NACs are plant-specific transcription factors (TFs) involved in multiple aspects of development and stress. In rice, three NAC TF encoding genes, namely ONAC020, ONAC026 and ONAC023 express specifically during seed development, at extremely high levels. They exhibit significantly strong association with seed size/weight with the sequence variations located in the upstream regulatory region. Concomitantly, their expression pattern/levels during seed development vary amongst different accessions with variation in seed size. The alterations in the promoter sequences of the three genes, amongst the five rice accessions, correlate with the expression levels to a certain extent only. In terms of transcriptional properties, the three NAC TFs can activate and/or suppress downstream genes, though to different extents. Only ONAC026 is localized to the nucleus while ONAC020 and ONAC023 are targeted to the ER and cytoplasm, respectively. Interestingly, these two proteins interact with ONAC026 and the dimers localize in the nucleus. Trans-splicing between ONAC020 and ONAC026 results in three additional forms of ONAC020. The transcriptional properties including activation, repression, subcellular localization and heterodimerization of trans-spliced forms of ONAC020 and ONAC026 are different, indicating towards their role as competitors. The analysis presented in this paper helps to conclude that the three NAC genes, which are associated with seed size, have independent as well as overlapping roles during the process and can be exploited as potential targets for crop improvement.

NAC transcription factors (TFs) are known for their role in development and stress. This article attempts to functionally validate the role of rice SS1/ ONAC025 (LOC_Os11g31330) during seed development. The gene is seed‐specific and its promoter directs reporter expression in the developing endosperm and embryo in rice transgenic plants. Furthermore, rice transgenic plants ectopically expressing SS1/ ONAC025 have a plantlet lethal phenotype with hampered vegetative growth, but increased tillers and an altered shoot apical meristem structure. The vegetative cells of these plantlets are filled with distinct starch granules. RNAseq analysis of two independent plantlets reveals the differential expression of reproductive and photosynthetic genes. A comparison with seed development transcriptome indicates differential regulation of many seed‐related genes by SS1/ ONAC025. Genes involved in starch biosynthesis, especially amylopectin and those encoding seed storage proteins, and regulating seed size are also differentially expressed. In conjunction, SS1/ ONAC025 shows highest expression in japonica rice. As a TF, SS1/ ONAC025 is a transcriptional repressor localized to endoplasmic reticulum and nucleus. The article shows that SS1/ ONAC025 is a seed‐specific gene promoting grain filling in rice, and negatively affecting vegetative growth.

Many quantitative trait loci (QTLs) have been identified by molecular genetic studies which control grain size by regulating grain width, length, and/or thickness. Grain width 2 (GW2) is one such QTL that codes for a RING-type E3 ubiquitin ligase and increases grain size by regulating grain width through ubiquitin-mediated degradation of unknown substrates. A natural variation (single-nucleotide polymorphism at the 346^th position) in the functional domain-coding region of OsGW2 in japonica rice genotypes has been shown to cause an increase in grain width/weight in rice. However, this variation is absent in indica rice genotypes. In this study, we report that reduced expression of OsGW2 can alter grain size, even though natural sequence variation is not responsible for increased grain size in indica rice genotypes. OsGW2 shows high expression in seed development stages and the protein localizes to the nucleus and cytoplasm. Downregulation of OsGW2 by RNAi technology results in wider and heavier grains. Microscopic observation of grain morphology suggests that OsGW2 determines grain size by influencing both cell expansion and cell proliferation in spikelet hull. Using transcriptome analysis, upregulated genes related to grain size regulation have been identified among 1,426 differentially expressed genes in an OsGW2_RNAi transgenic line. These results reveal that OsGW2 is a negative regulator of grain size in indica rice and affects both cell number and cell size in spikelet hull.