Background Plants have the unique capability to form embryos from both gametes and somatic cells, with the latter process known as somatic embryogenesis. Somatic embryogenesis (SE) can be induced by exposing plant tissues to exogenous growth regulators or by the ectopic activation of embryogenic transcription factors. Recent studies have revealed that a discrete group of RWP-RK DOMAIN-CONTAINING PROTEIN (RKD) transcription factors act as key regulators of germ cell differentiation and embryo development in land plants. The ectopic overexpression of reproductive RKDs is associated with increased cellular proliferation and the formation of somatic embryo-like structures that bypass the need for exogenous growth regulators. However, the precise molecular mechanisms implicated in the induction of somatic embryogenesis by RKD transcription factors remains unknown. Results In silico analyses have identified a rice RWP-RK transcription factor, named Oryza sativa RKD3 (OsRKD3), which is closely related to Arabidopsis thaliana RKD4 (AtRKD4) and Marchantia polymorpha RKD (MpRKD) proteins. Our study demonstrates that the ectopic overexpression of OsRKD3, which is expressed preferentially in reproductive tissues, can trigger the formation of somatic embryos in an Indonesian black rice landrace (Cempo Ireng) that is normally resistant to somatic embryogenesis. By analyzing the transcriptome of induced tissue, we identified 5,991 genes that exhibit differential expression in response to OsRKD3 induction. Among these genes, 50% were up-regulated while the other half were down-regulated. Notably, approximately 37.5% of the up-regulated genes contained a sequence motif in their promoter region, which was also observed in RKD targets from Arabidopsis. Furthermore, OsRKD3 was shown to mediate the transcriptional activation of a discrete gene network, which includes several transcription factors such as APETALA 2-like (AP2-like)/ETHYLENE RESPONSE FACTOR (ERF), MYB and CONSTANS-like (COL), and chromatin remodeling factors associated with hormone signal transduction, stress responses and post-embryonic pathways. Conclusions Our data show that OsRKD3 modulates an extensive gene network and its activation is associated with the initiation of a somatic embryonic program that facilitates genetic transformation in black rice. These findings hold substantial promise for improving crop productivity and advancing agricultural practices in black rice.
Many studies have reported the great potency of black rice as functional food for human diet. Cempo Ireng is one of Indonesian black rice cultivars with the highest content of anthocyanin. However, it also suffers from long harvest period. This experiment aims at investigating the behavior of the flowering genes in order to gain basic information to develop this cultivar. We sampled the leaves' blades of Black Rice 'Cempo Ireng' at 48, 55, 68, 81, and 90 DAP then performed RNA isolation and cDNA synthesis, amplification of targeted flowering genes, and a semi-quantitative analysis to see the expression of flowering genes. Our results showed that the flowering genes Hd3a and RFT1 were redundantly up-regulated to induce flowering in black rice Cempo Ireng under a neutral day condition in a tropical region. We also noted that the patterns of FT-like genes and flowering regulatory genes including FT-L5, OsCOL4 were expressed together with two major flowering genes. FT-like genes were temporally co-expressed with two flowering genes Hd3a and RFT1, whereas the Hd1 had a unique expression pattern. Meanwhile, OsCOL4 as the flowering repressor was only detected in the early stage when the flowering gene Hd3a began to express. The results suggest that black rice Cempo Ireng has similar and conserved flowering pathway under a neutral day condition as indicated in the common rice flowering models.
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