Regulation of SlAGO1 by SlmiR-168a increased plant tolerance to K+ deficiency stress
To elucidate whether the regulation of SlAGO1 bySlmiR-168a was responsible for differences in low K+ tolerance, transformants of 35S:SlmiR-168aand 35S:rSlAGO1 were obtained. SlmiR-168a -resistant constructs (rSlAGO1 ) were generated using a point mutation (Fig. 3a). This mutation did not change the native protein sequence ofSlAGO1 . The rSlAGO1 and pre-SlmiR-168a fragments were amplified by PCR for overexpression vector construction (Fig. 3b and c). 35S:SlmiR-168a and 35S:rSlAGO1 transformants were obtained for subsequent experiments. The root morphology of WT,35S:SlmiR-168a , and 35S:rSlAGO1 all showed a larger root system after 7 days of development at the normal K+concentration (4 mM) (Fig. S5). However, in the presence of a low K+ concentration (0.5 mM), the roots of35S:SlmiR-168a plants appeared stronger than WT and35S:rSlAGO1 plants after 7 days of treatment. Microexamination revealed that the number of root hairs was obviously increased in35S:SlmiR-168a plants following low K+treatment at 7 days compared with that in WT and 35S:rSlAGO1plants (Fig. 4a). Leaf development was also observed under K+ deficiency stress (Fig. 4b). The leaf margins of JZ18 control plants turned yellow under low K+conditions, and those of 35S:rSlAGO1 plants showed increased yellowing; in contrast, 35S:SlmiR-168a plants did not exhibit yellowing of the leaves. Analysis of the root-shoot ratio (Fig. 4c) showed that under low K+ conditions, root-shoot ratios of 35S:SlmiR-168a plants did not differ significantly compared with that in WT plants. However, 35S:rSlAGO1 plants exhibited a decreased root-shoot ratio compared with WT and 35S:SlmiR-168aplants. Additionally, the chlorophyll content was highly increased in35S:SlmiR-168a plants but decreased in WT and 35S:rSlAGO1plants under K+ deficiency stress (Fig. 4d). Chlorophyll contents in 35S:rSlAGO1 plants were also lower than that in WT plants under both normal K+ and low K+ conditions. Analysis of K+contents in roots (Fig. 4e) showed that under normal K+ concentration conditions, 35S:SlmiR-168aplants showed increased K+ contents with development time, particularly after 7 days of treatment. 35S:SlmiR-168aplants also showed higher K+ contents than WT plants after 7 days, whereas 35S:rSlAGO1 plants exhibited lower K+ contents than WT plants. Under low K+ concentration conditions, 35S:SlmiR-168aplants exhibited improved root and leaf growth, whereas35S:rSlAGO1 showed weaker root and leaf growth. Moreover,35S:SlmiR-168a plants exhibited higher K+contents in roots under K+ deficiency stress.