRT-PCR. Leaf regions examined were the tips of leaves, middle of the leaves and the base of the leaves. Error bars signify standard errors of means from three independent experiments, bars with the same letters are statistically similar, where p < 0.05.

Figure 6

Expression of ZmRD22A and ZmRD22B in various regions of leaves in response to water deficit treatments. (A) Transcript accumulation of ZmRD22A in various leaf regions of the 2^nd youngest leaves of maize seedlings subjected to water deficit were measured by qRT-PCR. Leaf regions examined were the tips of leaves, middle of the leaves and the base of the leaves. (B) Expression of ZmRD22A in various leaf regions of the 2^nd youngest leaves of maize seedlings in response to water deficit. Expression was evaluated by qRT-PCR in the same leaf regions as in (A). Error bars signify standard errors of means from three independent experiments, bars with the same letters are statistically similar, where p < 0.05.