Identification of DEMETER-like DNA demethylase gene family in citrus and their role in drought stress-adaptive responses.

DEMETER-Like DNA demethylases (DMLs) are epigenetic regulators of many developmental and biological processes in plants. No comprehensive information about the DML gene family in citrus is available to date. Here, a total of three DML genes in the genomes of Citrus sinensis (named CsDML1-3) and C. clementina (named CcDML1-3) were identified and analyzed. They encode hydrophilic and relatively large proteins, with prediction of nuclear localization, containing the conserved domains and motifs typical of plant DMLs. Protein interaction network analysis suggested that they interact primarily with proteins related to the maintenance of DNA methylation and remodeling of chromatin. Analysis of their promoter regions led to the identification of several cis-acting regulatory elements involved in stress response, including drought, heat and cold stresses. The presence of several miRNA targets and potential phosphorylation sites suggest that their expression is also regulated at post-transcriptional and post-translational levels. RNA-Seq data and quantitative real-time PCR analysis showed a low and drought-regulated gene expression of the citrus DMLs in different plant tissues. CsDML1 and CsDML3 were also differentially regulated by deficit irrigation in fruits at different developmental stages, with a positive and significant correlation found between CsDML1 and PHYTOENE SYNTHASE (PSY) and between CsDML3 and ATP CITRATE LYASEs (ACLs) and ZETA-CAROTENE DESATURASE (ZDS) gene expression. These results indicate that the citrus DMLs are potentially functional enzymes involved in developmental processes and drought stress-adaptive responses, providing a useful reference for further investigation of their functions and applications on the citrus improvement.

Genome-wide characterization and expression analysis of citrus NUCLEAR FACTOR-Y (NF-Y) transcription factors identified a novel NF-YA gene involved in drought-stress response and tolerance.

Nuclear factor Y (NF-Y) is a ubiquitous transcription factor found in eukaryotes. It is composed of three distinct subunits called NF-YA, NF-YB and NF-YC. NF-Ys have been identified as key regulators of multiple pathways in the control of development and tolerance to biotic and abiotic factors. The present study aimed to identify and characterize the complete repertoire of genes coding for NF-Y in citrus, as well as to perform the functional characterization of one of its members, namely CsNFYA5, in transgenic tobacco plants. A total of 22 genes coding for NF-Y were identified in the genomes of sweet orange (Citrus sinensis) and Clementine mandarin (C. clementina), including six CsNF-YAs, 11 CsNF-YBs and five CsNF-YCs. Phylogenetic analyses showed that there is a NF-Y orthologous in the Clementine genome for each sweet orange NF-Y gene; this was not observed when compared to Arabidopsis thaliana. CsNF-Y proteins shared the same conserved domains with their orthologous proteins in other organisms, including mouse. Analysis of gene expression by RNA-seq and EST data demonstrated that CsNF-Ys have a tissue-specific and stress inducible expression profile. qRT-PCR analysis revealed that CsNF-YA5 exhibits differential expression in response to water deficit in leaves and roots of citrus plants. Overexpression of CsNF-YA5 in transgenic tobacco plants contributed to the reduction of H2O2 production under dehydration conditions and increased plant growth and photosynthetic rate under normal conditions and drought stress. These biochemical and physiological responses to drought stress promoted by CsNF-YA5 may confer a productivity advantage in environments with frequent short-term soil water deficit.