Identification and gene expression analysis of the CNGC gene family in apple

【Objective】As non-selective cation channels, the cyclic nucleotide-gated channel (CNGC) gene family regulates calcium uptake to participate in plant signaling, biotic stress signaling, abiotic stress signaling, and growth. High- quality apple varieties such as Fuji, Golden Delicious and Gala, which are mainly grown internationally, cannot survive the winter in Gansu province, China. Xinjiang wild apples are known for their strong cold resistance, but they are also characterized by high acidity and poor fruit quality; therefore, they are commonly used as rootstocks for grafting. Despite the key roles of the CNGC gene family in plant survival and environmental response, little is known about the activity and function of this gene family in apples.【Methods】In this study, the cyclic nucleotide-binding domain (cNMP_binding, PF00027) and ion transport protein domain (Ion_trans, PF00520) of CNGC were used to analyze and screen four apple CNGC gene sequences with tools including Pfam, CDD, HMMER, and SMART. The protein length, molecular weight, isoelectric points (pI), instability index, and hydrophobicity (GRAVY) were predicted by the ExPASy Server. The subcellular localization of CNGC was determined using the WoLF PSORT online prediction tool. Four apple and Arabidopsis CNGC protein sequences were aligned using the ClustalX 2.01 program. The phylogenetic tree of these CNGCs was constructed using MEGA7.0 with the maximum likelihood (ML) method and 1000 bootstrap replicates. The exon- intron structure of CNGC genes was analyzed online using the Gene Structure Display Server (GSDS2.0). The protein sequences of CNGCs were scanned for conserved motifs using the online MEME tool. Cis-acting regulatory elements in the 2000 bp upstream sequence of the CNGC genes were predicted using the Signal Scan search program in the PlantCARE database. Collinearity analysis was performed using MCScanX, and intraspecific gene duplications were visualized using TBtools-Ⅱ. The CodonW1.4.4 software was applied to analysis codon usage index, ENc- plot, PR2-plot, Neutrality-plot and RSCU analysis by the Python3.7.0 and R4.4.2 software. We downloaded RNA-seq data related to four aspects of apple CNGC genes from the NCBI-SRA database: flower regulation, fruit epidermal wax biosynthesis, hormone treatment, and biotic and abiotic stress expression. The expression of twenty-two CNGC genes in Gala apple under salt stress was analyzed by quantitative real-time polymerase chain reaction (qRT- PCR).【Results】Sixty- seven CNGC gene family members were identified across the genomes of four apple species: Fuji (14), Golden Delicious (13), Gala (22), and Xinjiang wild (18) apples. The four apple CNGCs are basic proteins with an average isoelectric point (pI) of 8.63 (ranging from 5.53 to 9.57).Analysis of protein properties showed that the length ranges from 548 (MdjCNGC1.2) to 1733 (MsCNGC1.2B) amino acids (aa), with an average of 812 aa, and the average molecular weight is 93.53 kDa (ranging from 63.24 to 198.90 kDa).A maximum likelihood phylogenetic analysis categorized the CNGC genes from four apple species and Arabidopsis into four distinct subfamilies (Groups Ⅰ-Ⅳ). Genes within the same subfamily exhibit similar domain structures, gene architectures, and motif distributions. Group Ⅰ subfamily comprises 7 to 9 exons, Group Ⅱ subfamily comprises 7 to 15 exons, Group Ⅲ subfamily comprises 6 exons, Group Ⅳa subfamily comprises either 6 or 12 exons, and Group Ⅳb subfamily comprises 6 to 20 exons. All protein sequences of the CNGC family in four apple varieties possess the two characteristic domain motifs: motif 1 (Ion_trans, PF00520) and motif 2 (cNMP_binding, PF00027). All the identified MsCNGC, MdfCNGC, and MdjCNGC genes were unevenly distributed across 12 chromosomes. By contrast, MdgCNGC genes were distributed on only 10 chromosomes, and whole genome duplication contributed to the expansion of the CNGC gene family in the four apple species. Analyses of the ENc-plot, PR2-plot, and Neutralityplot showed that codon usage of the CNGC gene family in four apples was mainly driven by natural selection, alongside other factors such as base mutations. RSCU analysis showed that high-frequency codons in the four apple CNGC genes mostly end with A/U, while low-frequency codons end with G/C. Codons with the highest RSCU values mainly encode arginine (Arg, AGA). Subcellular localization assays showed that all CNGC proteins from the four apples were localized to the plasma membrane. The majority of cis-acting regulatory elements were light-responsive, and others are associated with growth, development, low temperature, drought, and hormone responses, indicating the CNGC gene family potentially plays roles in these biological processes. Furthermore, among the tissue- specific expression patterns detected in flower samples of Golden Delicious, MdjCNGC7.3 was highly up-regulated at the flower bud stage, MdjCNGC9.1 was highly up-regulated at the flower popcorn stage, and MdjCNGC7.2 was highly up- regulated at the full bloom stage. Furthermore, MdjCNGC7.1, MdjCNGC7.2, Md-jCNGC7.3, and MdgCNGC7.2B exhibited significant responses to different hormone stimuli. Transcriptome analysis showed that MdfCNGC1.3 was significantly up-regulated following apple stem grooving virus infection, while MdjCNGC7.2 and MdjCNGC7.3 maintained higher expression levels than the control across multiple time points after inoculation with Pythium ultimum in apple seedlings. Transcriptome analysis showed that after 60 days of exposure to 5 ℃, the expression level of MdfCNGC7.1 was significantly higher than that of the control group. qRT-PCR analysis indicates that salt stress significantly induces four genes, MdgCNGC1.2A, MdgCNGC7.1A, MdgCNGC5A, and MdgCNGC9.1A, in the leaves and roots of‘Gala’apple seedlings.【Conclusion】In this study, a total of 18 genes from Malus sieversii, 14 genes from Malus domestica‘Fuji’, 13 genes from Malus domestica‘Golden Delicious’, and 22 genes from Malus domestica‘Gala’were identified as members of the CNGC gene family. This study used bioinformatics methods to analyze the physicochemical properties, gene structure, chromosomal localization, phylogenetic relationship, collinearity, codon usage bias, subcellular localization and cis-acting regulatory element characteristics of the CNGC gene family in four apples. Furthermore, transcriptomic data of apple tissues, hormone- related stress, and both biotic and abiotic stressors were subjected to additional analysis. The qRT-PCR analysis also showed that CNGC genes were highly expressed after salt stress treatment, while their expression was significantly lower under controlled conditions. This study examines the CNGC gene family across four apple varieties. It aims to conduct an indepth analysis of CNGC- related data, thereby establishing a foundational dataset for subsequent trials on stress resistance in apple varieties.