DISCUSSION
Drought severely affects plant growth and development. The WRKY TF family is involved in a multitude of biotic and abiotic stress responses (Rushton et al. , 2010; Parinita et al. , 2011; Chenet al. , 2012; Jiang et al. , 2014). A multiple sequence alignment confirmed that PtrWRKY75 contains the highly conserved WRKYGQK domain at the N-terminus homologous to that of AtWRKY75 (Figure 1). Previous studies have shown that AtWRKY75 accelerates leaf senescence mediated by SA and ROS accumulation in Arabidopsis(Guo et al. , 2017). However, little was known about the role of WRKY75 in drought tolerance, especially in poplar.
Earlier studies have reported that low concentrations of ROS can serve as signal molecules to regulate stress responses, for example, stomatal closure and the induction of defense gene expression (Desikan et al. , 2001; Rizhsky et al. , 2002). We observed thatPtrWRKY75 transcription responded to dehydration stress and exogenous SA treatment (Figure 1E, F). Under exogenous SA treatment, the ROS content increased to a greater degree and more rapidly in the OE lines than in WT (Figure 2A). This result is consistent with previous findings that SA promotes ROS production (Khokon et al. , 2011; Mori et al. , 2001). Reactive oxygen species are a crucial component in the regulation of stomatal closure (Maija et al. , 2016; Song et al. , 2014) and responses to adverse environmental conditions, such as drought (Carmody et al. , 2016). In our study, the stomatal aperture was significantly smaller in the OE lines than in WT after SA treatment (Figure 2C). Other studies have shown that drought stress leads to an increase in the SA concentration, for example, in Phillyrea angustifolia(Munné-Bosch and Peñuelas, 2003) and barley roots (Bandurska and Stroiński, 2005). In accordance with these reports, the SA content increased in both WT and OE plants under dehydration, and to a higher level (approximately double) in OE lines than in WT (Figure 3A). The higher expression level of PtrPAL1 in transgenic plants (Figure 3C), together with the results of the EMSA experiments (Figure 3D) and promoter transactivation assays (Figure 3E), indicate that WRKY75 promotes the production of SA by binding to the promoter ofPtrPAL1 , thereby inducing its expression. These results show that the higher drought tolerance of the transgenic lines overexpressingPtrWRKY75 results from SA-induced stomatal closure via ROS production.
Stomata play a vital role in water conservation and gas exchange between leaf tissues and the atmosphere. Some 90% of water loss (transpiration) occurs through stomata, and stomatal closure is the first step in water retention by plants under drought stress (Martin-St Paul et al. , 2017). Stomatal conductance and transpiration are positively correlated as they are opposite to drought tolerance (Baloch et al. , 2011). Compared with WT, the OE poplar lines showed a lowerG s and transpiration rate under well-watered conditions (Figure 4B, C). However, no significant difference in photosynthesis rate was observed between the OE lines and WT under well-watered conditions (Figure 4A), so that the WUE was higher in OE lines than in WT (Figure 4D). In reality, the photosynthetic rate is not always associated with G s, as indicated by decreases in Rubisco activity (Von Caemmerer et al. , 2004; Xuet al. , 2010). In the present study, the OE plants showed a lower water loss rate under drought at normal temperature compared with WT plants (Figure 5F). Improvement in the WUE of plants can enhance drought tolerance without penalizing yield under drought conditions (Karaba et al. , 2007). Electrolyte leakage, which is symptomatic of plasma membrane damage (Wang et al. , 2011; Shi et al. , 2013), was higher in WT than in OE lines under drought stress (Figure 5G), suggesting that WT plants suffered greater membrane damage under drought stress. This further supported the conclusion that overexpression of PtrWRKY75in poplar enhances its drought tolerance. We observed that drought stress caused reductions in photosynthetic activity and chlorophyll content in all plants, whereas the photosynthetic rate and chlorophylla content were higher in OE lines than in WT under drought stress (Figure 5B, 6C; Figure S3A–C).
A decrease in G s prevents excessive water loss under drought stress, thereby reducing the water demand of plants (Martin-St Paul et al. , 2017). However, it also decreases photosynthesis and biomass accumulation (Tardieu, 2012). Transgenic poplar plants showed no difference in growth and development from the WT plants under well-watered conditions (Figure 6D). Under drought conditions, the OE lines showed an increased growth rate and greater biomass production (Figure 6D,E), which may be attributed to the improved leaf RWC (Figure 6B). Maximal PSII quantum yield (F v/F m), which reflects the potential maximum light energy conversion efficiency of plants, declines in plants under drought stress (Ke et al. , 2016). The OE lines in this study showed higher PSII efficiency than that of the WT (Figure S3D), indicating that there was less damage to chloroplasts in the OE lines. We conclude that PtrWRKY75 is a promising gene target for increasing the tolerance of poplar to drought stress.