Categorizing developmental processes of embryo and endosperm based on gene expression pattern
To further investigate the relationship between developmental stages and gene expression pattern, we performed PCA on the complete dataset of embryo and endosperm (Figure 2d). PCA distinguished the samples into two distinct groups based on tissue identity, validating the purity of our tissue samples. Particularly, the first component (PC1; 77.1% variance explained) clearly separated the embryo from endosperm samples, while the second component (PC2; 12.6% variance explained) discriminated the stages of development for all of the tissues.
As the PCA proposed the specificity of gene expression across the tissues and developmental stages, we further classified the transcriptomic data into a dendrogram by hierarchical clustering (HCL) analysis, and uncovered four distinguishing groups within embryo and endosperm, with each group corresponding to a specific developmental stage (Figure 2e-h).
For embryo samples, both genotypes showed an almost identical dendrogram (Figure 2e, f). The first cluster is formed at 5 DAF, representing the stage around differentiation (Em-S1). During this stage, coleoptile and SAM emerge and first leaf primordium becomes visible on the opposite side of the coleoptiles, as visualized by electron microscopy (Figure S4). Similarly, another adjacent cluster from 10-15 DAF represents the embryo enlargement stage (Em-S2), as characterized by the formation of second and third leaf primordia, and organ enlargement (particularly the scutellum). A third cluster at 20DAF corresponds to the maturation phase (Em-S3) that shows no obviously morphological change. The fourth cluster from 25 DAF to 60 DAF represents the period of seed dormancy (Em-S4).
By contrast, the duration of developmental stage in endosperm varied with genotypes. Firstly, endosperms from WT showed four primary clusters (Figure 2g, h), but those from NB showed only three clusters, suggesting abnormal development during middle and later stages (Figure 2i, j). Secondly, within the endosperm, the upper and bottom parts of WT showed identical duration. By contrast, for NB, the time span of endosperm filling in the bottom endosperm (NB_EnB) was prolonged (15-25 DAF), as compared to the upper endosperm (NB-EnU) that had the identical duration of second cluster (15-20 DAF) to the WT. Consequently, the maturation and dormancy stage were delayed in the bottom endosperm and displayed as a merged cluster (30-60 DAF) of stages 3 and 4, as compared to the upper part, where stages 3 and 4 cluster had the same duration (25-60 DAF) as WT. Collectively, these findings indicate a disturbance in the developmental process of the bottom endosperm of NB by its proximity to the embryo.
Therefore, normal type of WT instead of the disturbed NB is used for a general description of the developmental signatures of rice grain. The four clearly defined clusters in WT endosperm showed that the differentiation (En-S1) stage last from 5-10 DAF in which it completes the cellularization, with the aleurone cells starting to become morphologically distinct from the starchy endosperm (Figure S4). Then storage accumulation stage (En-S2) occurs at 15-20 DAF, followed by the maturation (En-S3) phase of the endosperm at 25-30 DAF. The biological processes involved in dormancy (En-S4) are induced from 45-60 DAF. Altogether, these results suggested a divergent staging mechanism of developmental characteristics between embryo and endosperm based on transcriptomic data.