The NAC gene family is one of the largest families of transcriptional regulators in plants, and it plays important roles in the regulation of growth and development as well as in stress responses. Genome-wide analyses have been performed in diverse plant species, but there is still no systematic analysis of the NAC genes of Coffea canephora Pierre ex A. Froehner. In this study, we identified 63 NAC genes from the genome of C. canephora. The basic features and comparison analysis indicated that the NAC gene members increased via duplication events during the evolution of the plant. Phylogenetic analysis divided the NAC proteins from C. canephora, Arabidopsis and rice into 16 subgroups. Analysis of the expression patterns of CocNACs under cold stress and coffee bean development indicated that 38 CocNACs were differentially expressed under cold stress; six genes may play important roles in the process of cold acclimation, and four genes among 54 CocNACs showing a variety of expression patterns during different developmental stages of coffee beans may be positively related to the bean development. This study can expand our understanding of the functions of the CocNAC gene family in cold responses and bean development, thereby potentially intensifying the molecular breeding programs of Coffea spp. plants.

WRKY transcription factors are known to play roles in diverse stress responses in plants. Low temperatures limit the geographic distribution of Coffea canephora Pierre ex A.Froehner. The WRKYs of C. canephora are still not well characterized, and the response of C. canephora WRKYs (CcWRKYs) under cold stress is still largely unknown. We identified 49 CcWRKYs from the C. canephora genome to gain insight into these mechanisms. These CcWRKYs were divided into three groups that were based on the conserved WRKY domains and zinc-finger structure. Gene expression analysis demonstrated that 14 CcWRKYs were induced during the cold acclimation stage, 17 CcWRKYs were preferentially upregulated by 4 °C treatment, and 12 CcWRKYs were downregulated by cold stress. Subsequently, we carried out a genome-wide analysis to predict 14,513 potential CcWRKY target genes in C. canephora. These isolated genes were involved in multiple biological processes, and most of them could be grouped by the response to stimulus. Among the putative CcWRKY target genes, 235 genes were categorized into response to the cold process, including carbohydrate metabolic, lipid metabolic, and photosynthesis process-related genes. Furthermore, the qRT-PCR and correlation analysis indicated that CcWRKY might control their putative targets that respond to cold stress. These results provide a basis for understanding the molecular mechanism for CcWRKY-mediated cold responses.

WUSCHEL-related homeobox (WOX) genes play significant roles in plant development and stress responses. Difficulties in somatic embryogenesis are a significant constraint on the uniform seedling production and genetic modification of Coffea arabica, hindering efforts to improve coffee production in Yunnan, China. This study comprehensively analyzed WOX genes in three Coffea species. A total of 23 CaWOXs, 12 CcWOXs, and 10 CeWOXs were identified. Transcriptomic profile analysis indicated that about half of the CaWOX genes were actively expressed during somatic embryogenesis. The most represented CaWOXs were CaWOX2a, CaWOX2b, CaWOX8a, and CaWOX8b, which are suggested to promote the induction and development of the embryogenic callus, whereas CaWOX13a and CaWOX13b are suggested to negatively impact these processes. Co-expression analysis revealed that somatic embryogenesis-related CaWOXs were co-expressed with genes involved in embryo development, post-embryonic development, DNA repair, DNA metabolism, phenylpropanoid metabolism, secondary metabolite biosynthesis, and several epigenetic pathways. In addition, qRT-PCR showed that four WOX genes responded to cold stress. Overall, this study offers valuable insights into the functions of CaWOX genes during somatic embryogenesis and under cold stress. The results suggest that certain WOX genes play distinct regulatory roles during somatic embryogenesis, meriting further functional investigation. Moreover, the cold-responsive genes identified here are promising candidates for further molecular analysis to assess their potential to enhance cold tolerance.

In numerous coffee-producing areas, coffee plants are routinely exposed to a low chilling temperature on a seasonal cycle. Despite the well-established significance of NAC transcription factors in mediating plant responses to abiotic stresses, their functions in Coffea arabica remain underexplored. This study identified 161 CaNAC genes and classified them into 15 distinct subgroups distributed across 22 chromosomes, with chromosome 11 harboring the largest number of these genes. Furthermore, a total of 1077 cis-elements were detected in the promoter regions of the 161 CaNAC genes. Among these, MYB-binding sites and ABA-responsive elements (ABREs) were the most prevalent. RNA-seq analysis under chilling stress revealed 16,767 differentially expressed genes, which were grouped into four clusters. GO enrichment analysis highlighted biological processes such as the abscisic acid-activated signaling pathway, response to cold, and response to salicylic acid, providing fundamental insights into the transcriptional response of C. arabica to chilling stress. Expression pattern analysis of CaNACs under chilling stress showed that 38 CaNACs were differentially expressed; 15 genes, including CaNAC46/49/116/125, were downregulated, while 12 genes, including CaNAC56/64, were upregulated. This study enhances our understanding of the CaNAC gene family’s role in cold responses, potentially bolstering molecular breeding programs for C. arabica.