Objective : This study aimed to evaluate the drought resistance of two histidine kinase PagHK3a gene knockout lines of poplar '84k' (Populus alba × P. glandulosa '84k'), and explore the molecular regulatory mechanism of PagHK3a gene in poplar response to drought stress.

Method : Plantlets of wild-type (WT) and gene knockout strains (C1 and C2) sub-cultured for 25 days were treated with 5% PEG and the mature leaves of the plantlets were gathered 3 hours later. Quantitative real-time PCR (qPCR) was used to detect the expression levels of PagHK3a gene, PagHK3a downstream related genes, drought stress response genes and antioxidant related genes in leaves of the three lines. Meanwhile, the drought stress treatment was carried out using the potted plants of each line by weighing method with three levels: normal watering greenhouse (soil relative water content: 70%-75%), moderate drought (40%-45%) and severe drought (25%-30%). After four weeks of drought stress treatment, transient photosynthetic parameters, contents of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), activities of peroxidase (POD) and superoxide dismutase (SOD), plant height and ground diameter of WT and C1, C2 were measured.

Result : Under moderate drought conditions, the height growth of both C1 and C2 lines was significantly higher than that of WT, but under severe drought conditions, the height growth of both lines was not significantly different from that of WT. Analysis of photosynthetic parameters revealed that the stomatal conductance and intercellular carbon dioxide concentration of C1 and C2 were significantly higher than those of WT under moderate drought condition, but only C2 was significantly higher than that of WT under severe drought stress condition. Under moderate drought stress, the instantaneous net photosynthetic rate of C1 was significantly higher than that of WT, and under severe drought stress, the instantaneous net photosynthetic rate of C2 was significantly higher than that of WT. At the same time, the results of biochemical indexes showed that MDA and H₂O₂ contents in knockout lines C1 and C2 were significantly lower than those in WT under moderate drought stress, but only C2 was significantly lower than that in WT under severe drought stress. Comparative analysis of the antioxidant enzyme activity of each line found that, under normal water supply conditions, the POD enzyme activity of C1 strain and the SOD activity of C2 strain were significantly higher than that of WT. And under moderate drought stress, the two protective enzyme activities of C1 strain were significantly higher than that of the control, while C2 was significantly higher than that of WT only under severe drought stress. In addition, under 5% PEG treatment, the expression levels of histidine kinase gene PagHK3a and its downstream response regulatory proteins PagRR2 and PagRR15 in the leaves of gene knockout lines C1 and C2 were significantly down-regulated compared with those of WT. The expression levels of drought stress response gene PagNAC3 and peroxidase synthesis gene POD1 were significantly up-regulated, while the expression level of superoxide dismutase synthesis gene SOD4 was not significantly different from that of WT.

Conclusion : In PagHK3a knockout lines, expression of PagHK3a was down-regulated, expression of its downstream genes PagRR2 and PagRR15 in the cytokinin signaling pathway was also significant down regulated and the expression of stress response gene PagNAC3 and POD synthesis gene POD1 were significantly up regulated; meanwhile the PagHK3a knockout lines had stronger gas exchange ability, lower MDA and H₂O₂ content and larger plant height growth under moderate drought conditions, therefore had stronger drought stress resistance than WT.