A novel method to qualify the effect of embryo on endosperm development
As shown in Figure 2, the duration of middle stage in the bottom part of the NB grain (Figure 2j) was prolonged relative to that of its upper counterpart (Figure 2i) as well as those of the WT (Figure 2g, h), indicating a strong influence of the embryo on the bottom endosperm proximate to it. To quantitively evaluate the embryo effect, we developed a novel comparison system by comparing the upper and bottom endosperms of NB, using WT as a reference (Figure 6).
Briefly, this system has three key components: (i) Position effect. The comparison between the upper (WT_EnU) and bottom (WT_EnB) endosperms of WT reflects the difference in position between upper and bottom endosperms within the WT grain, where nutrients and signals move freely without being blocked by the notched line as in NB (Figure 6a). (ii) Compound effect. Due to the notched line, movement of nutrients and signals are severely restricted between the two endosperms, trapping the influence of the embryo in the bottom endosperm. Comparison between the bottom part (NB_EnB) and upper part (NB_EnU) of the NB grain shows the compound effect of position and embryo (Figure 6b). (iii) Embryo effect. Finally, by eliminating the position effect, we can precisely calculate the influence of embryo on the endosperm via NB(EnB/EnU)/ WT(EnB/EnU) (Figure 6c).
To exemplify the working principle of this comparison method, we selected three metabolites including glutelin, asparagine (Asn), and Zn to show the influence of embryo (Figure 6). Firstly, taking in account the position effect and compound effect, the up-regulation of Asn and down-regulation of glutelin and Zn was observed in both genotypes. However, when eliminating the effect of position, embryo has a positive effect on the content of Zn and a negative effect Asn and glutelin, verifying the influence of embryo on endosperm filling.