| In this study, a model plant Arabidopsis thaliana was used as materials to study effects of high pH stress on Arabidopsis thaliana seedlings growth. The main results were obtained as follows:1. On the pH6.5 medium, the relative primary root growth was 12% higher compared to the controls. When the pH values were greater than 8.0, the growth of primary roots was completely inhibited. Therefore, the primary root of plant was more sensitive to the high alkali stress.2. We found that growth inhibition of the primary roots at severe high pH stress was caused by programmed cell death (PCD). By analyzing the death rate of Arabidopsis ecotype Col at pH8.0, we found that the death peak was detected at the third day of the treatment, where about 67.5% primary roots of the stressed plants were died. TUNEL and DNA ladder examination proved high pH induced cell death was PCD. The stress induced PCD may offer increased tolerance to adapt the adverse environment conditions by adjusting root system development.3. To further investigate the factors that mediate PCD, DAB staining was first conducted in primary roots of the Col seedlings. We discovered that H2O2 may participate in the PCD process, with increasing pH values, increased H2O2 staining appeared earlier. For example, when pH was 6.5, 7.0, 7.5 and 8.0, strong accumulation of H2O2 in the stressed plant roots was detected at 36h, 30h, 24h and 18h after treatment, respectively. The DAB staining was mainly at meristematic zone of the growth tips, this result was consistent with the results using TUNEL analysis. The results indicated that stress induced increase in active oxygen may play important roles in activating the processes of PCD in meristematic cells of Arabidopsis seedling primary roots. Under the pH8.0 condition, the genes related to active oxygen, like At3g03670, At4g08770, At3g49960 and At1g69920 were analyzed. The results showed that expression levels of these genes were increased. Moreover, transcription of these genes reached maximum at the time earlier than H2O2, further suggesting that the active oxygen species may be involved in Arabidopsis seedling primary root PCD process.4. To monitor the contents of cytokinin and auxin, the transgenic lines expressing ARR5::GUS and DR5::GUS were used. GUS staining results showed that cytokinin contents were not affected by high pH, suggesting that cytokinin may not involve in the response to high alkali. But the strong GUS staining was detected in the stressed plants possessing DR5::GUS construct. When pH was increased to 8.0, GUS activity was remarkably reduced with the prolonged treatment, and nearly no GUS staining was observed at 120 h after treatment, indicating that the auxin contents were reduced in seedling primary root tips under high alkali stress. Together, the data showed that auxin may play a critical role in plant adaption to high alkali stress.5. At 14 days after stressed on the pH8.0 medium, the Col leaves still maintained green, and the lateral roots and adventitious roots formed. It was likely that the Col seedlings remodeled the root system development by PCD of the primary roots, conferring the ability of the plants to adapt the stress environment.In addition, when transferred the stresses plants to normal conditions, increased numbers of lateral roots and adventitious roots were also observed. The number of lateral roots of the Col seedling was nearly 50% more than that of the control plants. The results suggests that the seedlings, after the high alkali stress signal was sensed, could effectively activate a signaling cascade to adjust themselves to adapt the stress conditions at various levels, such as gene expression, PCD and induction of lateral roots etc.Our results discovered a new adaptive mechanism for the plants in response to the high pH stress. The findings would provide novel insights into how the plants response and adapt to the alkaline conditions, it will be helpful for our further research on crop improvement for enhanced tolerance in alkali and saline conditions.
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