Transcriptomic analysis reveals the role of FOUR LIPS in response to salt stress in rice.

KEY MESSAGE: An R2R3-MYB transcription factor FOUR LIPS associated with B-type Cyclin-Dependent Kinase 1;1 confers salt tolerance in rice. The Arabidopsis FOUR LIPS (AtFLP), an R2R3 MYB transcription factor, acts as an important stomatal development regulator. Only one orthologue protein of AtFLP, Oryza sativa FLP (OsFLP), was identified in rice. However, the function of OsFLP is largely unknown. In this study, we conducted RNA-seq and ChIP-seq to investigate the potential role of OsFLP in rice. Our results reveal that OsFLP is probably a multiple functional regulator involved in many biological processes in growth development and stress responses in rice. However, we mainly focus on the role of OsFLP in salt stress response. Consistently, phenotypic analysis under salt stress conditions showed that osflp exhibited significant sensitivity to salt stress, while OsFLP over-expression lines displayed obvious salt tolerance. Additionally, Yeast one-hybrid assay and electrophoretic mobility shift assay (EMSA) showed that OsFLP directly bound to the promoter region of Oryza sativa B-type Cyclin-Dependent Kinase 1;1 (OsCDKB1;1), and the expression of OsCDKB1;1 was repressed in osflp. Disturbing the expression of OsCDKB1;1 remarkably enhanced the tolerance to salt stress. Taken together, our findings reveal a crucial function of OsFLP regulating OsCDKB1;1 in salt tolerance and largely extend the knowledge about the role of OsFLP in rice.

Arabidopsis F-BOX STRESS INDUCED 4 is required to repress excessive divisions in stomatal development.

During the terminal stage of stomatal development, the R2R3-MYB transcription factors FOUR LIPS (FLP/MYB124) and MYB88 limit guard mother cell division by repressing the transcript levels of multiple cell-cycle genes. In Arabidopsis thaliana possessing the weak allele flp-1, an extra guard mother cell division results in two stomata having direct contact. Here, we identified an ethylmethane sulfonate-mutagenized mutant, flp-1 xs01c, which exhibited more severe defects than flp-1 alone, producing giant tumor-like cell clusters. XS01C, encoding F-BOX STRESS-INDUCED 4 (FBS4), is preferentially expressed in epidermal stomatal precursor cells. Overexpressing FBS4 rescued the defective stomatal phenotypes of flp-1 xs01c and flp-1 mutants. The deletion or substitution of a conserved residue (Proline166) within the F-box domain of FBS4 abolished or reduced, respectively, its interaction with Arabidopsis Skp1-Like1 (ASK1), the core subunit of the Skp1/Cullin/F-box E3 ubiquitin ligase complex. Furthermore, the FBS4 protein physically interacted with CYCA2;3 and induced its degradation through the ubiquitin-26S proteasome pathway. Thus, in addition to the known transcriptional pathway, the terminal symmetric division in stomatal development is ensured at the post-translational level, such as through the ubiquitination of target proteins recognized by the stomatal lineage F-box protein FBS4.

DNA methylation is involved in the regulation of pepper fruit ripening and interacts with phytohormones.

There is growing evidence to suggest that epigenetic tags, especially DNA methylation, are critical regulators of fruit ripening. To examine whether this is the case in sweet pepper (Capsicum annuum) we conducted experiments at the transcriptional, epigenetic, and physiological levels. McrBC PCR, bisulfite sequencing, and real-time PCR demonstrated that DNA hypomethylation occurred in the upstream region of the transcription start site of some genes related to pepper ripening at the turning stage, which may be attributed to up-regulation of CaDML2-like and down-regulation of CaMET1-like1, CaMET1-like2, CaCMT2-like, and CaCMT4-like. Silencing of CaMET1-like1 by virus-induced gene silencing led to DNA hypomethylation, increased content of soluble solids, and accumulation of carotenoids in the fruit, which was accompanied by changes in expression of genes involved in capsanthin/capsorubin biosynthesis, cell wall degradation, and phytohormone metabolism and signaling. Endogenous ABA increased during fruit ripening, whereas endogenous IAA showed an opposite trend. No ethylene signal was detected during ripening. DNA hypomethylation repressed the expression of auxin and gibberellin biosynthesis genes as well as cytokinin degradation genes, but induced the expression of ABA biosynthesis genes. In mature-green pericarp, exogenous ABA induced expression of CaDML2-like but repressed that of CaCMT4-like. IAA treatment promoted the transcription of CaMET1-like1 and CaCMT3-like. Ethephon significantly up-regulated the expression of CaDML2-like. Treatment with GA3 and 6-BA showed indistinct effects on DNA methylation at the transcriptional level. On the basis of the results, a model is proposed that suggests a high likelihood of a role for DNA methylation in the regulation of ripening in the non-climacteric pepper fruit.