Tree Genetics and Molecular Breeding 2025, Vol.15 http://genbreedpublisher.com/index.php/tgmb © 2025 GenBreed Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved.
Tree Genetics and Molecular Breeding 2025, Vol.15 http://genbreedpublisher.com/index.php/tgmb © 2025 GenBreed Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. GenBreed Publisher is an international Open Access publisher specializing in tree genetics and molecular breeding, trees genetic diversity and conservation genetics registered at the publishing platform that is operated by Sophia Publishing Group (SPG), founded in British Columbia of Canada. Publisher GenBreed Publisher Edited by Editorial Team of Tree Genetics and Molecular Breeding Email: edit@tgmb.genbreedpublisher.com Website: http://genbreedpublisher.com/index.php/tgmb Address: 11388 Stevenston Hwy, PO Box 96016, Richmond, V7A 5J5, British Columbia Canada Tree Genetics and Molecular Breeding (ISSN 1927-5781) is an open access, peer reviewed journal published online by GenBreed Publisher. The journal publishes all the latest and outstanding research articles, letters and reviews in all aspects of tree genetics and molecular breeding, include studies in tree genetics and molecular breeding, include studies in crop/fruit/forest/ornamental/horticultural trees genetic diversity, conservation genetics, molecular genetics, evolutionary genetics, population genetics, physiology, biochemistry, transgene, genetic rule analysis, QTL analysis, vitro propagation; fruit/forest/ornamental/horticultural trees breeding studies and advanced breeding technologies. All the articles published in Tree Genetics and Molecular Breeding are Open Access, and are distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. GenBreed Publisher uses CrossCheck service to identify academic plagiarism through the world’s leading plagiarism prevention tool, iParadigms, and to protect the original authors’ copyrights.
Tree Genetics and Molecular Breeding (online), 2025, Vol. 15 ISSN 1927-5781 http://genbreedpublisher.com/index.php/tgmb © 2025 GenBreed Publisher, an online publishing platform of Sophia Publishing Group. All Rights Reserved. Sophia Publishing Group (SPG), founded in British Columbia of Canada, is a multilingual publisher Latest Content Identification and Functional Validation of Key Genes Regulating Flowering Time in Tea Jie Huang, Minghui Zhao, Jiayao Zhou Tree Genetics and Molecular Breeding, 2025, Vol. 15, No. 3, 89-97 Cloning and Functional Analysis of Key Genes Involved in Anthocyanin Biosynthesis in Morella rubra Zhen Liu, Dandan Huang, Wenfang Wang Tree Genetics and Molecular Breeding, 2025, Vol. 15, No. 3, 98-107 Integrated Transcriptome and Metabolome Analysis Reveals the Genetic Regulation of Aroma Biosynthesis in Citrus Xingzhu Feng Tree Genetics and Molecular Breeding, 2025, Vol. 15, No. 3, 108-116 Functional Genomics of Root Development in Populus and Its Ecological Implications Minghua Li, Hongpeng Wang, Shiying Yu Tree Genetics and Molecular Breeding, 2025, Vol. 15, No. 3, 117-127 Epigenetic Variation and Oil Accumulation in Camellia oleifera: A Case from High- and Low-Altitude Regions Jianmin Zheng, Lian Chen, Chuchu Liu Tree Genetics and Molecular Breeding, 2025, Vol. 15, No. 3, 128-137
Tree Genetics and Molecular Breeding 2025, Vol.15, No.3, 89-97 http://genbreedpublisher.com/index.php/tgmb 89 Research Insight Open Access Identification and Functional Validation of Key Genes Regulating Flowering Time in Tea Jie Huang 1, Minghui Zhao 1, Jiayao Zhou 2 1 Tropical Medicinal Plant Research Center, Hainan Institute of Tropical Agricultural Resources, Sanya, 572025, Hainan, China 2 Cuixi Academy of Biotechnology, Zhuji, 311800, Zhejiang, China Corresponding email: jiayao.zhou@cuixi.org Tree Genetics and Molecular Breeding, 2025, Vol.15, No.3 doi: 10.5376/tgmb.2025.15.0011 Received: 10 Apr., 2025 Accepted: 13 May, 2025 Published: 21 May, 2025 Copyright © 2025 Huang et al., This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Huang J., Zhao M.H., and Zhou J.Y., 2025, Identification and functional validation of key genes regulating flowering time in tea, Tree Genetics and Molecular Breeding, 15(3): 89-97 (doi: 10.5376/tgmb.2025.15.0011) Abstract This study discusses the genetic basis and molecular mechanism of when tea plants flower, and summarizes the progress made in identifying and verifying key genes, such as transcription factors, genes in hormone signaling pathways, and genes that can integrate flowering signals. Research has found that some major regulatory genes, such as FT, SOC1, GI and CO, play a key role in integrating light duration and temperature signals. Some non-coding RNAs, such as miRNA and lncRNA, are also involved in the detailed regulation of flowering time. Gene editing tools like CRISPR-Cas9 have also begun to be used to verify the functions of these genes. This study aims to provide a foundation for molecular breeding of tea plants, enabling people to more accurately control the flowering time and thereby increase yield and planting efficiency. Keywords Tea (Camellia sinensis); Flowering time; Gene regulation; Non-coding RNA; Functional validation 1 Introduction The tea (Camellia sinensis) is an important economic crop. When it flowers will directly affect the formation of seeds, the progress of breeding and the yield of tea (Liu, 2024). If the flowering time is appropriate, it can not only balance the growth and fruiting of tea, but also improve the success rate of pollination and increase the number of seeds, thereby making the yield of tea gardens higher (Lin et al., 2022; Naik et al., 2025). However, flowering consumes a lot of water and nutrients. If tea flower too early or too late, it may affect normal growth and eventually lead to a decline in tea quality and yield (Tian et al., 2021; Xu et al., 2022). Tea is perennial plants, which are different from annual crops. Its flowering time is influenced by many factors, such as genetics, hormone levels, light duration, temperature and nutrition, etc. (Fan et al., 2024; Naik et al., 2025). Weather changes, low temperatures in winter or rain may all cause unstable flowering time of tea, thereby affecting the yield (Lin et al., 2022; Fan et al., 2024). In addition, the flowering of tea is also closely related to the dormancy of winter buds. Currently, scientists are still studying the signals and genes involved (Liu et al., 2022; Xu et al., 2022). Although exogenous hormones or chemical agents can regulate the flowering period, the working principles and safety of these methods are not yet clear (Ionescu et al., 2016; Lin et al., 2022). This study identified some key genes that affect the flowering time of tea, conducted functional verification on them, and combined transcriptome analysis, hormone signaling pathways and experimental verification to find out the important factors regulating the flowering period. These findings are helpful for better controlling the flowering time of tea. This study aims to provide theoretical support and molecular tools for future breeding and increased production. 2 Biological Basis of Flowering in Tea Plants 2.1 Floral transition phases in Camellia sinensis The flowering process of the tea plant (Camellia sinensis) generally includes several stages: flower induction, flower bud differentiation and flower organ differentiation. Through morphological observation, researchers can clearly distinguish the time points of flower induction and flower bud differentiation. The gene expression of different varieties varies at these stages. At present, many genes related to flowering have been identified, such as
Tree Genetics and Molecular Breeding 2025, Vol.15, No.3, 89-97 http://genbreedpublisher.com/index.php/tgmb 90 PRR7, GI, GID1B, GID1C related to flower induction, and LFY, PNF, PNY, etc. related to flower bud formation (Liu et al., 2020a). There are also some key genes, such as SOC1, HD3A and LFY, which play major roles throughout the flower formation process (Xu et al., 2022). Furthermore, transcriptome analysis also revealed that the expression levels of some transcription factors, such as WRKY, ERF, bHLH, MYB and MADS-box, would increase during flowering period changes, indicating that they are important for flowering period regulation (Liu et al., 2017). 2.2 Seasonal and developmental cues influencing flowering The flowering of tea plants is jointly influenced by seasonal factors (such as light duration and temperature) and the plant’s own hormones. Studies have found that during the development of tea plant flowers, many genes are related to the biological clock and the autonomous flowering pathway. Hormone levels also change significantly during the flowering period. For example, the contents of auxin and gibberellin fluctuate up and down, and the related synthesis and signaling pathway genes also change accordingly (Xu et al., 2022). Some specific genes, such as CsFLC1 (a member of the MADS-box family), have high expression levels during winter dormancy and flowering periods and may be involved in regulating seasonal flowering (Liu et al., 2022). Furthermore, miR156 controls the flowering time by influencing SPL-like genes (such as CsSPL1), and the higher the expression of miR156, the later the flowering time. The genes of the PMEI family (such as CsPMEI2 and CsPMEI4) can also accelerate the flowering process, and they function through the autonomous flowering pathway (Wang et al., 2022). 2.3 Anatomical and morphological features of tea flowering Tea plants have bisexual flowers, which means that the same flower contains both stamens and pistils. The structure of flowers is the same as that of general angiosperms, including sepals, petals, stamens and pistils. During the development of floral organs, many secondary metabolites accumulate, such as flavonoids and anthocyanins. These substances are mainly concentrated in the petals and stamens (Chen et al., 2018). During pollen development, the content changes of certain flavonols (such as derivatives of kaempferol) are highly related to the viability of pollen (Shi et al., 2021). When tea plants flower, nitrogen and sugars in the leaves are redistributed and transported to the flowers, which need these substances to complete development (Fan et al., 2019). In addition, the formation of flowers is also closely related to ethylene, other hormone signals, and the regulation of certain transcription factors (Liu et al., 2020b). 3 Environmental and Hormonal Control of Flowering Time 3.1 Influence of photoperiod and temperature The flowering time of tea plants is influenced by environmental factors such as the duration of light (photoperiod) and temperature. Research has found that many genes controlling flowering are related to circadian rhythms and autonomous flowering pathways. Among them, the SOC1 gene plays a very crucial role in the development process of flowers (Xu et al., 2022). There are also FLC genes like CsFLC1, which have high expression levels during plant dormancy and flowering in winter. This suggests that it may be the key for plants to sense low temperature and control flowering time (Liu et al., 2022). Environmental signals affect proteins like FT through photosensitive proteins and biological clocks. FT moves to the top of plants and initiates flowering (Freytes et al., 2021). Temperature changes and vernalization processes can also regulate these signals, thereby making the flowering time more adaptable to seasonal changes (Lee et al., 2023). 3.2 Roles of phytohormones During the flowering process of tea plants, the content of hormones in their bodies and the expression of genes related to hormones will also change. Studies have found that more than 200 genes related to hormone synthesis and nearly 200 genes related to hormone signal transduction are involved during flower development (Xu et al., 2022). Hormones, such as gibberellin (GA), abolic acid (ABA), ethylene and auxin (IAA), can affect flower formation by regulating key genes such as SOC1, FT and LFY (Liu et al., 2017; Liu et al., 2022). The use of exogenous hormones can also affect flowering. For example, spraying ethephon can cause some flowers and flower buds to drop and regulate the expression of the ethylene receptor gene CsETR (Zhang et al., 2022). In
Tree Genetics and Molecular Breeding 2025, Vol.15, No.3, 89-97 http://genbreedpublisher.com/index.php/tgmb 91 addition, the use of hydrocyanamide in tea oil tea can make it flower earlier. This effect is related to hormones, MAPK pathway, oxygen metabolism, etc. in the plant (Lin et al., 2022). 3.3 Environmental stressors and their impact on flowering When the environment is not ideal, such as drought, high salt content or reduced soil nutrients, the flowering time of tea will also be affected. These pressures can alter the flowering process by regulating photoperiod and hormone signaling pathways (Lee et al., 2023). Under difficult conditions, plants may flower earlier or later to increase the success rate of reproduction (Cho et al., 2017). These environmental signals and photoperiodic control mechanisms are interconnected, and genes like FT act as the “bridge” in the middle, simultaneously participating in responding to stress and controlling flowering (Riboni et al., 2014). In addition, environmental stress may also indirectly affect the expression of genes related to flowering by altering glucose metabolism, reactive oxygen species levels, etc. within plants (Liu et al., 2017; Cho et al., 2018; Lin et al., 2022). 4 Identification of Flowering Time Genes in Tea 4.1 Genome and transcriptome resources for gene discovery In recent years, genomic information and a large amount of transcriptome data of the tea (Camellia sinensis) have laid the foundation for identifying the genes that control the flowering time. By conducting transcriptome sequencing on tea plants of different varieties and at different developmental stages, researchers identified a total of 92 core genes related to flower development, covering the entire process from flower bud formation to full flower opening (Xu et al., 2022). Meanwhile, through genomic alignment, they also identified 401 and 356 genes related to flowering respectively from small-leaf and large-leaf tea plants, all of which are candidates for subsequent functional studies (Liu et al., 2020a). 4.2 Homology-based identification using model species Researchers also identified multiple homologous genes in tea plants by leveraging known flowering genes in model plants such as Arabidopsis thaliana and rice. For instance, CsFLC1 in tea plants belongs to the MADS-box gene family. It is very similar to FLC in Arabidopsis thaliana. Experimental results show that it can affect the flowering time of tea plants and is also related to the dormancy of buds in winter (Figure 1) (Liu et al., 2022). In addition, CsWRKY7 is also very similar to AtWRKY7 and AtWRKY15 of Arabidopsis thaliana. Overexpression of it will cause delayed flowering of plants and reduce the expression of key genes such as FT, AP1 and LFY at the same time (Chen et al., 2019). The regulatory modules miR156 and SPL also have similar functions in tea plants. For example, Csn-miR156d can regulate FT, AP1, FUL and SOC1 by targeting CsSPL1, thereby delaying the flowering time. Figure 1 Expression patterns of CsFLC1 (Adopted from Liu et al., 2022) Image caption: (A) Expression of CsFLC1 in axillary or flower buds of tea plant throughout the year. (B) Different tissue expression of CsFLC in tea plant. (C) Various parts of flowers of tea plant. (D) Expression patterns of CsFLC1 in different parts of flowers in tea plant. (E) GUS staining of pCsFLC1::GUS transgenic Arabidopsis thaliana (Adopted from Liu et al., 2022)
Tree Genetics and Molecular Breeding 2025, Vol.15, No.3, 89-97 http://genbreedpublisher.com/index.php/tgmb 92 4.3 High-throughput screening strategies (RNA-seq, WGCNA, GWAS) Nowadays, high-throughput technologies such as RNA-seq and WGCNA have been widely used to screen genes related to flowering in tea plants. Researchers analyzed transcriptome data of different developmental stages and different varieties and found that genes such as SOC1, LFY, GI and PRR7 showed obvious expression trends at the flower induction and flower bud differentiation stages (Liu et al., 2020a). Furthermore, differential expression analysis also identified hundreds of genes related to hormone synthesis and signal transduction, constituting a relatively complex hormone regulatory network. It is speculated that genes such as MYC, FT, SOC1 and LFY are the cores among them (Xu et al., 2022). Although GWAS methods are not widely used in tea plants, previous studies, through genome-wide association analysis combined with functional validation, have discovered genes like CsMADS27 that play an important role in the dormancy and germination processes of tea plants (Hao et al., 2024). 5 Key Gene Families Involved in Flowering Regulation 5.1 CONSTANS-like and FT/TFL1 family genes CONSTANS (CO) and FLOWERING LOCUS T (FT) are very important regulatory genes in the photoperiodic pathway. Research has found that in tea plants and their close relatives, these two genes and their similar genes play a key role in the regulatory network of circadian rhythms and photcycles, and can regulate flower bud differentiation and flowering time. For instance, Unigene0001842 (that is, CO) and Unigene0084708 (that is, FT) have been identified as the core genes regulating the flowering time of tea plants. They can also work together with genes such as GI and PRR that regulate circadian rhythms to help initiate flower bud differentiation (Guo et al., 2022). In addition, genes like HD3A in the FT/TFL1 family also occur during the flower development process of tea plants, while SOC1, as a downstream integration factor, plays a leading role throughout the flowering process (Xu et al., 2022). 5.2 MADS-box genes and floral integrators The MADS-box gene is also very important in the regulation of flowering in tea plants. CsFLC1 is a gene similar to FLC in the MADS-box family, and its expression level is very high when plants are dormant and preparing to flower in winter. It can affect the expression of flower-related genes such as SOC1, AGL42, SEP3 and AP3, and can also change the flowering time by regulating hormone signals (Liu et al., 2022). There is another key gene, CsMADS27, which also belongs to the MADS-box family. It can control the dormancy and germination of tea plants. This gene is regulated by CsCBF1 and CsZHD9, and controls the expression of downstream CsDJC23 (Hao et al., 2024). These MADS-box genes can also work together with transcription factors such as WRKY, MYB, and bHLH to jointly regulate flower development and secondary metabolism (Liu et al., 2017; Sun et al., 2019). 5.3 Circadian clock and vernalization-related genes The flowering of tea plants is also regulated by some genes in the circadian rhythm and vernalization pathway. Genes such as PRR7, GI and LHY are closely related to flower bud differentiation, and their expression levels increase under stress (Liu et al., 2020a; Guo et al., 2022). CsFLC1 can not only control flowering but also regulate the dormancy of tea plants in winter, indicating that the spring pathway also plays an important role in tea plants (Liu et al., 2022). Furthermore, flowering integration genes such as SOC1 and LFY, like an intersection point, can integrate various signals from photoperiod, hormones and vernalization, and ultimately precisely control when flowering occurs (Xu et al., 2022). 6 Regulatory Networks and Molecular Pathways 6.1 Integrative regulatory models in tea flowering When tea plants flower is determined by many levels of molecular mechanisms. These mechanisms include miRNA, transcription factors, hormone signals and energy metabolism, etc. miR156 and miR172 can control the expression of key genes such as SPL and AP2, thereby affecting the expression of SOC1. This forms two main pathways: miR156-SPL and miR172-AP2, which jointly participate in the regulation of flowering time (Wang, 2014; Fan et al., 2024). In addition, genes such as PRR, LHY, GI, CO and FT can receive light and temperature
Tree Genetics and Molecular Breeding 2025, Vol.15, No.3, 89-97 http://genbreedpublisher.com/index.php/tgmb 93 signals in the environment and pass on this information to promote flower bud formation (Guo et al., 2022). The flowering of tea plants also requires a large amount of energy and carbohydrates. These substances, in combination with hormone signals, jointly construct a very complex regulatory network (Tang et al., 2023). 6.2 Crosstalk between hormone signaling and gene expression Different plant hormones play different roles in the flowering process of tea plants. Some hormone levels increase, such as zeaxanthin (cZ), brassinolide (BL), salicylic acid (SA), ACC and jasmonic acid (JA), which can accelerate flowering. Hormones such as ABA, tZR, dh-Z and IP can delay flowering (Fan et al., 2024). These hormones can also interact with some key genes, helping to control the process of flower bud differentiation and flowering. The study also found that hormones in old leaves can guide flower bud development by regulating energy metabolism and rhythm genes (Guo et al., 2022). When the bud begins to grow, zeaxin plays a core role. It can activate many growth-promoting factors and help the bud grow rapidly (Tang et al., 2023). 6.3 Epigenetic mechanisms and chromatin remodeling In addition to genes and hormones, epigenetics and chromatin conditions can also affect the flowering of tea plants. Small RNA molecules like miR156 and miR172 can participate in epigenetic regulation by controlling the genes they target (Wang, 2014; Fan et al., 2024). In addition, some transcription factors, such as bHLH, MYB and NAC families, also regulate the expression of genes related to secondary metabolism and flowering, change the structure of chromatin, and thereby affect the activity level of genes (Tai et al., 2018). These mechanisms work together to ensure that tea plants can enter the flowering stage at the appropriate time (Li et al., 2015; Wang et al., 2025). 7 Case Study: Functional Characterization of CsaFT1 in Tea Flowering Regulation 7.1 Identification and cloning of CsaFT1 CsaFT1 is a FLOWERING gene in tea plants. It belongs to the FT (FLOWERING LOCUS T) family and is an important member that regulates the flowering time of plants. Researchers identified this gene by analyzing the genome and transcriptome of the tea plant and obtained its complete cDNA sequence using molecular cloning technology. In multiple studies, FT genes are considered to play a key role in the development of tea plant flowers and are expressed at different developmental stages, indicating that they are involved in flower bud initiation and the formation of floral organs (Figure 2) (Liu et al., 2020a; Xu et al., 2022). Figure 2 Putative gene regulatory network of flowering in tea plants (Adopted from Liu et al., 2020a) Image caption: Arrows indicate positive control, perpendicular lines indicate negative control, orange frames indicate physiological processes, other frames indicate proteins, white letters without frames indicate mRNA and black letters without frames indicate genes (Adopted from Liu et al., 2020a)
Tree Genetics and Molecular Breeding 2025, Vol.15, No.3, 89-97 http://genbreedpublisher.com/index.php/tgmb 94 7.2 Expression pattern and functional assays The expression level of CsaFT1 in tea plants is significantly increased before flowering and during the flower bud formation stage. Functional tests show that after introducing CsaFT1 into Arabidopsis thaliana, the plants can flower earlier and also increase the expression levels of flower-related genes such as SOC1 and LFY. These results suggest that CsaFT1 can indeed promote plant flowering (Liu et al., 2020a). Furthermore, some plant hormones, such as gibberellin and abscisic acid, also affect the expression of CsaFT1, indicating that it is also involved in the hormone-regulated flowering mechanism (Xu et al., 2022). 7.3 Implications for breeding early- or late-flowering cultivars The research on CsaFT1 provides a new direction for regulating the flowering time of tea plants. By controlling the expression of this gene, it is possible to achieve breeding of early or late flowering traits, thereby growing more suitable tea varieties in different regions or seasons. This not only helps balance the vegetative growth and flowering of tea plants, but also increases the yield and quality of tea (Liu et al., 2020a; Xu et al., 2022). In the future, CsaFT1 can be used as a molecular marker to breed new tea varieties that adapt to different climates and tea-picking times, bringing a more sustainable development path to the tea industry. 8 Applications in Tea Breeding and Crop Improvement 8.1 Marker-assisted selection for flowering traits With the sequencing of the tea genome and the pan-genome completed, scientists have identified many important genes and allelic variations related to flowering time. These achievements have laid the foundation for the development of molecular markers and the realization of label-assisted selection (MAS). By combining genotype and phenotype analysis, researchers can more accurately identify early-flowering or late-flowering varieties, thereby accelerating the breeding progress and improving the breeding efficiency (Chen et al., 2023; Li et al., 2023). 8.2 Molecular breeding strategies for climate adaptation Nowadays, climate change is becoming increasingly obvious, which also has an impact on the growth and flowering time of tea. Molecular breeding methods, such as genomic selection, genetic modification and gene editing, can help breed new varieties that are more adaptable to extreme weather conditions like drought and high temperatures. By screening genes related to stress resistance and flowering time and combining genomic breeding technology, scientists are expected to cultivate high-yield and high-quality tea varieties more quickly (Mukhopadhyay et al., 2015; Lubanga et al., 2022; Ramakrishnan et al., 2023). 8.3 Integration with phenology-based cultivation practices If these molecular breeding achievements are combined with the planting and management methods of tea gardens, the flowering time can be better controlled. For instance, by using molecular markers to identify early-flowering or late-flowering varieties and combining them with local weather and growth records, more reasonable planting and tea-picking times can be arranged, thereby enhancing the yield and quality of tea. Meanwhile, this method can also help tea gardens better cope with climate change and maintain stable production (Ranatunga, 2019; Li et al., 2023; Zakir et al., 2023). 9 Concluding Remarks In recent years, research on the molecular mechanism of when tea flower has made rapid progress. Scientists have identified many key genes, such as CsFLC1 and CsMADS27 in the MADS-box family, which play a significant role in regulating flowering time, winter dormancy and germination. Through genomic and transcriptomic analysis, classic flowering genes such as SOC1, LFY, FT, GI, and PRR7 were also discovered. The combination of miR156d and CsSPL1 is also closely related to the flowering time and the development of flower organs. In addition, hormones such as gibberellin, abolic acid and ethylene, along with their signaling pathways, also affect flowering and dormancy. These hormones, together with transcription factors (such as MYB, WRKY, bHLH, etc.), form a complex regulatory network.
Tree Genetics and Molecular Breeding 2025, Vol.15, No.3, 89-97 http://genbreedpublisher.com/index.php/tgmb 95 However, it is still not very clear how tea integrates external signals (such as light duration and temperature) to regulate flowering. The specific expression and function of these genes in different varieties, at different developmental stages or under environmental pressure still require more experiments to verify. In particular, there are still many questions that have not been clarified regarding how key genes interact with each other, how hormones and transcription factors work together, and the role of epigenetics in the regulatory process. Nowadays, with the increasing amount of tea genomic data and the maturation of gene editing tools like CRISPR, it is expected that in the future, the genes that control the flowering time can be precisely regulated. This will help regulate the growth rhythm of tea and increase the yield. In the future, new tea varieties that are more adaptable to climate and have better control over flowering time can be bred through molecular marker breeding, gene transformation or editing, thereby promoting the efficient and sustainable development of the tea industry. Acknowledgments The authors appreciate the modification suggestions from two anonymous peer reviewers on the manuscript of this study. Conflict of Interest Disclosure The authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest. 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Tree Genetics and Molecular Breeding 2025, Vol.15, No.3, 98-107 http://genbreedpublisher.com/index.php/tgmb 98 Feature Review Open Access Cloning and Functional Analysis of Key Genes Involved in Anthocyanin Biosynthesis in Morella rubra Zhen Liu 1, Dandan Huang 2, Wenfang Wang 1 1 Institute of Life Sciences, Jiyang Colloge of Zhejiang A&F University, Zhuji, 311800, Zhejiang, China 2 Hainan Institute of Biotechnology, Haikou, 570206, Hainan, China Corresponding email: wenfang.wang@jicat.org Tree Genetics and Molecular Breeding, 2025, Vol.15, No.3 doi: 10.5376/tgmb.2025.15.0012 Received: 17 Apr., 2025 Accepted: 19 May, 2025 Published: 30 May, 2025 Copyright © 2025 Liu et al., This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Liu Z., Huang D.D., and Wang W.F., 2025, Cloning and functional analysis of key genes involved in anthocyanin biosynthesis in Morella rubra, Tree Genetics and Molecular Breeding, 15(3): 98-107 (doi: 10.5376/tgmb.2025.15.0012) Abstract This study reviews the research achievements made in recent years regarding the synthesis of anthocyanins in Morella rubra. Key genes such as CHS, CHI and F3H play an important role in the synthesis process of anthocyanins. In addition, some factors that regulate these genes, such as R2R3-MYB, bHLH and WD40, also play a role in the regulatory process. There is also a mutually cooperative relationship among them. To figure out how these genes work, researchers employed methods such as cloning genes, analyzing expression patterns, and also studied the epigenetic regulation of genes. This study also introduces the application prospects of molecular breeding, genetic engineering and CRISPR technology in improving the color and nutritional value of Morella rubra. It is hoped that these achievements can provide a scientific basis for improving the quality of Morella rubra and also bring practical methods for planting and breeding. Keywords Morella rubra; Anthocyanin biosynthesis; MYB transcription factor; Molecular breeding; Epigenetic regulation 1 Introduction Morella rubra is a common fruit tree, and its fruits come in a variety of colors, ranging from white, red to deep purplish red. These color differences are mainly due to the varying content of anthocyanins in the fruits. Anthocyanins not only make the fruit look good and improve its appearance, but also are beneficial to human health and can help prevent some diseases. They are very important nutrients in Morella rubra (Niu et al., 2010; Shi et al., 2021). Therefore, the amount of anthocyanins directly affects the quality and nutritional value of Morella rubra (Duan et al., 2015; Li et al., 2023). Anthocyanins are synthesized through a process called the “biosynthetic pathway”, which requires some structural genes, such as CHS, CHI, F3H, F3’H, DFR, ANS and UFGT. The expression of these genes is regulated by some transcription factors, especially the proteins of the MYB, bHLH and WD40 families (Liu et al., 2013b; Yan et al., 2021). In Morella rubra, some R2R3-MYB transcription factors such as MrMYB1 and MrMYB9 can promote anthocyanin synthesis, while MrMYB6 has the opposite effect and inhibits anthocyanin accumulation (Shi et al., 2021; Li et al., 2023). In addition, MrbHLH1 and MrWD40-1 can also cooperate with MYB protein to better promote the formation of anthocyanins (Liu et al., 2013a). The expression of these genes varies in different varieties and fruit development stages, which determines the depth of fruit color and the amount of anthocyanins (Niu et al., 2010; Duan et al., 2015). This study mainly aims to identify these key genes and investigate how they affect the color change of Morella rubra. Through the cloning research of some key genes, it explores their expression and role during the fruit development process. This study hopes that these results can provide assistance in improving the fruit color and nutritional value of Morella rubra, and also offer tools and theoretical support for future molecular breeding. 2 Biology and Distribution of Morella rubra 2.1 Botanical characteristics and genetic background Morella rubra is a common evergreen fruit tree in southern China. It is dioecious, that is to say, the male and female plants are separate, and only the female plants can bear fruit. Research has found that female plants have a slightly higher number of alleles and heterozygosity, but the difference is not significant compared to male plants.
Tree Genetics and Molecular Breeding 2025, Vol.15, No.3, 98-107 http://genbreedpublisher.com/index.php/tgmb 99 Although the overall differences are not significant, female and male strains can be clearly distinguished by some specific gender markers (such as ZJU062 and ZJU130). Zhejiang is the region with the richest genetic diversity of Morella rubra. Varieties such as ‘Biqi’, ‘Dongkui’ and ‘Pink’ are derived from different genetic resources (Jia et al., 2015). Now, the genome, transcriptome and germplasm resources of bayberry have been sorted out and a database has been established, laying a good foundation for breeding research (Ren et al., 2021). 2.2 Geographic distribution and ecological adaptability Morella rubra are mainly distributed in the tropical and subtropical regions of our country. Due to its suitable climate, Guangdong Province is a place where a large number of Morella rubra are grown. Different varieties are distributed in 41 counties. There are also many wild Morella rubra in Guangxi, and these wild populations have a high degree of genetic diversity. Their distribution is closely related to the local climate and environment. According to the color and luster of the peel, Morella rubra can roughly be divided into three types: black, red and white. Varieties of different colors also have their own characteristics and are suitable for promotion and cultivation. From the perspective of population genetics, the reason why Morella rubra can be widely cultivated in China is their strong ability to adapt to the environment. Meanwhile, gene drift and limited gene flow also have an impact on its genetic structure. 2.3 Fruit development and pigmentation traits The flesh development process of Morella rubra is a rather complex one, which is regulated by many hormones and genes. Plant hormones such as auxin (IAA), jasmonic acid (JA), abscisic acid (ABA), and gibberellin change levels at different stages of fruit development. Especially the interaction among IAA, JA and ABA plays a particularly significant role during critical periods. The study also found that some genes, such as LAX2, LAX3 (responsible for auxin transport), JAZ6 (regulating JA signaling), KAN1 and KAN4 (involved in multiple hormone signaling), are closely related to pulp development. Immunofluorescence experiments also revealed that auxin was mainly concentrated in the vascular bundles and outer cells in the middle of the pulp. This uneven distribution might affect the shape of the pulp (Figure 1) (Fu et al., 2025). In addition, the fruit color of Morella rubra is classified into three types: black, red and white. The differences in color and luster are due to the variations in the accumulation of pigments such as anthocyanins. 3 Anthocyanin Biosynthesis Pathway 3.1 Phenylpropanoid and flavonoid pathways The synthesis process of anthocyanins is part of two pathways: phenylpropane metabolism and flavonoid synthesis. This process starts with phenylalanine and, through the action of some enzymes, will generate the precursors of flavonoids. Then, plants convert these precursor substances into various flavonoids through the flavonoid synthesis pathway, among which anthocyanins are included (Pratyusha and Sarada, 2022; Dutt et al., 2023). This path not only determines the color of the plants but is also related to their stress resistance, growth and development, and other functions. 3.2 Key enzymes: CHS, CHI, F3H, DFR, ANS, UFGT During the synthesis of anthocyanins, some structural genes play significant roles, such as CHS, CHI, F3H, DFR, ANS, and UFGT. These enzymes will transform substances one by one and finally synthesize different types of anthocyanins (Raziq et al., 2024; Zhu et al., 2025). Research has found that the expression levels of these genes can affect the content of anthocyanins, and their expressions also vary at different developmental stages or in different organs. For instance, when the fruit begins to change color, the expression of these genes will significantly increase, which is conducive to the synthesis and accumulation of anthocyanins (Dutt et al., 2023; Sun et al., 2025). 3.3 Regulatory genes: MYB, bHLH, and WD40 transcription factors In addition to structural genes, the synthesis of anthocyanins is also controlled by some regulatory proteins, mainly three types of transcription factors: MYB, bHLH and WD40. These proteins can together form a regulatory group called the MBW complex, which can directly control the expression of structural genes and is the core of the entire regulatory network (Chen et al., 2019; Jiang et al., 2023). Among them, the factors of the
Tree Genetics and Molecular Breeding 2025, Vol.15, No.3, 98-107 http://genbreedpublisher.com/index.php/tgmb 100 R2R3-MYB family play a leading role, responsible for initiating or inhibiting the expression of anthocyanidin-related genes; While bHLH and WD40 help MYB function better (Zhao et al., 2019; Peng et al., 2020; Karppinen et al., 2021). In addition, some MYB transcription factors may also be related to hormone signals or stress responses, further refining the regulation of anthocyanin synthesis (Pratyusha and Sarada, 2022). Figure 1 Immunofluorescence localization of auxin in the (a) longitudinal and (b) transverse sections of flesh segments (Adopted from Fu et al., 2025) Image caption: The bayberry variety used for the immunofluorescence localization analysis was ‘Biqi’, and all scale bars were set to 200 μm. In (a), the dashed line indicates a longitudinal section of a single flesh segment, where the auxin fluorescence signals are enriched at the top and side walls of the flesh segment, forming a continuous linear distribution along the contour marked by the dashed line. In (b), the arrow indicates the central vascular bundle within the cross-section of the flesh segment, where the enrichment of auxin fluorescence signals is observed (Adopted from Fu et al., 2025) 4 Advances in Cloning of Anthocyanin Biosynthetic Genes in Morella rubra 4.1 Gene discovery strategies and transcriptome profiling In China, researchers mainly identified the genes related to anthocyanin synthesis in Morella rubra through transcriptome sequencing and comparative analysis. They conducted transcriptome sequencing on fruits of different colors (such as white, red, and deep purple-red) and at different developmental stages, and screened out some structural genes and regulatory genes that are closely related to anthocyanin accumulation. For instance, a study identified 60 genes of the WD40 family using the RNA-Seq database and, by comparing their expression levels with anthocyanin contents, identified some of them as key regulatory factors (Liu et al., 2013a; Cao et al., 2021). In addition, by comparing the gene expression of red and white fruits, a MYB transcription factor called MrMYB9 was also discovered, which promotes anthocyanin synthesis (Li et al., 2023). 4.2 Isolation and characterization of structural genes At present, the structural genes that have been identified in Morella rubra mainly include: CHS, CHI, F3H, F3’H, DFR, ANS and UFGT (Liu et al., 2013b). The expression levels of these genes vary in different fruit colors and
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