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.