{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T04:25:47Z","timestamp":1787199947353,"version":"build-2736575974"},"reference-count":36,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2026,8,11]],"date-time":"2026-08-11T00:00:00Z","timestamp":1786406400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Central Government Financial Forestry Science and Technology Extension and Demonstration Project","award":["Yunnan [2024] TG35"],"award-info":[{"award-number":["Yunnan [2024] TG35"]}]},{"award":["Yunnan [2024] TG35"],"award-info":[{"award-number":["Yunnan [2024] TG35"]}],"id":[{"id":"https:\/\/ror.org\/036h6g940","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Yunnan Special Fund for Scientific and Technological Innovation of Tropical Crops","award":["RF2026"],"award-info":[{"award-number":["RF2026"]}]},{"name":"Yunnan Province Industrial Innovation Talent Special Support Project","award":["yfgrc202520"],"award-info":[{"award-number":["yfgrc202520"]}]},{"name":"Yunnan Province Key Research and Development Program","award":["202603AS090013"],"award-info":[{"award-number":["202603AS090013"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Plants"],"abstract":"<jats:p>To characterize the transcriptomic dynamics underlying shell hardening in Macadamia integrifolia, RNA sequencing was performed on pericarp and shell tissues collected at three key developmental stages. A total of 18 libraries were generated, yielding 125.19 Gb of clean data. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that husk-associated DEGs were mainly enriched in photosynthesis-related functions, whereas shell-associated DEGs were enriched in phenylpropanoid biosynthesis, secondary metabolism, and cell wall organization. Weighted gene co-expression network analysis (WGCNA) identified module M8 as being strongly associated with the middle and late stages of shell development. Genes in this module were mainly enriched in phenylpropanoid metabolism, redox-related processes, and extracellular region functions. The hub genes within M8 included those encoding a glutaredoxin family protein, a WNK-type protein kinase, a tRNA methyltransferase-related protein, an EFR3 membrane-associated protein, a pectin modification-related protein, and a WAT1-related protein. Collectively, these results reveal clear tissue-specific transcriptomic patterns during pericarp and shell development and identify candidate co-expression networks that may contribute to shell hardening.<\/jats:p>","DOI":"10.3390\/plants15162442","type":"journal-article","created":{"date-parts":[[2026,8,11]],"date-time":"2026-08-11T13:46:16Z","timestamp":1786455976000},"page":"2442","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Integrated Transcriptome and WGCNA Analyses Reveal Candidate Regulatory Networks Associated with Shell Hardening in Macadamia integrifolia"],"prefix":"10.3390","volume":"15","author":[{"given":"Qingyi","family":"Long","sequence":"first","affiliation":[{"name":"Yunnan Institute of Tropical Crops, Jinghong 666100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yang","family":"Li","sequence":"additional","affiliation":[{"name":"Yunnan Institute of Tropical Crops, Jinghong 666100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jianjian","family":"Geng","sequence":"additional","affiliation":[{"name":"Yunnan Institute of Tropical Crops, Jinghong 666100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lidan","family":"Gong","sequence":"additional","affiliation":[{"name":"Yunnan Institute of Tropical Crops, Jinghong 666100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chao","family":"Wu","sequence":"additional","affiliation":[{"name":"Yunnan Institute of Tropical Crops, Jinghong 666100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing","family":"Ma","sequence":"additional","affiliation":[{"name":"Yunnan Institute of Tropical Crops, Jinghong 666100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tingyu","family":"Li","sequence":"additional","affiliation":[{"name":"Yunnan Institute of Tropical Crops, Jinghong 666100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nanhua","family":"Zi","sequence":"additional","affiliation":[{"name":"Yunnan Institute of Tropical Crops, Jinghong 666100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3713-7265","authenticated-orcid":false,"given":"Xinghao","family":"Tu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Tropical Fruit Biology, Ministry of Agriculture & Rural Affairs, South Subtropical Crops Research Institute of Chinese Academy of Tropical Agricultural Sciences, Zhanjiang 524091, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liang","family":"Tao","sequence":"additional","affiliation":[{"name":"Yunnan Institute of Tropical Crops, Jinghong 666100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Nock, C.J., Baten, A., Barkla, B.J., Furtado, A., Henry, R.J., and King, G.J. (2016). Genome and transcriptome sequencing characterises the gene space of Macadamia integrifolia (Proteaceae). BMC Genom., 17.","DOI":"10.1186\/s12864-016-3272-3"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Dardick, C., and Callahan, A.M. (2014). Evolution of the fruit endocarp: Molecular mechanisms underlying adaptations in seed protection and dispersal strategies. Front. Plant Sci., 5.","DOI":"10.3389\/fpls.2014.00284"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"209","DOI":"10.21273\/JASHS05324-23","article-title":"Difference in kernel shape and endocarp anatomy promote dehiscence in pistachio endocarp","volume":"148","author":"Zhang","year":"2023","journal-title":"J. Am. Soc. Hortic. Sci."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Khan, M.K.U., Muhammad, N., Jia, Z., Peng, J., and Liu, M. (2022). Mechanism of stone (hardened endocarp) formation in fruits: An attempt toward pitless fruits, and its advantages and disadvantages. Genes, 13.","DOI":"10.3390\/genes13112123"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"110030","DOI":"10.1016\/j.scienta.2021.110030","article-title":"Comparative transcriptome profiling provides insights into endocarp lignification of walnut (Juglans regia L.)","volume":"282","author":"Wu","year":"2021","journal-title":"Sci. Hortic."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Yu, A., Zou, H., Li, P., Yao, X., Guo, J., Sun, R., Wang, G., Xi, X., and Liu, A. (2023). Global transcriptomic analyses provide new insight into the molecular mechanisms of endocarp formation and development in iron walnut (Juglans sigillata Dode). Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24076543"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Dardick, C.D., Callahan, A.M., Chiozzotto, R., Schaffer, R.J., Piagnani, M.C., and Scorza, R. (2010). Stone formation in peach fruit exhibits spatial coordination of the lignin and flavonoid pathways and similarity to Arabidopsis dehiscence. BMC Biol., 8.","DOI":"10.1186\/1741-7007-8-13"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Li, Y., Liao, B., Wang, Y., Luo, H., Wang, S., Li, C., Song, W., Zhang, K., Yang, B., and Lu, S. (2022). Transcriptome and metabolome analyses provide insights into the relevance of pericarp thickness variations in Camellia drupifera and Camellia oleifera. Front. Plant Sci., 13.","DOI":"10.3389\/fpls.2022.1016475"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Tao, L., Long, Q., Chen, J., Zhang, Q., Guo, G., He, F., Cai, H., Geng, J., Song, X., and Zeng, H. (2025). Co-analysis of transcriptome and metabolome reveals flavonoid biosynthesis in macadamia pericarp across developmental stages. Foods, 14.","DOI":"10.3390\/foods14213618"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Shi, R., Tao, L., Tu, X., Zhang, C., Xiong, Z., Horowitz, A.R., Asher, J.B., He, J., and Hu, F. (2021). Metabolite profiling and transcriptome analyses provide insight into phenolic and flavonoid biosynthesis in the nutshell of Macadamia ternifolia. Front. Genet., 12.","DOI":"10.3389\/fgene.2021.809986"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"515","DOI":"10.1093\/jxb\/erv533","article-title":"Secondary cell walls: Biosynthesis and manipulation","volume":"67","author":"Kumar","year":"2016","journal-title":"J. Exp. Bot."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Nakano, Y., Yamaguchi, M., Endo, H., Rejab, N.A., and Ohtani, M. (2015). NAC\u2013MYB-based transcriptional regulation of secondary cell wall biosynthesis in land plants. Front. Plant Sci., 6.","DOI":"10.3389\/fpls.2015.00288"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Li, P., Wang, H., Liu, P., Li, Y., Liu, K., An, X., Zhang, Z., and Zhao, S. (2021). The role of JrLACs in the lignification of walnut endocarp. BMC Plant Biol., 21.","DOI":"10.1186\/s12870-021-03280-3"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Chen, J., Xie, L., Lin, Y., Zhong, B., and Wan, S. (2024). Transcriptome and weighted gene co-expression network analyses reveal key genes and pathways involved in early fruit ripening in Citrus sinensis. BMC Genom., 25.","DOI":"10.1186\/s12864-024-10651-1"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Xie, N., Guo, Q., Li, H., Yuan, G., Gui, Q., Xiao, Y., Liao, M., and Yang, L. (2024). Integrated transcriptomic and WGCNA analyses reveal candidate genes regulating mainly flavonoid biosynthesis in Litsea coreana var. sinensis. BMC Plant Biol., 24.","DOI":"10.1186\/s12870-024-04949-1"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Yao, Y., Xiong, E., Qu, X., Li, J., Liu, H., Quan, L., Lu, W., Zhu, X., Chen, M., and Li, K. (2023). WGCNA and transcriptome profiling reveal hub genes for key development-stage seed size\/oil content between wild and cultivated soybean. BMC Genom., 24.","DOI":"10.1186\/s12864-023-09617-6"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"16887","DOI":"10.1038\/s41598-020-73958-5","article-title":"A universal method for high-quality RNA extraction from plant tissues rich in starch, proteins and fiber","volume":"10","author":"Vennapusa","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2825","DOI":"10.1038\/s41598-022-06810-7","article-title":"An optimized method to obtain high-quality RNA from different tissues in Lilium davidii var. unicolor","volume":"12","author":"Wang","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"e1364","DOI":"10.1002\/wrna.1364","article-title":"RNA-Seq methods for transcriptome analysis","volume":"8","author":"Hrdlickova","year":"2017","journal-title":"Wiley Interdiscip. Rev. RNA"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"61","DOI":"10.2144\/000114133","article-title":"Library construction for next-generation sequencing: Overviews and challenges","volume":"56","author":"Head","year":"2014","journal-title":"BioTechniques"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"e1054","DOI":"10.1002\/cpz1.1054","article-title":"RNA-Seq data analysis: A practical guide for model and non-model organisms","volume":"4","author":"Simpson","year":"2024","journal-title":"Curr. Protoc."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"i884","DOI":"10.1093\/bioinformatics\/bty560","article-title":"fastp: An ultra-fast all-in-one FASTQ preprocessor","volume":"34","author":"Chen","year":"2018","journal-title":"Bioinformatics"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1038\/nmeth.3317","article-title":"HISAT: A fast spliced aligner with low memory requirements","volume":"12","author":"Kim","year":"2015","journal-title":"Nat. Methods"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Yang, Y., He, S., Xu, L., Wang, M., Chen, S., Bai, Z., Yang, T., Zhao, B., Wang, L., and Zhang, H. (2025). Transcriptome and WGCNA reveal the potential genetic basis of photoperiod-sensitive male sterility in soybean. BMC Genom., 26.","DOI":"10.1186\/s12864-025-11314-5"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"550","DOI":"10.1186\/s13059-014-0550-8","article-title":"Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2","volume":"15","author":"Love","year":"2014","journal-title":"Genome Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"923","DOI":"10.1093\/bioinformatics\/btt656","article-title":"featureCounts: An efficient general-purpose program for assigning sequence reads to genomic features","volume":"30","author":"Liao","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"D20","DOI":"10.1093\/nar\/gkae979","article-title":"Database resources of the National Center for Biotechnology Information in 2025","volume":"53","author":"Sayers","year":"2025","journal-title":"Nucleic Acids Res."},{"key":"ref_28","first-page":"100141","article-title":"clusterProfiler 4.0: A universal enrichment tool for interpreting omics data","volume":"2","author":"Wu","year":"2021","journal-title":"Innovation"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"634","DOI":"10.1093\/clinchem\/hvaf043","article-title":"MIQE 2.0: Revision of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments Guidelines","volume":"71","author":"Bustin","year":"2025","journal-title":"Clin. Chem."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Yang, Q., Yang, Z., Zeng, H., Zou, M., Song, X., Wan, J., Wang, Z., Chen, J., and Luo, L. (2024). Evaluation and validation of reliable reference genes for quantitative real-time PCR Analysis of the gene expression in Macadamia integrifolia. Forests, 15.","DOI":"10.3390\/f15111966"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"101515","DOI":"10.1016\/j.xpro.2022.101515","article-title":"Protocol for qPCR analysis that corrects for cDNA amplification efficiency","volume":"3","author":"Damgaard","year":"2022","journal-title":"STAR Protoc."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"242","DOI":"10.1038\/s41467-021-27937-7","article-title":"Signatures of selection in recently domesticated macadamia","volume":"13","author":"Lin","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_33","first-page":"10","article-title":"Tissue-specific transcriptomics reveals a central role of CcNST1 in regulating the fruit lignification pattern in Camellia chekiangoleosa, a woody oil-crop","volume":"2","author":"Yan","year":"2022","journal-title":"For. Res."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Sheng, Y., Yao, X., Liu, L., Yu, C., Wang, K., Wang, K., Chang, J., Chen, J., and Cao, Y. (2023). Transcriptomic time-course sequencing: Insights into the cell wall macromolecule-mediated fruit dehiscence during ripening in Camellia oleifera. Plants, 12.","DOI":"10.3390\/plants12183314"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"361","DOI":"10.1007\/s00709-020-01568-z","article-title":"Anatomy and lignin deposition of stone cell in Camellia oleifera shell during the young stage","volume":"258","author":"Wang","year":"2021","journal-title":"Protoplasma"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"313","DOI":"10.1186\/s13059-021-02531-8","article-title":"A systems genetics approach reveals PbrNSC as a regulator of lignin and cellulose biosynthesis in stone cells of pear fruit","volume":"22","author":"Wang","year":"2021","journal-title":"Genome Biol."}],"container-title":["Plants"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2223-7747\/15\/16\/2442\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T04:13:07Z","timestamp":1787199187000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2223-7747\/15\/16\/2442"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,11]]},"references-count":36,"journal-issue":{"issue":"16","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["plants15162442"],"URL":"https:\/\/doi.org\/10.3390\/plants15162442","relation":{},"ISSN":["2223-7747"],"issn-type":[{"value":"2223-7747","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,11]]}}}