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.