Sugars and starch
During embryo and endosperm development, sucrose content gradually increased, peaking at 15 DAF and then decreased thereafter (Figures 4a, S6a). Glucose and fructose levels decreased during seed development (Figures S7a, S7b, S8a, S8b). The ratio of glucose to sucrose mediates endosperm differentiation (Olsen, 2020). It was higher in the embryo and endosperm at 5-10DAF, and then decreased gradually (Figures S7c, S8c), corresponding to the transition from differentiation to storage accumulation. These sugars were unevenly distributed in rice seed, with the embryo generally having higher contents (Figures 4a, S6a, S7a, S7b, S8a, S8b). Genes participating in sugar metabolism were differentially expressed between embryo and endosperm. For the five genes encoding sucrose synthase, two of them (OsSUS1 and OsSUS6 ) were mainly expressed in embryo, while another three (OsSUS2 ,OsSUS3 , OsSUS4 ) in endosperm (Figures 4a, S6a).
During grain filling, starch content increased gradually, peaking at 20 DAF and 30 DAF in the embryo and endosperm, respectively (Figures 4b, S6b). Compared with embryo, endosperm contained higher starch content, which is consistent with previous report (Juliano & Bechtel, 1985). Starch synthesis-related genes were coordinately expressed in the embryo and endosperm, similar to that in wheat (Xiang et al., 2019). Moreover, these genes were primarily expressed in endosperm at 5-20 DAF. By contrast, they displayed a gradual increase in embryo at 5-20 DAF.
T6P and its target protein SnRK1
Trehalose-6-phospate (T6P) signals the availability of sucrose in plant cells through the feast-famine kinase, SnRK1, which targets the genes responsible for metabolism, growth, and development (Dingkuhn et al., 2020; O’Hara, Paul, & Wingler, 2013; Paul, Oszvald, Jesus, Rajulu, & Griffiths, 2017). To better understand the role of this signaling pathway in rice seed, we analyzed T6P content and SnRK1 activities (Figures 4c, 4d, S6c, S6d). T6P level in embryo and endosperm showed a gradual decrease in the development course. Compared with endosperm, embryo has higher content at early stage (5-10DAF) (Figures 4c, S6c). For example, at 5 DAF, T6P was 3.98 and 6.61 nmol g−1DW in endosperm and embryo, respectively. In addition, TPS genes were expressed in both embryo and endosperm, being predominantly expressed at 5-20 DAF.
The SnRK1 activity in endosperm maintained a low level at 5-20 DAF, and then increased rapidly at later stages (25-60 DAF). Different from the endosperm, SnRK1 in the embryo was at a lower level, being higher only at 5 DAF (Figures 4d, S6d). SnRK1A was specifically expressed in the embryo, while two SnRK1B genes (SnRK1B andOSK35 ) were mainly expressed in endosperm. In addition,SnRK1A was broadly expressed across the time points in embryo. By contrast, SnRK1B and OSK35 were more prevalent in endosperm at 5-20 DAF. Interestingly, in contrast to the strong correlations between the actual level of T6P and the activity of its regulating genes, those for the SnRK1 activity showed no significant correlation, indicating a strong post-translational regulation.