Starch degradation are affected in OEmiR399 transgenic Arabidopsis plants under freezing stress
The soluble sugar contents of Arabidopsis llines were analysed. The results showed that at low temperature, the soluble sugar contents of OEmiR399 transgenic Arabidopsis plants were markedly higher than that of WT (Fig. 9A). The results of starch contents showed that at low temperature, the starch contents of OEmiR399 transgenic Arabidopsis plants were significantly lower than that of WT(Fig. 9B). These findings implied that the higher soluble sugar contents in OEmiR399 transgenic Arabidopsis plants were accumulated through degrading starch. To further validated tae-miR399 fuction on starch degradation under freezing stress, we detected the amylase activities in Arabidopsis lines. The results showed that α-amylase activity of OEmiR399 transgenic Arabidopsis plants was only higher than that of WT at 4℃ (Fig. 10A). Meanwhile, not only β-amylase activity of OEmiR399 transgenic Arabidopsis plants was higher than that of WT at 4℃, but also significantly higher than that of WT at -10℃ (Fig. 10B). The expressions of genes encoding α-amylase (AtAPL1 andAtAPL3 ) and β-amylase (AtBAM1 and AtBAM3 ) were also detected, the results showed that low temperature observably induced the expression of amylase genes in all Arabidopsis lines (Fig. 10C, D, E and F). However, not all of these amylase genes were affected by overexpression of tae-miR399. Before cold treatment, only the expression of AtAPL1 in OEmiR399 transgenic Arabidopsis plants was higher than that in WT(Fig. 10C). At 4℃,the expressions of all amylase genes in OEmiR399 transgenic Arabidopsis plants were higher than that of WT with variety degrees (Fig. 10). At -10℃, only the expression of AtBAM3 in OEmiR399 transgenic Arabidopsis was higher than that in WT, while the expressions ofAtAPL1 and AtBAM1 slightly lower than that of WT (Fig. 10C, E and F). To explore whether GWD in OEmiR399 transgenic Arabidopsis also regulates starch metabolism under freezing stress, we detected the expression of its coding genes AtSEX1 and AtSEX4 in Arabidopsis lines. The results showed that under low temperature stress, the expression of AtSEX1 in all Arabidopsis lines was not induced (Fig. 10G). And there was no significant difference in the expression level of AtSEX1 among all Arabidopsis lines (Fig. 10G). The expression of AtSEX4 was different from that of AtSEX1 . At low temperature, the expression of AtSEX4 in all Arabidopsis lines was significantly up-regulated (Fig. 10H). And at 24°C and 4°C, the expression of AtSEX4 in OEmiR399 transgenic Arabidopsis plants was significantly higher than that in WT(Fig. 10H). However, at -10°C, there was no significant difference in the expression level ofAtSEX4 between WT and OEmiR399 lines(Fig. 10H).