OsWRKY45 alleles play different roles in abscisic acid signalling and salt stress tolerance but similar roles in drought and cold tolerance in rice

Abstract

Although allelic diversity of genes has been shown to contribute to many phenotypic variations associated with different physiological processes in plants, information on allelic diversity of abiotic stress-responsive genes is limited. Here it is shown that the alleles OsWRKY45-1 and OsWRKY45-2 play different roles in abscisic acid (ABA) signalling and salt stress adaptation in rice. The two alleles had different transcriptional responses to ABA and salt stresses. OsWRKY45-1-overexpressing lines showed reduced ABA sensitivity, whereas OsWRKY45-1-knockout lines showed increased ABA sensitivity. OsWRKY45-1 transgenic plants showed no obvious difference from negative controls in response to salt stress. In contrast, OsWRKY45-2-overexpressing lines showed increased ABA sensitivity and reduced salt stress tolerance, and OsWRKY45-2-suppressing lines showed reduced ABA sensitivity and increased salt stress tolerance. OsWRKY45-1 and OsWRKY45-2 transgenic plants showed differential expression of a set of ABA- and abiotic stress-responsive genes, but they showed similar responses to cold and drought stresses. These results suggest that OsWRKY45-1 negatively and OsWRKY45-2 positively regulates ABA signalling and, in addition, OsWRKY45-2 but not OsWRKY45-1 negatively regulates rice response to salt stress. The different roles of the two alleles in ABA signalling and salt stress may be due to their transcriptional mediation of different signalling pathways.

Fig. 1.

Expression patterns of OsWRKY45-1 and OsWRKY45-2 in response to ABA and abiotic stresses in rice cultivars IRAT109 and Zhenshan 97. Bars represent the mean (three technical replicates) ±standard deviation. The ‘1’ or ‘2’ indicates that a significant difference was detected between treated plants and untreated control (ck) at P <0.01 or P <0.05, respectively.

Fig. 2.

Modulating OsWRKY45-1 and OsWRKY45-2 expression influenced rice response to ABA treatment. Control, negative siblings from corresponding transgenic segregating populations. (A) Rice seedlings before ABA treatment. Plants grown on MS medium for 10 d. (B) Phenotypes of transgenic plants after ABA treatment. Rice seedlings were grown on MS medium containing ABA for 7–10 d (until the transgenic and control plants showed marked differences in growth rate) and then phenotypes were recorded. Bars represent the mean (8–10 plants) ±standard deviation. The ‘1’ or ‘2’ indicates that a significant difference was detected between a positive transgenic line and its negative control at P <0.01 or P <0.05, respectively. Similar results were obtained in two biological repeats and only data from one repeat are presented. Because of the varying durations of ABA treatment (7–10 d) in different groups of plants, the control plants with the same genetic background in the OsWRKY45-1-oe and OsWRKY45-2-oe groups exhibited different lengths of shoot and root.

Fig. 3.

Modulating the expression of OsWRKY45-2 but not OsWRKY45-1 influenced rice response to salt stress. Rice seedlings were irrigated with a solution containing 200 mM NaCl at the 4- to 5-leaf stage. After 4–6 d of stress (until almost all the leaves of one group in the pot lost their green colour and some leaves died), the survival rates were recorded. Negative, negative siblings from corresponding transgenic segregating populations; positive, positive transgenic plants. Bars represent the mean (three technical replicates with each replicate containing 16–20 plants) ±standard deviation. The ‘1’ indicates that a significant difference was detected between a positive transgenic line and its negative control at P <0.01. Similar results were obtained in three biological repeats and only data from one repeat are presented. Because of the varying durations of salt stress (4–6 d) in different groups of plants, the control plants with the same genetic background in the OsWRKY45-1-oe and OsWRKY45-2-oe groups exhibited different survival rates.

Fig. 4.

Modulating OsWRKY45-1 and OsWRKY45-2 expression influenced rice response to cold stress. Rice seedlings were kept at 4 °C for 4–6 d (until almost all the leaves of one group in the pot became completely rolled and some leaves died) and then transferred to room temperature (25–28 °C) for recovery. Negative, negative siblings from corresponding transgenic segregating populations; positive, positive transgenic plants. (A) Phenotypes of transgenic plants before and after cold treatment. (B) Survival rate of cold-treated plants. Bars represent the mean (two or three replicates with each replicate containing 16–20 plants) ±standard deviation. The ‘1’ or ‘2’ indicates that a significant difference was detected between a positive transgenic line and its negative control at P <0.01 or P <0.05, respectively. Similar results were obtained in three biological repeats and only data from one repeat are presented.

Fig. 5.

Modulating OsWRKY45-1 and OsWRKY45-2 expression influenced rice response to drought stress. Water was withheld from rice seedlings at the 4- to 5-leaf stage for 3–5 d (until almost all the leaves of one group in the pot became completely rolled). After 3 d of recovery, the survival rates were recorded. Negative, negative siblings from corresponding transgenic segregating populations; positive, positive transgenic plants. Bars represent the mean (two or three replicates with each replicate containing 16–20 plants) ±standard deviation. The ‘1’ indicates that a significant difference was detected between a positive transgenic line and its negative control at P <0.01. Similar results were obtained in the three biological repeats and only data from one repeat are presented. Because of the varying durations of drought stress (3–5 d) in different groups of plants, the control plants with the same genetic background in the OsWRKY45-1-oe and OsWRKY45-2-oe groups exhibited different survival rates.

Fig. 6.

Modulating OsWRKY45-1 and OsWRKY45-2 expression influenced the expression of ABA- and abiotic stress-related genes. Bars represent the mean (three technical replicates) ±standard deviation. The ‘1’ or ‘2’ indicates that a significant difference was detected between a transgenic line and its corresponding wild-type (WT) plant at P <0.01 or P <0.05, respectively.