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. 2026 Feb 25;26:460. doi: 10.1186/s12870-026-08209-2

Integrative analysis of endogenous phytohormones and transcriptomics reveals mechanism of light-induced callus formation in tea (Camellia sinensis) cuttings

Ying Gao 1,2, Xinyu Liu 2, Shuwei Yu 1,2, Yuantao Yao 2, Wei Zhang 4, Fan’an Zhang 2, Yusheng Li 3, Hanyue Wang 3, Lubin Song 1,2,✉, Xiaojia Zhang 1,2,✉
PMCID: PMC12980969  PMID: 41742034

Abstract

Backgroud

Cutting is the mainly asexual propagation method for tea plant, and moreover, callus formation is a key point for the survival rate of tea cuttings. As reported, light is crucial for callus formation of tea cuttings via influencing phytohormone contents and plant hormone signal transduction. However, the molecular mechanism of light-induced callus formation is not clear.

Results

In this study, callus growth was analyzed in short tea cuttings exposed to light conditions with/without shading. The results showed the callus formation proportion exposed to light was twice that of shading treatment on the 21st day. To discover the regulatory process of light-induced callus formation, transcriptome and phytohormone analyses of stems were performed at the 7th and 14th day. KEGG results displayed that “starch and sucrose metabolism” and “early auxin hormone signaling” were enriched by up-regulated genes in S7d_VS_L7d, while “motor proteins” and “photosynthesis-antenna proteins” were enriched by up-regulated genes in S14d_VS_L14d. Hormonal analysis displayed that the tZ, IAA and ABA were higher with light treatment in the L14d, and the tZ and IAA content increased from 7d to 14d, while ABA content decreased. Integrated transcriptomics results, the key genes of tZ and IAA biosynthesis were also up-regulated as the tZ and IAA content, and the ABA cleavage genes-abscisic acid 8’-hydroxylase (ABA8ox) were highly expressed at 14d under light treatment. Moreover, the CKs signal transduction were also promoted by light, corresponding with the expression level of genes related to cell cycle and division in cuttings under this condition. Weighted gene co-expression network analysis (WGCNA) of DEGs in turquoise module and protein-protein interaction (PPI) analysis of DEGs in L14d_vs_S14d revealed that CsMYB82 is the hub gene during light-induced callus formation, and the interaction of CsMYB82 and CsGL3 was verified via yeast two-hybrid (Y2H) assay and bimolecular fluorescence complementation (BiFC) assay. Structural analyses illustrated that CsMYB82 can interact CsGL3 via hydrogen bonds and salt bridges.

Conclusions

Through the phytohormones and transcriptome analysis, carbohydrate metabolism and phytohormones metabolism were closely correlated with light treatments. And the change of phytohormones might result in the interactions of CsMYB82 and CsGL3, leading to the cell fate transition and highly expression of genes related with cell cycle and motor proteins. This study illustrates light is an important factor for callus formation according to a complex and orderly process, and also provides an understanding of the asexual reproduction mechanism of tea plants.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08209-2.

Keywords: Light, Cell fate transition, Auxin accumulation, Cytokinin accumulation, Callus formation

Introduction

Tea (Camellia sinensis), as a beverage crop without alcohol, is popular and important in the world [1]. The widely spread nature of tea is associated with healthy attributes and unique flavors, which mainly depend on the elite varieties [2]. Due to the high self-incompatibility of tea plants, cross pollination can lead to a complex genetic variation, which is not conducive to preserving the characteristics of elite tea varieties [3]. The asexual propagation of tea plants, mainly through cuttings, has become the mainstream method for distributing tea varieties, and the key to the survival capability of cuttings lies in the formation of callus [4].

During the formation of adventitious roots, cells undergo early signaling, and then auxin accumulation, and cell fate transition under hormones or stresses [5]. The callus of cuttings is formed in the cell fate transition through regeneration-competent cell formation, fate transition, cytokinin accumulation and continuous cell division [5, 6]. The callus formation of cuttings is not only a complex and orderly process, but also affected by exogenous hormones and light of environment [4, 7, 8].

Light, as a vital environmental factor for most organisms, plays an important part in plant growth and development [9]. During cottage propagation, light is involved in carbohydrate metabolism, which is beneficial for the survival of stem cuttings [10]. The survival rate of tea cuttings under light was above 90% after 90 days, while those cultured in darkness all died [11]. Additionally, light is involved in metabolite biosynthesis, especially phytohormones. For tea cuttings, blue light could promote the contents of abscisic acid (ABA), trans-zeatin (tZ), indole-3-carboxylic acid (ICA), 5-deoxystrigol (5DS), and gibberellin 9 (GA9) for callus formation [4]. Lastly, the changes in light intensities/wavelengths are also accompanied by the alteration of expression level of genes correlated with hormone signal transduction, such as YUC, AUX/IAA, ARF, ARR, and SAUR in plant [9, 12, 13]. Therefore, light is considered as a crucial environmental factor for callus formation in cuttings via metabolite biosynthesis, and plant hormone signal transduction.

In this experiment, light (10000 lx) and shading (two layers of shade net, 2500 lx) treatment was performed on tea cuttings to investigate the development process and molecular mechanism of callus formation. RNA sequencing and hormonal analysis were conducted for exploring the process and mechanism of light-induced callus formation in tea cuttings. As for further obtaining hub genes during callus formation, WGCNA were also performed, and the dynamic changes in phytohormones and expression profile of genes in hormonal signaling pathway were analyzed. Furthermore, CsMYB82 and CsGL3 associated with cell fate transition were identified through protein-protein interaction analysis. This study provides important and valuable insights for the callus formation mechanism of tea cuttings.

Results

Callus formation conditions of tea cuttings with different light treatments

White tiny callus at the bottom of stems could be seen with the naked eye aſter incubation for 21 days under light (Fig. 1A). The proportion of cuttings with callus was up to 66.67%, significantly higher than other treatments (Fig. 1C). The cuttings with treatment L-S, S and S-L hardly formed callus, and the proportion of cuttings with callus was from 32.22% to 35.56% (Fig. 1B&C). These results indicate that light promotes callus formation in tea cuttings.

Fig. 1.

Fig. 1

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Observation of the stems of tea cuttings. A The cutting branches with light (L) and shading (S) treatment at 7 and 21 days after insertion, and white callus remarked by red circle; B Callus proportion of different treatments, L: tea cuttings under light exposure, L-S: tea cuttings under light exposure for the first week and then moved to shading, S: tea cuttings under shading, S-L: tea cuttings under shading for the first week and then moved to light; C Microscopic observation of the stem of L14d; D Microscopic observation of the stem of S14d; All bars = 500 μm

RNA sequencing and differential analysis of tea plant cuttings under different light treatments

In order to study the transcriptional differences during callus formation in different stages, DEGs between lighting and shading treatment were identified. A total of 16,997 DEGs were identified between lighting and shading treatment at three time points (Fig. 2A). Venn analysis (Fig. S1) revealed 7,046 DEGs between L7d_VS_CK0 and 7,490 DEGs between L14d_VS_L7d during callus formation in tea cuttings under lighting treatment, while the number of DEGs of S7d_VS_CK0 and S14d_VS_S7d were 6,882 and 2,963, respectively. There were only 165 DEGs in L7d_VS_S7d, while 4600 DEGs were obtained in L14d_VS_S14d. Clustering and PCA analysis (Fig. 2A&B) also indicated that the expression profile of DEGs in CK0 was obviously different from others, and the expression profile of DEGs in L7d was similar to S7d, while L14d was distinguished from S14d.

Fig. 2.

Fig. 2

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Analysis of differentially expressed genes. A Clustering analysis of differentially expressed genes; B PCA analysis of differentially expressed genes

KEGG analysis (Fig. 3) was performed to identify the function of up- and down-regulated DEGs between lighting and shading treatments. In S7d_VS_L7d, the 110 up-regulated DEGs were mainly enriched in “starch and sucrose metabolism” and “plant hormone signal transduction”, while 55 down-regulated DEGs were mainly enriched in “phenylpropanoid biosynthesis”. Through GSEA (Fig. S2) of “starch and sucrose metabolism” and “plant hormone signal transduction” between S7d and L7d, the core genes trehalose-phosphate phosphatase J (TPPJ), glucan endo-1,3-beta-glucosidase 11 (E1311), beta-amylase 3 (BAM3) and beta-glucosidase 41 (BGL41) were identified in “starch and sucrose metabolism”, and the core genes pathogenesis-related leaf protein 6 (PR6), carboxylesterase 18 (CXE18), auxin-induced protein 22D (AUX22D), indole-3-acetic acid-amido synthetase GH3.1 (GH3.1), methyl jasmonate esterase 1 (MJE1), MYB family transcription factor EFM (EFM), ethylene-responsive transcription factor C3 (ERFC3) and ethylene-responsive transcription factor 98 (ERF98) were identified in “plant hormone signal transduction” (Fig. 4). The core genes highly expressed in L7d indicate that carbohydrates and auxin signaling transductions, such as, AUX22D and GH3.1, play important roles at early stages of callus formation under light.

Fig. 3.

Fig. 3

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KEGG analysis of differentially expressed genes. A KEGG analysis of up-regulated genes of S7d_VS_L7d; B KEGG analysis of down-regulated genes of S7d_VS_L7d; C KEGG analysis of up-regulated genes of S14d_vs_L14d; D KEGG analysis of down-regulated genes of S14d_vs_L14d

Fig. 4.

Fig. 4

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Heatmaps of core genes via GSEA. A Core genes in pathway “starch and sucrose metabolism”, including TPPJ (trehalose-phosphate phosphatase J), E1311 (glucan endo-1,3-beta-glucosidase 11), BAM3 (beta-amylase 3) and BGL41 (beta-glucosidase 41); B Core genes in pathway “plant hormone signal transduction”, including PR6 (pathogenesis-related leaf protein 6), CXE18 (carboxylesterase 18), AUX22D (auxin-induced protein 22D), GH3.1 (indole-3-acetic acid-amido synthetase GH3.1), MJE1 (methyl jasmonate esterase 1), EFM (MYB family transcription factor EFM), ERFC3 (ethylene-responsive transcription factor C3) and ERF98 (ethylene-responsive transcription factor 98); C Core genes in pathway “motor proteins”, including KNs (kinesin-like protein genes), TBBs (tubulin beta chain genes), TBAs (tubulin alpha chain genes) and MYOs (myosin genes); D Core genes in pathway “photosynthesis-antenna proteins”, including CBs (chlorophyll a-b binding protein genes), LHCAs (photosystem I chlorophyll a/b-binding protein genes) and CA (chlorophyll a/b binding protein)

In S14d_VS_L14d, the most obviously enriched pathways of 2795 up-regulated genes include “motor proteins” and “photosynthesis-antenna proteins”, while “plant-pathogen interaction” and “plant hormone signal transduction” were mainly enriched by 1805 down-regulated DEGs. GSEA analysis (Fig. S2) showed that 47 DEGs of S14d_VS_L14d obtained core enrichment in “motor proteins” pathway, mainly including 34 kinesin-like protein genes (KNs), 4 tubulin beta chain genes (TBBs), 3 tubulin alpha chain genes (TBAs), and 3 myosin genes (MYOs) which are critical for cell division and indicate that cell division rate is higher in L14d than S14d. As for “photosynthesis-antenna proteins” pathway, 8 core genes were obtained, including 5 chlorophyll a-b binding protein genes (CBs), 2 photosystem I chlorophyll a/b-binding protein genes (LHCAs), and 1 chlorophyll a/b binding protein gene (CA) (Fig. 4), illustrating that light is important for callus formation. These findings indicate that light is beneficial for callus formation via promoting photosynthesis and cell division.

Hormonal analysis of tea cuttings under different light treatments

In order to detect the difference in endogenous phytohormones of tea cuttings under different light treatments, the changes of phytohormone content were comprehensively analyzed. A total of 48 phytohormones and metabolites were identified (Fig. 5), including abscisic acids (ABA, ABA-GE, ABA-ald), auxins (IAA, IBA, TRA, IAA-Glu, IAA-Ala, IAA-Asp), cytokinins (tZR, tZ, DHZ7G, DHZR, DHZROG, 2MeScZ, 2MeSiP, 2MeScZR, tZRMP, cZ, cZR, iPRMP, etc.), ethylene (ACC), gibberellins (GA19, GA29), jasmonic acids (JA, JA-Phe, JA-Val, JA-ILE, OPDA), and salicylic acids (SAG, Phe, SA, MeSAG, t-CA).

Fig. 5.

Fig. 5

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Clustering analysis of different phytohormones. A Clustering analysis of all differential phytohormones; B Clustering analysis of up-regulated phytohormones of L14d_vs_S14d; C Clustering analysis of down-regulated phytohormones of L14d_vs_S14d

In the beginning of the experiment, the contents of ABA, IAA and tZR were higher, and the contents decreased during cottage propagation, especially in tea cuttings under shading. At day 7, the contents of ABA, ABA-ald, IAA, and iPRMP were higher in tea cuttings under light than those under shading, whereas those of tZ, DHZROG, DHZ7G, tZRMP and tZR were lower in tea cuttings under light than those under shading. Compared to S14d, levels of ABA, ABA-ald, IAA, iPRMP, tZ, DHZROG, DHZ7G, tZRMP and tZR were obviously higher in the L14d, while those of IAA-Asp, IBA, 2MeSiP, cZ, cZR, 2MeScZ and OPDA were lower. Interestingly, the ABA and IAA level were all lower in L7d and L14d than CK0 in the callus formation process, but the tZ and IAA level was higher in L14d than L7d, while the ABA content slightly decreased in L14d compared with L7d. These results suggest that light could regulate the phytohormones level, such as, IAA, CK and ABA. Among them, the phytohormones IAA, CK are closely related with callus formation in tea cuttings, while the ABA content decreased as the callus formation.

Analysis of hormonal metabolism

To explore why the contents of tZ, IAA and ABA were higher with light treatment in the L14d, the key genes related with the synthesis and decomposition of CK, IAA and ABA were identified (Fig. 6). In the zeatin biosynthesis, the key genes adenylate isopentenyltransferase (IPT) and cytochrome P450 (CYP) were all up-regulated during callus formation under light. On the contrary, although the expression of IPTs increased at 7 d, but decreased at 14 d in shading treatment. As for the decomposition of CK, 3 cytokinin dehydrogenase (CKX), 9 UDP-glycosyltransferase (UGT) and 1 zeatin O-glucosyltransferase (ZOG) were found, in which most genes were lower expressed in light treatment compared with shading treatment (Fig. 18 A). All of these indicate that light can promote the zeatin biosynthesis, and CK is important for callus formation.

Fig. 6.

Fig. 6

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Heatmaps of key genes in CK, IAA and ABA metabolism. A Key genes in CK metabolism, including adenylate isopentenyltransferase (IPT), cytochrome P450 (CYP), cytokinin dehydrogenase (CKX), 9 UDP-glycosyltransferase (UGT) and zeatin O-glucosyltransferase (ZOG); B Key genes in IAA metabolism, including tryptophan aminotransferase-related protein (TAR), indole-3-pyruvate monooxygenase YUCCA (YUC), aldehyde dehydrogenase (ALDH) and 2-oxoglutarate-dependent dioxygenase DAO (DAO); C Key genes in ABA metabolism, including 9-cis-epoxycarotenoid dioxygenase (NCED) and abscisic acid 8’-hydroxylase (ABA8ox)

The genes tryptophan aminotransferase-related protein (TAR) and indole-3-pyruvate monooxygenase YUCCA (YUC), critical for IAA synthesis, were both down-regulated in L7d, and both highly expressed in L14d, but still be down-regulated in S14d. And 3 aldehyde dehydrogenase (ALDH) genes correlated with IAA decomposition showed also higher expression in the L14d than S14d, while 1 ALDH and 2-oxoglutarate-dependent dioxygenase DAO (DAO) presented an opposite trend (Fig. 6B). All of these suggest that auxin biosynthesis mainly happen in later stage of callus formation and is hindered without light.

As for ABA, the 2 key genes of synthesis, 9-cis-epoxycarotenoid dioxygenase (NCED), and 3 key genes of cleavage, abscisic acid 8’-hydroxylase (ABA8ox) all displayed higher expression level in the L14d than S14d (Fig. 6C). Additionally, one of the NCED gene was down-regulated at day 14, while the ABA8ox genes were all up-regulated at day 14, indicating that the ABA may be not needed at the later stage of callus formation.

Analysis of plant hormone signal transduction pathways

In order to further investigate the involvement of phytohormones-related genes in the callus formation of tea cuttings under different light treatment, the ABA, IAA and CK related signal transduction pathways were outlined (Fig. 7). Altogether, 58 DEGs of signal transduction pathways were obtained of C. sinensis cuttings across different phases. In auxin signal transduction, 38 DEGs were annotated. AUX1 was significantly up-regulated in L14d. Similarly, TIR1 were also up-regulated in L14d. The expression profile of AUX/IAA family genes obviously fluctuated in different treatments. Three DEGs of GH3 family were identified, while 11 DEGs of SAUR family were identified. These results suggest that AUX1, TIR1, GH3, AUX/IAA, and SAUR family genes of auxin signal transduction pathway have an important influence on callus formation.

Fig. 7.

Fig. 7

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Heating maps of DEGs associated with plant hormone signal transduction in different grades of C. sinensis cuttings. From top to bottom, the three panels show DEGs involved in the signal transduction pathways of auxins, CTKs, and ABA, respectively. The red squares are upstream and the blue squares are downstream

Moreover, 13 DEGs were obtained in the abscisic acid signal transduction pathway. Among them, 2 DEGs from the PYR/PYL family were obviously up-regulated in S14d than L14d, while one gene was down-regulated. On the contrary, 5 genes from the PP2C family obtained significantly higher expression in L14d than S14d, while 3 DEGs were down-regulated, following by the higher expression level of both ABF and SnRK2 genes in S14d. In the cytokinin signal transduction pathway, the expression level of AHP, B-ARR and A-ARR genes increased in L14d. Interestingly, both B-ARR and A-ARR genes were highly expressed in S7d but were down-regulated in S14d. The expression of genes related with cell division and cell cycle, such as cyclin-dependent kinase B2-2 (CKB22), kinesin-like protein KIN-14I (KN14I), kinesin-like protein KIN-4 C (KN4C), G2/mitotic-specific cyclin-2 (CCNB2), and mitotic spindle checkpoint protein MAD2 (MAD2), were also significantly up-regulated in L14d (Fig. S3). These results suggest that abscisic acid signal transduction is inhibited in L14d, while cytokinin signal transduction is promoted for callus formation.

Screening hub genes highly related with callus formation by WGCNA

A total of 12 modules were identified by WGCNA based on the expression profiles of 16,997 genes (Fig. 8A). Interestingly, there were 4304 DEGs in turquoise module, which were higher expressed in L14d than other samples (Fig. 8B). In the turquoise module, there were 36 DEGs correlated with KEGG pathway “plant hormone signal transduction” (Fig. 9A), while 53 transcription factors were obtained (Fig. 9B).

Fig. 8.

Fig. 8

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WGCNA analysis of differentially expressed genes. A Module clustering analysis of differentially expressed genes; B Expression pattern of differentially expressed genes in the turquoise module

Fig. 9.

Fig. 9

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Clustering analysis of differentially expressed genes in the turquoise module. A Clustering analysis of hormone related-genes in the turquoise module; B Clustering analysis of transcription factors in the turquoise module

In the turquoise module, the number of DEGs with kTotal above 1000 were 128. Among them, 5 transcription factors were obtained, including ethylene-responsive transcription factor ERF003 (ERF03), transcription factor AS1 (AS1), AP2-like ethylene-responsive transcription factor ANT (ANT), scarecrow-like protein 28 (SCL28), and transcription factor MYB82 (MYB82), while LRR receptor-like serine/threonine-protein kinase IRK (IRK) and SCL28 were related with “plant hormone signal transduction”. The expression levels of partial genes were verified by qRT-PCR (Fig. S4).

In order to further explore the key genes during callus formation in tea cuttings, PPI analysis of DEGs of L14d_vs_S14d was conducted. A total of 4,336 pairs of interacted proteins with combined score above 700 were predicted. MYB82 were also detected and correlated with GL3/EGL1 through weight value and corresponding expression profile (Fig. 10). Therefore, CsMYB82 was screened for further analysis.

Fig. 10.

Fig. 10

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Cytoscape of PPI network of DEGs in L14d_vs_S14d

GL3 interacts with MYB82

On the basis of qRT-PCR, the expression level of CsGL3 and CsMYB82 were significantly up-regulated in L7d and L14d, compared to S7d and S14d, respectively (Fig. 11A). Y2H assay was conducted to identify the correlated relationship of CsGL3 and CsMYB82. The results of Y2H indicated that CsMYB82 was interacted with CsGL3 (Fig. 11B). BiFC assay was conducted to further verify the interaction. The YNE-CsMYB82 and YCE-CsGL3 fusion proteins in tobacco leaves produced a marked fluorescence signal (Fig. 11C), while the negative control of YNE-YCE did not show yellow fluorescence signal.

Fig. 11.

Fig. 11

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Verification of interaction between CsGL3 and CsMYB82. A Verification of the expression level of CsGL3 and CsMYB82 by qRT-PCR, where different letters indicate the significant difference at p < 0.05; B Yeast two-hybrid assays to detect the interaction between CsGL3 and CsMYB82. CsMYB82 was used to fuse to GAL4 activation domain (AD) as prey and CsGL3 was used to fuse to GAL4 DNA-binding domain (BD) as bait; C The bimolecular fluorescence complementation (BiFC) assays indicate that CsGL3 interacts with CsMYB82. nYFP-CsMYB82 and cYFP-CsGL3 were co-expressed in tobacco protoplasts prepared from tobacco leaf; D Three-dimensional structure of CsGL3 (green) and CsMYB82 (blue); E The salt bridges and hydrogen bonds between CsGL3 and CsMYB82; F The hydrogen bonds between CsGL3 and CsMYB82; G The salt bridges and hydrogen bonds between CsGL3 and CsMYB82. Scale bar = 50 μm

To explore the interaction information, AlphaFold software was utilized for establishing the three-dimensional model of CsGL3 and CsMYB82 (Fig. 11D). In the model with high confidence (0.9368), the side chains of THR135 and GLN137 in CsMYB82 can interact with the C-terminal side chains ASP149 and ARG132 of CsGL3 via hydrogen bonds (Fig. 11F). CsMYB82 can also interact with N-terminal of CsGL3 via hydrogen bonds and salt bridges. The hydrogen bonds formed by side chains of GLU18, MET68, ASP70, GLN116, ASN146, LYS24, ARG67, MET68, LYS73, ARG109, ARG113 and GLN116 of CsMYB82 and ARG486, ARG612, ARG604, ARG467, GLU520, SER532, ASN533, GLN608, VAL536, GLN608 and VAL538 of CsGL3, and the salt bridges formed by side chains of ASP70, ASP148, LYS24 and ARG109 of CsMYB82 and ARG612, ARG467, GLU520 and ASP534 of CsGL3 (Fig. 11E&G). Taken together, these phenomena demonstrate that CsGL3 interacts with CsMYB82 which is important for callus formation in tea cuttings.

Discussion

Transcriptome analysis of C. sinensis cuttings subjected to different treatments

Callus formation in tea cuttings is regulated by a complex network of pathways and genes [14]. Analyzing gene expression profile among different treatments is beneficial for identifying the molecular mechanisms responsible for growth and development differences [15]. Shen et al. [4] demonstrated that, compared with red light, blue and white light could improve callus formation in tea cuttings by promoting the expression of genes related with plant hormone signal transduction, auxin synthesis and transport. Contrarily, for Picea abies, red light could promote adventitious roots likely induced by regulating jasmonate (JA) biosynthesis and cytokinins accumulation [16]. In our experiment, the KEGG pathways “starch and sucrose metabolism” and “photosynthesis-antenna proteins” were enriched by up-regulated genes on 7th day and 14th day respectively, which illustrates that carbohydrate metabolism is important for callus formation in tea cuttings for the survival of tea cuttings to form callus at an early stage. At day 7, “plant hormone signal transduction” was also enriched by up-regulated genes, and GSEA results showed that core genes AUX22D and GH3.1 were obtained, which were key genes of early auxin signaling transduction. Moreover, the AUX22D encodes AUX protein which is auxin negative response factor and GH3.1 encodes auxin amide synthetase which can result in the auxin inactivation [17], suggested that auxin negative signaling is critical for the early stage of callus formation. At day 14, “motor proteins” was significantly enriched, meanwhile, genes related with cell cycle and cell division were highly up-regulated. These findings demonstrate that carbohydrate metabolism, auxin signaling transduction, and motor proteins are needed for callus formation in tea cuttings [11], and light can promote the expression of differentially expressed genes associated with these interconnected metabolic pathways for callus formation in cuttings.

Light induces callus formation by affecting endogenous phytohormone content

Phytohormones play critical roles in the process of plant growth and development by mediating cellular processes, including the process of rooting [18]. In this study, although the content of ABA and IAA decreased during cuttage propagation, the contents of ABA and IAA in tea cuttings under light were still significantly higher than those in tea cuttings under shading, indicating that light can help the tea cuttings to maintain the ABA and IAA content during callus formation in tea cuttings.

Among all phytohormones, exogenous IAA is well-known for promoting the formation of root primordium via inducing the periclinal division of pericycle cells [19]. The IAA content of L14d was also higher than L7d, meanwhile, the key genes expression of IAA biosynthesis, TARs and YUCs [20], were inhibited at L7d and highly expressed at L14d, meanwhile, the auxin negative response genes AUX22D and GH3.1 were enriched at early stage of callus formation, illustrating that auxin plays an important role in the later stage of callus formation.

The ABA content with light/shading treatment both decreased during callus formation, indicating that the effect of ABA is gradually weakening during callus formation in tea cuttings. Additionally, the ABA content was slightly higher in light condition than shading condition. As reported, application of ABA could enhance sugar synthesis and stimulate chlorophyll, carbohydrate and nitrogen content during adventitious root formation [21, 22]. In this study, the carbohydrate metabolism related KEGG pathways “starch and sucrose metabolism” and “photosynthesis-antenna proteins” were also enriched, suggesting that ABA might improve the survival of tea cuttings to form callus by enhancing carbohydrate metabolism in tea cuttings.

Similar to auxin, cytokinin also functions in mediating plant growth and development via promoting cell division and the growth of meristematic tissues [14]. Besides, optimum CK synthesis is necessary for callus formation [23] and normal adventitious root primordia formation [24]. At day 7, there were little differences in tZ content between light and shading treatments; by day 14, the tZ content significantly increased in tea cuttings under light with increased expression of key genes IPT and CYP [25, 26], whereas the tZ content significantly decreased in tea cuttings with high content of cis-zeatin (cZ) under shading, indicating that zeatin biosynthesis was not inhibited in the absence of light, but more cZ formed which may play housekeeping roles not organ development [27]. Meanwhile, as the CK content significantly increased, the auxin/CK ratio gradually decreased under light treatment, indicating that light could regulate the balance of phytohormones to form callus [28].

Light induces callus formation by affecting plant hormone signal transduction in tea cuttings

Thus far, research on adventitious root inducing has mainly focused on the synthesis, transport and signal transduction of phytohormones [14]. Among all phytohormones, auxin is a key hormone, and the auxin signaling pathway is involved in cell fate transition to regeneration-competent cells, constituting the first step of callus formation [5, 29]. Several types of auxin response factors are involved in the auxin signaling transduction pathway, such as those in the AUX/IAA family, ARF family, GH3 family, and SAUR family [30] fourteen auxin respective genes (ARFs, AUX/IAAs, and GH3s), and 3 auxin transporters (AUX22) have been found to be up-regulated during the callus formation process in tea cuttings [7]. In Arabidopsis, IAA14, interacting with ARF7 and ARF19, is involved in the callus formation of rooting [29]. Without exogenous auxin, strong light intensity up-regulated the expression of CpARF5-7 to promote rooting [31]. In this study, additional genes related with auxin signaling were identified, illustrating that the mechanism of light-mediated adventitious root development via the auxin signaling transduction pathway is important and complex [32].

As for ABA signal transduction, PYR/PYL are candidate ABA receptors that mediate the downstream genes of ABA signaling to regulate adventitious root development [33]. In our experiment, the expression of 2 PYR/PYLs and the downstream genes of SnRK2 and ABF were inhibited under light treatment, suggesting the weakening of ABA signaling.

CKs signal transduction also play a critical role in the callus formation process. ARRs acted as downstream of cytokinin signaling, including type-A/B/C ARRs [34]. And type-A ARRs also play important roles in light signaling pathways [35]. However, B-ARRs and A-ARRs processed higher expression under light treatment than under shading treatment on the 14th day, contrary to the situation on the 7th day. The above results indicated that CKs signal transduction may be also critical for the later stage of callus formation during the induction of adventitious roots in cuttings under light.

Importance of CsGL3 and CsMYB82 during callus formation in tea plant cuttings

During callus formation in tea cuttings, MYB82 were found to interact with GLABRA3 (GL3). MYB82 could regulate trichome development by interacting with GL3 [36]. GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3), encoding a bHLH transcription factor, mainly participate in root hair development, trichome branching and DNA endoreduplication [37]. The 27 cis-acting elements in promoter region of GL3s are responsive to SA, ABA, MeJA, and GA [37–39]. Research has also found that the expression of GL3 could be regulated by IAA to control metabolite biosynthesis [40]. And the auxin-responsive element (TGA-element) and cis-acting element involved in the abscisic acid responsiveness (ABRE-element) were both found in the promoter of CsGL3 gene (Fig. S5). In Arabidopsis, GL3 and EGL3 act together with the incomplete MYB protein CPC in the H position, blocking non-hair cell fate and leading to the hair cell decision [41]. During adventitious root regeneration, callus formation is considered as a vital step of cell fate transition phase which is triggered by auxin accumulation [5, 29]. In this experiment, the ABA content and ABA signaling were both weaken during callus formation, but the IAA content was higher in tea cuttings under light than under shading, while CsGL3 and CsMYB82 were both highly expressed under light treatment, especially on the 14th day, suggesting that CsGL3 and CsMYB82 might be regulated by IAA to participate in the cell fate transition to form callus.

Conclusions

In summary, light treatments, transcriptomics, and hormonal analysis were integrated to uncover the potential mechanisms of light-induced callus formation in tea cuttings.

The results indicate that light is a necessary factor to affect carbohydrate related metabolism-starch and sucrose metabolism, which was critical for the survival of tea cuttings at the early stage. And photosynthesis, as the energy source at later stage, was also closely correlated with light. Simultaneously, the metabolism of endogenous phytohormones, such as auxins, CKs, and ABA, are also affected by light. Integrated transcriptomics and hormonal analysis revealed that auxin signal transduction was more important at the early stage of callus formation, and negative auxin response factor inhibited IAA biosynthesis at the early stage. As the tZ and IAA content increasing, CKs signal transduction also played an important role at the later stage of callus formation, while ABA content and signaling became weaken during callus formation.

Subsequently, changes in hormone levels and ratios result in the increased expression of genes CsMYB82 and CsGL3, and the interaction of CsMYB82 and CsGL3 might participate in the cell fate transition of stems combined with carbohydrate metabolism and hormone signal transduction, leading to the up-regulated expression of genes involved in cell cycle and motor proteins. All of these observations suggest that light-induced callus formation in tea cuttings is a complex and hierarchical process. These findings of this paper provide a crucial foundation for understanding the molecular genetic regulation of light-induced callus formation in tea cuttings (Fig. 12).

Fig. 12.

Fig. 12

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A probable model diagram of light promoting callus formation in tea cutting. As for carbohydrate metabolism, genes in KEGG pathway “Starch and sucrose metabolism”-TPPJ, E1311, BAM3 and BGL41 at early stage and genes in KEGG pathway “Photosynthesis-antenna proteins”- CBs, LHCAs and CA at later stage were important for light induced callus formation; As for plant hormone signal transduction, core genes AUX22D and GH3.1 related with “auxin signal transduction” were obtained at early stage which negatively regulate auxin biosynthesis, and AHP, B-ARR and A-ARR related with “CK signal transduction” were up-regulated at later stage following by tZ accumulation; the accumulation of tZ and IAA at later stage resulted in the change of phytohormone levels and ratios under light treatment, leading to the interaction of CsGL3 and CsMYB82. Furthermore, various metabolisms and changes of phytohormones ratio led to the cell fate transition of stems, subsequently, the cell cycle and cell division related genes were highly expressed, such as, CKB22, CCNB2, and MAD2, KNs, TBBs, TBAs and MYOs. Lastly, all of these led to the light induced callus formation during cuttage

Materials and methods

Plant materials and light treatment

The 90 tea branches of multi-year-old tea plant were obtained from the Jiangshi tea garden of Linyi for each treatment, which were then cutted into 3–4 cm half-lignified cuttings with a plump axillary bud. The cultivar of tea cuttings was ‘Zhongcha108’. Tea cuttings were cultured with perlite (about 1.5 cm insertion depth) and cultured with (S)/without shading (L) (two layers of shade net) in the greenhouse of the Shandong Academy of Agricultural Sciences, where the relative humidity was kept at 70%. Four treatments for tea cuttings were set up in this experiment, including cuttings under light (L), and those under light for the first week and then moved to shading (L-S), those under shading (S), and those under shading for the first week and then moved to light (S-L). The samples were obtained at day 0 (CK0), day 7 (L7d&S7d) and day 14 (L14d&S14d). The samples of L14d and S14d were embedded in paraffin and stained with toluidine blue, and photos were taken by Nikon Eclipse C1. The lower part (about 1 cm) of 25 stems in the similar position was obtained from each sample, and stored at −80℃ for further study. The callus formation rates were calculated day 21. Samples were in triplicate in all groups of the experiment.

Phytohormone extraction and detection

The contents of auxin, CK, GA, JA, SA, ABA, BR, MLT, and ACC were detected by Wuhan Metware Biotechnology Co., Ltd. (Wuhan, China). Firstly, 50 mg powder of samples were mixed with 1 ml methanol/water/formic acid (15:4:1, v/v/v), and then added with 10 µL internal standard mixed solution (100 ng/mL). After vortex for 10 min and centrifugation for 5 min at 4 °C,12,000 r/min, the supernatant were taken and concentrated; Subsequently, 100 µL 80% methanol/water solution was used for redissolving, then filtered through a 0.22 µM filter, and placed in the injection bottle for LC-MS/MS analysis. A MWDB (Metware Database) database based on standard samples were constructed and qualitative analysis on mass spectrometry detection data were performed [42, 43].

An UPLC-ESI-MS/MS system were used for analyzing the sample extract. Ultra Performance Liquid Chromatography (UPLC) (ExionLC™ AD, AB Sciex Pte. Ltd., Shanghai, China) and Tandem Mass Spectrometry (MS/MS) (QTRAP® 6500+, AB Sciex Pte. Ltd., Shanghai, China) were used for data collection. As for UPLC analysis, a Waters ACQUITY UPLC HSS T3 C18 column (1.8 μm, 100 mm x 2.1 mm i.d.) was adopted, where the mobile phase A was ultrapure water with 0.04% acetic acid, while the mobile phase B was acetonitrile with 0.04% acetic acid. The gradient elution time program was as follows: in the first 1 min, the Phase A concentration was 95%, and which decreased from 95 to 5% in the next 7 min, was subsequently maintained at 5% for another 1 min, then shifted back to 95% in 0.1 min and held for another 3 min. The analysis parameters were set as: oven temperature 40 °C, total flow 0.35 mL/min, injection volume 2 µL [44–46].

The QTRAP® 6500 + LC-MS/MS System and scheduled multiple reaction monitoring (MRM)was used for phytohormones detection and analysis. Multiquant 3.0.3 software (Sciex) was utilized to quantify all metabolites. The mass spectrometry conditions mainly included the following: The electric spray ion source (ESI) temperature was 550 °C; the positive and negative ion mode voltage was 5500 V and − 4500 V, respectively; the Curtains Gas (CUR) was 35 psi [47–49].

RNA extraction and sequencing

An RNAprep Pure Plant Kit (Tiangen Biotech Co., Ltd., Beijing, China) was utilized on RNA extraction following by the manufacturer’s protocol. Nanodrop 8000 Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and Qubit 4.0 fluorometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) were used for analyzing the purity and concentration of RNA, respectively. RNA integrity was detected by a Qsep400 Bioanalyzer (BiOptic BIO-TECH. Inc., Jiangsu, China) using a Qubit 4 System Verification Assay Kit.

After the construction, quality-controlled and quantified of RNA libraries completed, RNA library sequencing was conducted by Wuhan Metware Biotechnology Co., Ltd. (Wuhan, China). Illumina Novaseq 6000 System was used for library sequencing with 150 bp paired-end reads, controlled by Q20 and GC content distribution. Cutadapt software (v1.9.3) was used for trimming 3’ adaptor and removing low-quality reads. Then, the clean reads were aligned to the reference genome (CSS_ChrLev_20200506_Genome.fas.gz) (http://tpia.teaplants.cn/) [50, 51] using HISAT2 software (v2.0.4). The expression level of genes was quantified using featureCounts to calculate the gene alignment statistics. Subsequently, FPKM (Fragments Per Kilobase Million) values for calculating gene expression levels were computed based on gene length. DESeq2 (v1.22.1) was used to conduct differential expressed genes (DEGs) analysis (FDR-adjusted P-values < 0.05 and |log2(fold change) |> 1). The enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was conducted on the differentially expressed genes. For gene set enrichment analysis GSEA was performed via the gsea tool [52].

Weighted gene co-expression network analysis (WGCNA) and protein-protein interaction (PPI) network analysis of DEGs

First, genes with low and unstable expression level were removed by the varFilter function of the R language ‘genefilter’ package (v1.90.0). WGCNA R package (v1.71) was used for dividing genes into different modules based on similar expression spectra [53, 54]. The analysis of protein interactions for DEGs was based on the STRING database. Diamond (v2.0.9) was utilized for aligning protein sequences of reference species with STRING database to obtain protein interaction relationships.

Cytoscape software (ver. 3.8.2) was used to construct the weighted gene co-expression network and predicted protein-protein interactions.

Quantitative real-time PCR analysis

qRT-PCR was employed to determine the expression of genes (Table S1) using CFX™ 384 Touch (Bio-Rad Laboratories, Inc, Hercules, CA, USA) and PerfectStartTM Green qPCR SuperMix (+ Dye II) (Beijing Transgen Biotechnology Co., Ltd, Beijing, China) as previously reported [55]. The PCR cycling conditions were as follows: 40 cycles at 94 °C for 30 s, 94 °C for 5 s, and 60 °C for 30 s. The relative gene expression levels were quantified by the 2−ΔΔC method using theβ-actin gene as the control [6]. qRT-PCR analysis was performed in triplicate.

Yeast two-hybrid (Y2H) assay

To verify the interaction between CsGL3 and CsMYB82, the ORFs of the CsGL3 and CsMYB82 genes were inserted into the pGBKT7 and pGADT7 vectors, respectively. The controls were empty pGADT7 with CsGL3- pGBKT7 and pGBKT7 vectors with CsMYB82- pGADT7 vectors. After co-transformed into the yeast strain AH109 and cultured on medium lacking Trp and Leu (-T-L) for 3 days at 30, the yeast cells were then moved to medium lacking Trp, Leu, His and adenine (-T-L-H-A) with X-gal for 5–7 days at 30 °C.

Bimolecular fluorescence complementation (BiFC) assay

The full-length cDNA sequences of candidate genes CsGL3 and CsMYB82 were cloned into the YCE and YNE vectors, respectively. YCE-CsGL3 was paired with YNE -CsMYB82 and nYFP- OsHAL3 was paired with YCE-OsHAL3 (positive control) then co-transformed into N. benthamiana leaves. The YFP fluorescence of tobacco leaves was imaged 48–72 h after infiltration using a Leica STELLARIS 8 confocal laser-scanning microscope by 488 nm excitation wavelength and 500–542 nm fluorescence.

Structural analyses of key proteins

The AlphaFold 2 software [56] was employed to model three-dimensional structures based on the protein sequence. Then, the HDOCK software [57] was utilized for protein-protein docking to yield complex structures screened by their respective confidence scores (> 0.7). Lastly, PyMOL software was used to delve into the binding site of the protein-protein complex [58].

Data analysis

All experiments were conducted independently at least three times. Duncan’s multiple range test by SPSS 20 (IBM Corp., Armonk, NY) software were utilized for data analysis and significant differences were identified by P-values < 0.05 between the different treatments.

Supplementary Information

Supplementary Material 1. (709.1KB, docx)

Acknowledgements

We would like to thank Linyi Jiangshi Tea Plant for their provision of tea branches.

Authors’ contributions

Y.G. mainly designing of the study, operating the experiments, interpretation of the results, writing the article; X.L. operating the experiments; S.Y., Y.Y., W.Z., F.Z., Y.L., and H.W. interpretation of the results; X.Z. designing of the study, operating the experiments, reviewing and polishing the article; L.S. designing of the study, preparation of the manuscript. All authors reviewed the manuscript.

Funding

This work was funded by the Shandong Provincial Natural Science Foundation (ZR2024QC270 and ZR2023QD085), Shandong-Chongqing Technology Collaboration Project (2024LYXZ025), Agricultural Science and Technology Innovation Project of the Shandong Academy of Agricultural Sciences (CXGC2024D15 and CXGC2025G10), the Technology System of Modern Agricultural Industry in Shandong Province (SDAIT-19-02) and the Project of Improved Agricultural Varieties in Shandong Province (2024LYXZ025).

Data availability

The short and long RNA-Seq data presented in the study are deposited in the NCBI repository, accession number PRJNA1279074. The other datasets generated and/or analysed during the current study are also available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Clinical trial

Not applicable.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Lubin Song, Email: songlubin@saas.ac.cn.

Xiaojia Zhang, Email: zhangxiaojia871230@163.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1. (709.1KB, docx)

Data Availability Statement

The short and long RNA-Seq data presented in the study are deposited in the NCBI repository, accession number PRJNA1279074. The other datasets generated and/or analysed during the current study are also available from the corresponding author on reasonable request.

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