2.1 Sequence analysis and expression profiles of OsWRKY08

The OsWRKY08 (Os05g50610) cDNA was cloned from screening of the 4℃-treated rice leaf (Oryza sativa cv. Nipponbare) library^[4], which encodes a protein of 337-amino acids. We clarified its sequence homology by using FASTA program with the sequence of OsWRKY08 as query and found it shares 80.9% and 74.1% similarities with AtWRKY71 (gene ID At1g29860) and AtWRKY28 (gene ID At4g18170), respectively. According to the phylogenetic tree of Zhang et al. ^[42], we also found that AtWRKY28 and AtWRKY71 are more closely related to OsWRKY08 than other WRKY TFs in Arabidopsis. Further sequence analysis indicated OsWRKY08, AtWRKY71 and AtWRKY28 had a typical WRKY domain (WRKYGQK) and a C₂H₂ zinc finger motif (Fig. 1), falling into Group II of WRKY superfamily according to the classification of Eulgem et al.^[1]. Through the sequence comparison of the three typical WRKY proteins in rice and Arabidopsis, we found a close relationship among OsWRKY08, AtWRKY71, and AtWRKY28, suggesting a similar function for them.

It was reported that the expression of several WRKY genes including AtWRKY28 responded to various abiotic stresses, such as high salinity, osmotic stress and several plant hormones^[27] (http://urgv.evry.inra.fr/CATdb). Also we examined the expression profiles of OsWRKY08 under various treatments. Our results showed that OsWRKY08 was induced strongly and rapidly following the treatment in 25% PEG8000, 300 mM NaCl, 3% H₂O₂, 100 μM ABA and 100 μM NAA solutions (Fig. 2a). OsWRKY08 transcripts peaked at 2 h and then decreased gradually following exposure to PEG8000 and NaCl solution (Fig. 2a). The expression of OsWRKY08 was increased and reached the maximal level of expression following ABA, NAA application at 2 h and oxidative stress (H₂O₂) application at 4 h (Fig. 2a). ABA and H₂O₂ were considered as two important response signals in the plant response to various stress conditions^[43]. OsWRKY08 was remarkably upregulated by ABA and H₂O_2, salt and osmotic stress, which implied a strong association of OsWRKY08 with some abiotic stresses.

Analysis of the expression profiles of OsWRKY08 was also performed by Northern blotting analysis. Tissue-specific analysis showed that OsWRKY08 was constitutively expressed in almost all the tissues and organs examined, including young roots, young panicles, young leaves, mature leaves, flag leaves and senescing leaves (Fig. 2b).

2.2 Overexpression of OsWRKY08 in Arabidopsis improves osmotic stress tolerance

To determine the in vivo function of OsWRKY08 in plant abiotic stress response, we generated transgenic Arabidopsis plants overexpressing the OsWRKY08 gene under the CaMV 35S promoter. Among eleven primary T1 transformants, seven plants showed the same phenotype as wild-type control plants, and four plants died at different stages. From the seven surviving transgenic lines we selected four individual overexpressing lines of OsWRKY08 using Northern blotting analyses for collecting the T3 generation seeds (Fig. 3). At the first abiotic stresses screen, #05 and #11 lines showed the same results as #02, and another three lines in which the expression level of OsWRKY08 was not higher than #06 showed similar results as #06. Therefore we used #02 and #06 lines to do the second and third abiotic stresses treatments.

For the abiotic tolerance assay, transgenic and wild type plants were germinated and grown on 1/2 × MS medium plates for 6 days, and then transferred to the new normal MS agar plates with or without 0.8 µM ABA, 2.0 µM NAA, 200 mM Manntiol, 100 mM NaCl or 3% sucrose for 7 days. As shown in Fig.4, the number of the lateral roots and primary root length of wild type plants were noticeably inhibited on MS agar plates with 100 mM NaCl, 200 mM Manntiol, 0.8 µM ABA, and without sucrose (Fig. 4). Except for the 200 mM Manntiol condition, the growth of 35S::OsWRKY08 plants was restrained as the wild type. The comparison of 35S::OsWRKY08 and wild type seedlings on MS agar plates with 200 mM Manntiol showed that the transgenic seedlings were stronger and healthier than the control seedlings (Fig. 4A). The number of the lateral roots (Fig. 4B) (P≤0.001) and relative primary root length (Fig. 4C) (P<0.01) of the 35S::OsWRKY08 transgenic lines were more numerous and longer than the wild type seedlings on MS agar plates with 200 mM Manntiol. These results indicated that overexpression of OsWRKY08 in Arabidopsis can greatly enhance plant tolerance to osmotic stress through generating more lateral roots and growing longer primary roots.

2.3 Altered expression of osmotic stress response-related genes in OsWRKY08 transgenic plants

To explore the molecular mechanism of the observed enhanced osmotic stress tolerance in the OsWRKY08 overexpressing transgenic Arabidopsis plants, we monitored the expression of osmotic stress responsive genes by RT-PCR analysis. Under normal conditions, the test marker genes including AtCOR47, AtRD21, AtKIN1, AtRD22, AtRD29A, and AtABI4^[44,45] showed two different cases: the expression level of AtRD21 and AtCOR47 in 35S::OsWRKY08 plants was higher than that in wild-type plants, whereas there was no significant induction of AtKIN1, AtRD29A, AtRD22, and AtABI4 in both transgenic and wild type plants (Fig.5). AtABI4, ABSCISIC ACID-INSENSITIVE 4 gene, encodes an AP2 transcription factor that binds to a CE1-like element present in lots of promoters of ABA and sugar regulated genes^[46]. AtRD29A, AtRD22, and AtKIN1 genes are drought-, cold-, and ABA- inducible genes that contain the ABA-responsive element (ABRE) and dehydration responsive element (DRE) in their promoter sequences^[44]. However, the reductions in the four genes expression in 35S::OsWRKY08 plants were slight compared with those in wild type plants (Fig.5). AtRD21 and AtCOR47 were involved in osmotic stress signaling by ABA-independent pathways^[47], and both of them were increased in 35S::OsWRKY08 plants (Fig.5). Thus, the increased expression of AtRD21 and AtCOR47 in transgenic plants suggests that OsWRKY08 may be involved in plant tolerance by ABA-independent pathways.

3  Discussion

To date, a great number of studies have credibly shown that WRKY proteins have regulatory functions in plant immune responses^[13,14]. Whether their function includes the direct regulation of abiotic stress tolerance remains to be demonstrated. As the first step towards understanding the effects on plant abiotic stress tolerance of these WRKY genes, several groups analyzed their expression profiles and found that a majority of members were differentially induced by drought, high salinity, cold, and heat^[26,27]. Consistently, many rice WRKY genes were inducible in drought, high salinity, cold, and heat stresses^[4]. Here, we focused on the OsWRKY08 which is up-regulated by osmotic stress and identified as an important transcriptional regulator of transgenic Arabidopsis osmotic stress tolerance.

The abiotic tolerance assay of the wild type and the 35S::OsWRKY08 transgenic Arabidopsis showed the growth (number of the lateral roots and primary root length) of 35S::OsWRKY08 plants was strongly enhanced compared with wild type plants upon mannitol treatment. However, the growth of 35S::OsWRKY08 plants was strongly inhibited compared with wild type plants in the NaCl treatment (Fig. 4). These results suggest that the function of OsWRKY08 is regulating the increase of osmotic stress tolerance and decrease of ionic stress tolerance. Several studies have proved there are ABA dependent and independent signaling pathways in response to osmotic stress^[44,45]. To confirm ABA-dependent or independent signaling pathways of OsWRKY08, the germination assay and ABA sensitivity assay were performed. The time course of germination rate (data not shown) and relative primary root length data upon ABA treatment (Fig. 4) revealed no significant difference between wild type and 35S::OsWRKY08 transgenic Arabidopsis. It seemed that the improved tolerance of osmotic stress was not potentiated by ABA-dependent signaling pathway in 35S::OsWRKY08 transgenic Arabidopsis. In addition, some studies have demonstrated that Auxin plays an important role in lateral root development. We therefore put the wild type and 35S::OsWRKY08 plants on MS medium with 2.0 µM NAA. The quantity of lateral roots on plates containing NAA showed no difference between 35S::OsWRKY08 #06 lines and the wild type (Fig. 4), and the lateral roots of 35S::OsWRKY08 #02 lines were less than those of the wild type. Taken together, these results indicate that the improved tolerance of osmotic stress was not affected by ABA and NAA.

It has been suggested that OsWRKY45, which was previously screened from rice seedlings treated by abiotic stress just as OsWRKY08^[4], could enhance drought tolerance in transgenic Arabidopsis probably through ABA-dependent pathway^[30]. In our study, we monitored the ABA-dependent stress marker genes such as RD22, RD29, KIN1, and ABI4 (Fig. 5) in 35S::OsWRKY08 transgenic Arabidopsis. The results revealed no significant difference between the wild type and 35S::OsWRKY08 transgenic Arabidopsis (Fig. 5). It seemed that the improved tolerance of osmotic stress was not potentiated by the ABA-dependent signaling pathway in 35S::OsWRKY08 transgenic Arabidopsis. The expression of OsWRKY08 responded to high salinity, osmotic, and oxidative damage, as do the TcWRKY53 in Thlaspi caerulescens^[32] and AtWRKY25/33 in Arabidopsis^[29]. TcWRKY53 negatively regulates the osmotic stress tolerance of transgenic tobacco^[32], and overexpression of AtWRKY25 or AtWRKY33 was sufficient to enhance Arabidopsis NaCl tolerance^[29]. We report here that overexpression of OsWRKY08 improves the osmotic stress tolerance of transgenic Arabidopsis and two ABA independent stress marker genes AtRD21 and AtCOR47^[47] were upregulated in transgenic Arabidopsis. AtRD21 and AtCOR47 were induced by drought and salt in abi (ABA-insensitive) or aba (ABA-deficient) Arabidopsis mutants^[44] but reduced in the los5 (low expression of osmotically responsive genes) and los6 Arabidopsis mutants^[48]. The expressional upregulation of both genes suggests that improved osmotic stress tolerance of 35S::OsWRKY08 transgenic Arabidopsis is through potentiation of the ABA-independent signaling pathway.