A WRKY Protein, MfWRKY40, of Resurrection Plant Myrothamnus flabellifolia Plays a Positive Role in Regulating Tolerance to Drought and Salinity Stresses of Arabidopsis

Abstract

WRKY transcription factors (TFs), one of the largest transcription factor families in plants, play an important role in abiotic stress responses. The resurrection plant, Myrothamnus flabellifolia, has a strong tolerance to dehydration, but only a few WRKY proteins related to abiotic stress response have been identified and functionally characterized in M. flabellifolia. In this study, we identified an early dehydration-induced gene, MfWRKY40, of M. flabellifolia. The deduced MfWRKY40 protein has a conserved WRKY motif but lacks a typical zinc finger motif in the WRKY domain and is localized in the nucleus. To investigate its potential roles in abiotic stresses, we overexpressed MfWRKY40 in Arabidopsis and found that transgenic lines exhibited better tolerance to both drought and salt stresses. Further detailed analysis indicated that MfWRKY40 promoted primary root length elongation and reduced water loss rate and stomata aperture (width/length) under stress, which may provide Arabidopsis the better water uptake and retention abilities. MfWRKY40 also facilitated osmotic adjustment under drought and salt stresses by accumulating more osmolytes, such as proline, soluble sugar, and soluble protein. Additionally, the antioxidation ability of transgenic lines was also significantly enhanced, represented by higher chlorophyll content, less malondialdehyde and reactive oxygen species accumulations, as well as higher antioxidation enzyme activities. All these results indicated that MfWRKY40 might positively regulate tolerance to drought and salinity stresses. Further investigation on the relationship of the missing zinc finger motif of MfWRKY40 and its regulatory role is necessary to obtain a better understanding of the mechanism underlying the excellent drought tolerance of M. flabellifolia.

Keywords: Myrothamnus flabellifolia; WRKY; drought tolerance; salinity tolerance; zinc finger.

Figure 1

Multiple sequence alignment (a) and phylogenetic analysis (b) of MfWRKY40 and several highly homologous WRKY proteins. Black and gray shade in (a) show identical and similar amino acids, respectively. The WRKY motif was indicated by red box. (b) Phylogenetic reconstruction using the neighbor-joining method. The accession numbers for the sequences used were as follows: AtWRKY40 (AT1G80840) of Arabidopsis thaliana; ArWRKY40 (GFZ11870.1) of Actinidia rufa; PtWRKY40 (AZQ19202.1) of Populus tomentosa; GaWRKY28 (AIY62465.1) of Gossypium aridum; GmWRKY28 (ABC26917.1) of Glycine max; AcWRKY40 (PSS01081.1) of Actinidia chinensis var. chinensis; HbWRKY18-like (XP_021659791.1) of Hevea brasiliensis; GhWRKY24 (AGV75937.1) of Gossypium hirsutum; CfWRKY (GAV83654.1) of Cephalotus follicularis; PaWRKY (PON74566.1) of Parasponia andersonii; ToWRKY (PON99223.1) of Trema orientale; and BgWRKY (BAG15874.1) of Bruguiera gymnorhiza.

Figure 2

Subcellular localization of MfWRKY40. YFP, yellow fluorescent protein.

Figure 3

Analysis of drought and salinity tolerance at seedling stage. (a,b) indicated morphology of transgenic and WT seedlings grown for nine days on 1/2 MS medium with varying concentrations of mannitol and NaCl. (c,d) indicated root length of corresponding plants under different treatments. Data are presented as mean and SD values (error bar) of three independent experiments. Asterisks indicate significant difference (* p < 0.05, ** p < 0.01, by independent sample t-test) comparing to WT.

Figure 4

Analysis of drought and salinity tolerance at the adult stage. (a,b) showed the change of growth status of transgenic and WT plants during the progress of drought and salinity treatments. (c–h) showed measurements of tolerance-related physiological indexes. Data are presented as mean and SD values of three independent experiments. Asterisks indicated significant difference (* p < 0.05, ** p < 0.01, by independent sample t-test) comparing to WT.

Figure 5

Analysis of ROS accumulation and activities of key antioxidant enzymes under drought and salt treatments. (a,b) showed the analysis of H₂O₂ and O₂⁻ accumulation by using histochemical staining with DAB and NBT, respectively. (c,d) showed the content of hydrogen peroxide (H₂O₂) and superoxide anion content (O₂⁻), respectively. (e,f) indicated activities of peroxidase (POD) and superoxide dismutase (SOD) in the leaves of transgenic and WT plants, respectively. Data are presented as mean and SD values of three independent experiments. Asterisks indicated significant difference (* p < 0.05, ** p < 0.01, by independent sample t-test) compared to WT.

Figure 6

Measurements of stomatal aperture. (a) Microscopy observation of the stomatal aperture of OE and WT plants treated by 300 mM mannitol. (b) Measurement of the stomatal aperture with or without mannitol treatment. Data are presented as mean and SD values of three independent experiments. Asterisks indicated significant difference (* p < 0.05, by independent sample t-test) comparing to WT.