tae-miR399-UBC24 module regulates starch degradation inArabidopsis thaliana in response to freezing stress
Pi can regulate plant carbohydrate metabolism, for example, in the dark, phosphorus deficient Arabidopsis will reduce photosynthesis, increase starch and sucrose content (Ciereszko, Johansson, & Kleczkowski, 2005; Muller, Morant, Jarmer, Nilsson, & Nielsen, 2007). OsSPX1 is a Pi-dependent OsPHR2 activity inhibitor, which plays an important role in the Pi signal pathway of rice (Z. Y. Wang et al., 2014), and the decreased expression of OsSPX1 affects the expression of genes involved in carbohydrate metabolism and sugar transport (K. Zhang et al., 2016). Starch degradation is involved in cold-induced soluble sugar accumulation (Ruelland et al., 2009). In this study, at 4°C and -10°C, the starch content of OEmiR399 Arabidopsis plants was significantly lower than that of WT, while the soluble sugar content was significantly higher than that of WT, which indicated that overexpressing tae-miR399 at low temperature promoted starch degradation in Arabidopsis (Fig. 9A and B). The amylase in plants is mainly divided into α-amylase and β-amylase. In this study, during cold acclimation, the activity of α-amylase and β-amylase in OEmiR399 Arabidopsis plants were higher than that in WT (Fig. 10A and B). However, during freezing stress, only β-amylase activity of OEmiR399 Arabidopsis plants was higher than that of WT (Fig. 10B). These results indicates that during cold acclimation, overexpressing tae-miR399 can promote both α-amylase and β-amylase activities to regulate starch degradation in Arabidopsis, while in freezing stress, overexpressing tae-miR399 only promote β-amylase to mediate starch degradation in Arabidopsis. It is worth noting that the expression of AtAPL3 and AtBAM3 in OEmiR399 Arabidopsis plants were similar to the changes in its own enzyme activity (Fig. 10D and F). These results indicate that the activities of α-amylase and β-amylase in OEmiR399 Arabidopsis plants may be mainly regulated byAtAPL3 and AtBAM3 . Starch phosphorylation is another way to degrade starch. It has been controversy about whether starch phosphorylase is involved in the starch degradation under low temperature stress. At 2°C, it was no significant change in the enzyme activity of starch phosphorylase in Arabidopsis thaliana (R. Yano, Nakamura, Yoneyama, & Nishida, 2005). However, the study by (Berrocal-Lobo et al., 2011) showed that the transcription level and protein level of SEX4 were significantly up-regulated at 4°C, which is consistent with the starch degradation trend. In this study, we found that AtSEX1 did not participate in the regulation of starch degradation in Arabidopsis under low temperature stress, but the expression of AtSEX4 in OEmiR399 Arabidopsis plants was induced during the cold acclimation (Fig. 10G and H). These results suggest that during cold acclimation, maybe starch phosphorylase also participate in the starch degradation of Arabidopsis.