INTRODUCTION
The seed (grain) of rice (Oryza sativa ) is a complex but delicate biological system, containing three genetically distinct tissues, the diploid embryo, the triploid endosperm, and the diploid maternal tissues (An et al., 2020). Rice is a main contributor of dietary calories and nutrients for nearly half of the global population (Wu, Müller, Gruissem, & Bhullar, 2020). Rice grain yield has to be sustainably improved both in quantity and quality, to solve formidable challenges including the ever-increasing population, climate change, and the quest for high-quality rice that has accompanied the rise in living standards (Sreenivasulu et al., 2015). A detailed understanding of the molecular and physiological mechanisms underpinning seed development may enable the design of effective strategies to boost rice quality and yield.
Milled rice is the main form of rice for consumption produced by milling during which the outer maternal layers, the embryo, the aleurone, and part of the starchy endosperm are removed. End use quality of rice seed is dominated by the physico-chemical properties of endosperm that are predominately packed with starch granules and protein bodies (Xi et al., 2014). Grain chalkiness affects grain quality due to perturbation of carbohydrate metabolism (Nakata et al., 2017) or protein synthesis (Ishimaru et al. 2019; Wada et al., 2019). Up to now, the majority of research on rice quality has been centered on the starchy endosperm, revealing that it is a rather sophisticated trait controlled by multiple genes and by environmental conditions (Fitzgerald, McCouch, & Hall, 2009; Ishimaru et al., 2019; Lin et al., 2017).
The composition, structure, and size of endosperm are affected by its interactions with other tissues, in particular the embryo, during the whole process of seed development. Endosperm provides nutrients to drive embryo growth, and physically restrains its size and development. The embryo is the germline component of the seed, and affects the allocation of sugars and other nutrients like amino acids and minerals to the endosperm. Concomitant development of embryo and endosperm under the constraint of maternal tissues (seed coat or pericarp) requires coordination of the two compartments (Doll et al., 2020; Lafon-Placette & Köhler, 2014). Growing evidence in rice as well as Arabidopsis (Arabidopsis thaliana ) and maize (Zea mays ) supports bidirectional communication between embryo and endosperm, as reviewed by Lafon-Placette and Köhler (2014); An et al. (2020) and Ingram (2020). Therefore, both embryo and endosperm and their interaction affect grain filling and quality.
Transcriptome analyses of seed development have already identified most genes expressed in the different compartments (Olsen, 2020). These comprehensive studies provided insights into the molecular networks and pathway interactions that function during the development of individual seed compartments for Arabidopsis (Belmonte et al., 2013), maize (Chen et al., 2014; Yi et al., 2019), wheat (Triticum aestivum ; Xiang et al., 2019), and barley (Hordeum vulgare ; Bian et al., 2019). In rice, Ishimaru et al. (2019) isolated the aleurone, dorsal, central and lateral tissues of developing endosperm by laser-capture microdissection (LCM), and profiled the gene expression by a 44 K microarray, revealing that high molecular weight heat shock proteins have a role in mediating redox, nitrogen and amino acid metabolism in the chalky tissue under heat stress. Wu et al. (2020) profiled gene expression activity in the nucellar epidermis, ovular vascular trace, endosperm and the aleurone at three distinct grain development stages, offering insights into the molecular aspects of grain development. Ram et al. (2020) employed an rapid LCM approach to individually collect pericarp, aleurone, embryo and endosperm at 10 DAF (days after anthesis). Subsequent RNA-Seq analysis identified 7760 differentially expressed genes, by which the authors built some key tissue-specific pathways responsible for nutrient partitioning.
However, these studies do not enable an integrated understanding of the cellular processes governing the formation of rice grain, because of the scarcity of information on embryo-endosperm communication and its influence on rice yield and quality, which is partly attributed to the complexity of seed structures (Doll et al., 2020).
Notched-belly grain is a misshapen type of rice grain, having a notched-line on its ventral side and thus being inferior in appearance and milling quality (Tong et al., 2018). Previously, we identified a notched-belly mutant (NB) of a japonica rice Wuyujing 3 using a chemical mutagen (Lin et al., 2014). It has high occurrence of white-belly grains, and the notched line is visible at 5 DAF, which separates the endosperm into two sub-compartments, the translucent upper part and the chalky bottom. Using this mutant, we developed a novel comparison system that can evaluate the embryo effect on endosperm composition, with a conclusion that embryo demonstrated a negative effect on the contents of proteins, amino acids, and minerals in the chalky endosperm (Lin et al., 2016). Complementary proteome and transcriptome profiling of the NB mutant suggested the transporters for metabolites and the signal messengers of hormones as potential signals for embryo-endosperm communications, confirming the value of the NB mutant for examining the role of embryo in grain filling and quality formation (Lin et al., 2017).
In this study, we used the NB mutant to characterize the genome-wide gene expression profile of embryo and endosperm samples between 0-60 DAF. In combination with physiological and histological investigations, we aimed to: (i) unravel the commonalities and specificities between embryo and endosperm in developmental processes as well as molecular and metabolic signatures; (ii) manifest the effect of embryo on endosperm development; and (iii) frame a holistic and dynamic picture of seed development by integrating the information of embryo-endosperm crosstalk. Comprehensive comparison of mRNAs datasets revealed a dragging effect of embryo on the developmental transition of the endosperm, which might be mediated by hormonal (GA and IAA) and T6P-SnRK1 signaling pathways. A new staging system is subsequently proposed by integrating the information about embryo-endosperm bidirectional communications. The findings obtained should offer insights into the biological processes and signatures of grain formation, hence providing a valuable resource for the manipulation of molecular and physiological processes responsible for cereal grain yield and quality.