RESUMO
Ran GTPases play essential roles in plant growth and development. Our previous studies revealed the nuclear localization of DlRan3A and DlRan3B proteins and proposed their functional redundancy and distinction in Dimocarpus longan somatic embryogenesis, hormone, and abiotic stress responses. To further explore the possible roles of DlRan3A and DlRan3B, gene expression analysis by qPCR showed that their transcripts were both more abundant in the early embryo and pulp in longan. Heterologous expression of DlRan3A driven by its own previously cloned promoter led to stunted growth, increased root hair density, abnormal fruits, bigger seeds, and enhanced abiotic stress tolerance. Conversely, constitutive promoter CaMV 35S (35S)-driven expression of DlRan3A, 35S, or DlRan3B promoter-controlled expression of DlRan3B did not induce the alterations in growth phenotype, while they rendered different hypersensitivities to abiotic stresses. Based on the transcriptome profiling of longan Ran overexpression in tobacco plants, we propose new mechanisms of the Ran-mediated regulation of genes associated with cell wall biosynthesis and expansion. Also, the transgenic plants expressing DlRan3A or DlRan3B genes controlled by 35S or by their own promoter all exhibited altered mRNA levels of stress-related and transcription factor genes. Moreover, DlRan3A overexpressors were more tolerant to salinity, osmotic, and heat stresses, accompanied by upregulation of oxidation-related genes, possibly involving the Ran-RBOH-CIPK network. Analysis of a subset of selected genes from the Ran transcriptome identified possible cold stress-related roles of brassinosteroid (BR)-responsive genes. The marked presence of genes related to cell wall biosynthesis and expansion, hormone, and defense responses highlighted their close regulatory association with Ran.
RESUMO
DNA methylation plays essential roles in gene regulation, chromatin structure stability, gene imprinting, X chromosome inactivation and embryonic development. However, the dynamics and functions of DNA methylation during the early stage of longan (Dimocarpus longan) somatic embryogenesis (SE) are still unclear. In this study, we carried out whole genome bisulphite sequencing and transcriptome sequencing analyses for embryogenic callus (EC), incomplete compact pro-embryogenic cultures (ICpEC) and globular embryos (GE) in an early SE system. At a global level, the DNA 5-methylcytosine content in EC, ICpEC and GE was 24.59, 19.65 and 19.74%, respectively, suggesting a global decrease in DNA methylation from EC to ICpEC and then a slight increase from ICpEC to GE. Differentially methylated region (DMR) analysis showed that hypomethylation mainly occurred in CHH contexts. Gene ontology and Kyoto encyclopedia of genes and genomes analysis of hypomethylated-CHH-DMR-associated genes revealed that zein biosynthesis, fatty acid biosynthesis, circadian rhythm and mitophagy pathways were involved in longan early SE. Expression patterns of DNA methyltransferase and demethylase genes during longan early SE suggested that the decrease in DNA methylation was probably regulated by DNA methyltransferase genes and the DNA demethylase gene REPRESSOR OF SILENCING 1 (ROS1). The correlation between DNA hypomethylation and gene expression revealed that decreased DNA methylation did not cause extensive changes in gene expression during early longan SE and that gene expression may be affected by methylation changes in gene and downstream regions. Inhibiting DNA methylation with 5-azacytidine treatment in EC promoted the formation of GE and enhanced the capability of longan SE. Our results suggest that DNA demethylation has important roles in longan SE development.