Genome-wide identification and expression analysis of growth-regulating factor (GRF) and GRF-interacting factor (GIF) gene families in Castanea mollissima

【Objective】Growth-regulating factors (GRFs) and GRF-interacting factors (GIFs) play important roles in plant growth, development, and response to environmental stresses. As an important woody food resource in East Asia, Chinese chestnut (Castanea mollissima) is affected by numerous en-vironmental stresses during its growth and development process. In this study, the Chinese chestnut GRF and GIF gene families were studied to provide a theoretical basis for analyzing the potential functions of GRF and GIF genes in Chinese chestnut development and response to environmental stresses. 【Methods】The GRF and GIF gene families in Chinese chestnut were systematically characterized using bioinformatics methods, including physicochemical properties, phylogenetic relationships, gene structure, conserved motifs, collinear and evolutionary relationships, chromosome distribution, duplication types, promoter cis-elements, and interactions with transcription factors. The expression profiles of CmGRFs and CmGIFs in five Chinese chestnut organs (seed kernel, female flowers, male flowers, ovules, and buds), as well as under three abiotic stresses (drought, freezing, heat shock) and infection by Drycomusk kuriphilus were analyzed using RNA- seq data in NCBI database. The expression of CmGRFs and CmGIFs during Chinese chestnut kernel development was validated through RT-qPCR experiments.【Results】A total of 10 CmGRFs and 3 CmGIFs members were identified in the Chinese chestnut genome through bioinformatics methods, and they were shown to be distributed on seven chromosomes. Analysis of the physicochemical properties of these CmGRFs proteins revealed that their lengths ranged from 236 to 602 amino acids, with an aliphatic index ranging from 45.51 to 68.71. The instability indexes of CmGRFs were all above 40, and the grand averages of hydropathicity indices were all less than zero, indicating that all the CmGRFs belonged to unstable hydrophilic proteins. The isoelectric points of CmGRFs were between 7.74 and 9.41, indicating that the CmGRFs were all alkaline proteins. Subcellular localization prediction showed that 10 CmGRFs were located in the nucleus. Similarly, CmGIFs were shown to be located on three chromosomes in the nucleus and considered to be unstable hydrophilic acidic proteins. Phylogenetic analysis divided the CmGRFs into four branches, and CmGIFs into three branches. Each CmGRF contained 2 to 9 conserved motifs and 2 to 5 introns. All three CmGIFs contain only one conserved motif, three introns, and four exons. Members of CmGRFs located in the same branch had similar gene structures and protein conserved motifs. A total of 270 and 86 cis-acting elements were identified in the promoter regions of CmGRFs and CmGIFs, respectively, mainly related to light response, environmental stresses, growth and development, and plant hormones. The collinear analysis within the Chinese chestnut genome indicated that segmental duplication was the main driving force for the expansion of the CmGRF and CmGIF gene families. The collinear analysis between Chinese chestnut and five other representative species (Arabidopsis thaliana, Quercus, Vitis vinifera, Oryza sativa, and Zea mays) suggested that the GRF and GIF families might have undergone independent duplication events after the differentiation between dicotyledonous and monocotyledonous plants. Transcriptome data analysis showed that CmGRF3 and CmGIF2 were consistently highly expressed in female flowers, while almost not in male flowers (FPKM＜1). Additionally, CmGRF3 and CmGIF2 exhibited extremely similar expression patterns, suggesting that they may work together and may be involved in the differentiation or development of male and female Chinese chestnut flowers. During the development of Chinese chestnut kernels in Yanshanzaofeng and Yanlong, the expression levels of CmGRF1 and CmGRF3 significantly changed, and CmGIF3 continued to be highly expressed, suggesting that they may be related to the development of Chinese chestnut kernels. The rapid changes in the expression levels of CmGRF2, CmGRF3, and CmGIF3 during freezing and heat shock stresses suggest that they may be involved in the response of Chinese chestnuts to these temperature stresses. Under drought stress, CmGRFs in Yanshanzaofeng and Dabanhong were almost not expressed, indicating that they may not be related to the response of Chinese chestnuts to drought stress. The infection of Drycosmusk kuriphilus significantly induced the expression of CmGRF2, CmGRF3, CmGRF7, and Cm-GIF1, indicating that they may be related to the response of Chinese chestnuts to D. kuriphilus infection. Furthermore, the expression levels of all CmGRFs and CmGIFs in Chinese chestnut kernels at 60, 70, 80, 90, and 100 days after flowering were validated by RT-qPCR experiments, and the results were consistent with RNA-seq data. In addition, the transcription factor families predicted to have the most interaction with the promoter regions of CmGRFs and CmGIFs included MYB, Dof, bHLH, C2H2, and TCP, indicating that they may play an important role in regulating the expression of CmGRFs and CmGIFs.【Conclusion】A total of 10 CmGRFs and 3 CmGIFs were identified and systematically analyzed in the Chinese chestnut genome. Some CmGRFs and CmGIFs showed significant changes in expression levels during different developmental stages of five Chinese chestnut organs (seed kernel, female flowers, male flowers, ovules, buds), as well as under three abiotic stresses (freezing, heat shock) and infection by D. kuriphilus. RT-qPCR was used to validate the expression levels of CmGRFs and CmGIFs at five developmental stages of Chinese chestnut kernels. This study provides a reference for further investigating the functions of CmGRFs and CmGIFs, and their potential roles in Chinese chestnut growth, development, and response to environmental stress.