
==== Front
BMC Plant Biol
BMC Plant Biol
BMC Plant Biology
1471-2229
BioMed Central London

5589
10.1186/s12870-024-05589-1
Research
Gibberellin 2-oxidase 1(CsGA2ox1) involved gibberellin biosynthesis regulates sprouting time in camellia sinensis
Qiu Ziyuan 1
Guo Wenhui 1
Yu Qian 1
Li Dongxue 1
Zhao Mengjie 1
Lv Han 1
Hua Xuewen 1
Wang Yu 2
Ma Qingping maqingpingtea@163.com

1
Ding Zhaotang dzttea@163.com

3
1 https://ror.org/03yh0n709 grid.411351.3 0000 0001 1119 5892 College of Agronomy, Liaocheng University, Liaocheng, 252000 China
2 https://ror.org/051qwcj72 grid.412608.9 0000 0000 9526 6338 College of Horticulture, Qingdao Agricultural University, Qingdao, 266109 China
3 grid.452757.6 0000 0004 0644 6150 Tea Research Institute, Shandong Academy of Agricultural Sciences, Jinan, 250100 China
17 9 2024
17 9 2024
2024
24 86925 5 2024
12 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Background

Tea is an important cash crop and buds are its main product. To elucidate the molecular mechanism of the sprouting time of tea plants, ‘Yuchunzao’, which was an early sprouting tea cultivar, was studied. ‘Echa 1’, sprout one week later than ‘Yuchunzao’ in spring, was used as the control.

Results

A total of 26 hormonal compounds and its derivatives in tea plants were qualified by using Ultra Performance Liquid Chromatography-Tandem mass spectrometry (UPLC-MS/MS). The result showed that GA20, GA3 and ICA were significantly different in ‘Yuchunzao’ than in ‘Echa 1’, with GA20 and GA3 up-regulated and ICA down-regulated. Based on the Illumina platform, transcriptome analysis revealed a total of 5,395 differentially expressed genes (DEGs). A diterpenoid biosynthesis related gene, gibberellin 2-oxidase 1 (CsGA2ox1), was downregulated in ‘Yuchunzao’ compared to ‘Echa 1’. CsGA2ox1 regulate the transformation of GA different forms in plants. The relative expression of CsGA2ox1 showed an adverse trend with the content of GA20 and GA3. Our results suggest that down regulation of CsGA2ox1 resulted in the accumulation of GA3 and GA20, and then promoted sprout of ‘Yuchunzao’.

Conclusion

This study provides theoretical basis of tea plants sprout and guides the tea breeding in practice.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-024-05589-1.

Keywords

Camellia sinensis (L.)
Endogenous hormone
Germination
Transcriptome
Gibberellin 2-oxidase 1 gene
Natural Science Fund of Shandong ProvinceZR2021QC159 National Natural Science Foundation of China32302607 Youth innovation team project of Shandong Province2023KJ208 the Open Project of Liaocheng University Landscape Architecture Discipline319462212 Innovation Training Program for College StudentsCXCY2023253 issue-copyright-statement© BioMed Central Ltd., part of Springer Nature 2024
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pmcIntroduction

Tea plant [Camellia sinensis (L.) O. Kuntze] is an important economic crop in China. New shoots of tea plants are the source of tea, which is one of the most popular drinks. The sprouting time of tea plants in spring determined the harvest season. However, even under the same cultivation conditions, tea varieties that germinate early show significant difference in sprouting time from those that germinate late [1]. Tea varieties that germinate early represent a long harvest period and high economic value [2–4]. Tea buds harvested in early spring have a better flavor and thus a high price [5]. In addition, because of the low temperatures in early spring, tea harvested at this time is less threatened by pests and diseases and is of better quality [6]. The price of tea produced in spring is usually higher than that in summer and autumn [3, 6]. Thus, the sprouting time of tea plants affected the economic income largely, so tea cultivars with early sprouting time in spring were desired.

Under prolonged cold stress in winter, tea buds are in dormancy [7]. Sprouting time is related to the time when the tea plant ends its dormancy. The sprout process is complex and influenced by many factors, photoperiod and temperature are environmental factors affecting winter dormancy in tea plants [8]. While, hormone levels, enzyme activity and carbohydrate levels are internal factors affecting the release of dormancy of tea buds [9], hormone levels are the key signaling regulators [1]. However, the regulation of plant growth and development by hormones is extremely complex, with multiple hormones interacting with each other. Previous studies have reported that some phytohormones could affect the sprouting of tea plants in spring, such as gibberellin (GA), cytokinin (CK), abscisic acid (ABA) and auxin [10, 11]. Of these, ABA could induce shoot dormancy, but GA, CK and auxin could break shoot dormancy of tea plants [11]. In a previous study, the expressions of several auxin transport related genes were consistent with the indole acetic acid (IAA) content changes and the active-dormant status transition in tea buds [8]. ABA levels and ABA/IAA ratio decreased while IAA levels increased in tea buds from dormancy to sprout stage [12], suggesting that the catabolic release of ABA as well as the accumulation of IAA promotes bud dormancy release. Tea bud sprouting was related to the ratio of hormones such as zeatin/ABA rather than the absolute amount of a particular hormone [13], and zeatin is one type of CK. In a previous study, zeatin, GA and IAA levels were significantly higher in the tea tree sprouting stage than in the dormant stage [1], indicating that these hormones have a positive effect on bud sprouting. In addition, exogenous application with the combination of GA and CK could also break dormancy early in tea shoots [14]. In plants, there are many GA types, and they have different activities and functions [15–17]. It is still unclear that which GA type contributes to the sprouting of tea plants. Although many studies have been conducted on dormancy and release of tea buds, the mechanism of bud sprouting is not yet known in tea plants.

In this study, an early sprouting tea cultivar ‘Yuchunzao’ and the control tea cultivar ‘Echa 1’ were used to interpret the sprouting mechanism of tea plants. Under the same conditions, ‘Yuchunzao’ sprouted approximately one week earlier than ‘Echa 1’. The content of endogenous hormonal compounds in the shoots of both varieties was examined by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. Meanwhile, the transcriptome sequencing analysis was carried out to find the differentially expressed genes related to sprouting of tea plants. The contents of endogenous hormonal compounds and the gene expressions of differentially expressed genes was compared to reveal the molecular mechanism of the early sprout of ‘Yuchunzao’. This study will provide a theoretical basis for regulating the sprouting time of tea plants.

Materials and methods

Plant materials

Two tea cultivars ‘Echa 1’ and ‘Yuchunzao’ were used as materials, and ‘Echa 1’ was used as the control. One year old plants of both tea cultivars were cultivated in the greenhouse (16 h light) with 25 ± 5 ℃ and 70% humidity. The apical shoots with one bud and two leaves for ‘Yuchunzao’ and the bud of ‘Echa 1’ at the same time on April 8, 2021 were picked and frozen in liquid nitrogen. These samples were stored at -80 ℃ for further transcriptome analysis and hormone detection. Three biological replicates of each cultivar were conducted. A total of ten buds were harvested for each biological replicate.

Extraction and detection of hormonal compounds

The samples were grind to powder with 30 Hz for 1 min. A total of 50 mg powder was extracted with methanol: water: formic acid (15:4:1 v/v). The concentrated extracts were re-dissolved in 100 µL of 80% methanol solution, and then filtered through a 0.22 μm PTFE membrane. Two microliter liquid was injected in an LC-ESI-MS/MS system (HPLC, Shim-pack UFLC SHIMADZU CBM30A system; MS, Applied Biosystems 6500 Triple Quadrupole). Waters ACQUITY UPLC HSS T3 C18 column (1.8 μm, 2.1 mm ×100 mm) was used for isolation of hormones. Mobile phase A was pure water and mobile phase B was acetonitrile with 0.05% formic acid. The gradients of the mobile phase were as follows: 95% A in 0 min and maintained for 1 min, down to 5% A in 8 min and maintained for 1 min, increased to 95% A immediately and maintained for 3 min. The temperature was performed at 40 oC and the flow rate was set at 0.35 mL/min. The effluent was alternately connected to the ESI-triple quadrupole-linear ion trap (QTRAP)-MS. The AB 6500 QTRAP LC/MS/MS system is equipped with an electrospray ionization (ESI) Turbo Ion Spray interface controlled by Analyst 1.6 software (AB Sciex). The ESI was used with gas temperature of 500 °C, the mass spectrometry voltage of 4500 V, and curtain gas flow rate of 35 psi. The collision-activated dissociation parameter is set to medium. Peak area was recorded for quantitative analysis.

Qualitative and quantitative analysis of hormonal compounds

A total of 26 hormonal compounds were detected in tea buds. All of the standards were purchased from Olchemim Ltd. (Olomouc, Czech Republic) and Sigma (St. Louis, MO, USA). Standards diluted at 0.01 ng/ml, 0.05 ng/ml, 0.1 ng/ml, 0.5 ng/ml, 1 ng/ml, 5 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml, 200 ng/ml, and 500 ng/ml were analyzed. Standards were dissolved and diluted using acetonitrile. The standard curve and regression equation were established with the concentration of the standards as the horizontal coordinate and the peak area as the vertical coordinate (Supplementary Table 1). The content of hormonal compounds in tea shoots was calculated according to the regression equation.

RNA extraction, complementary DNA (cDNA) library construction and high-throughput sequencing

Total RNA was extracted from tea leaves according to the instructions of Plant Quick RNA Isolation Kit (Huayueyang Biotech, Beijing, China). The concentration and purity of RNA was determined using a NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE). The integrity of RNA was evaluated using an Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The cDNA library was constructed according to the spcification of the NEBNext Ultra™ RNA Library Preparation Kit for Illumina (NEB, USA). An amount of 1 µg RNA was used to construct the cDNA library. The processes were as follows: the mRNA was purified in magnetic beads with Oligo (dT) and then randomly interrupted with fragmentation buffer. The first strand cDNA was synthesized using random hexamer primer, and then RNase H and DNA polymerase I were added. End-repaired, A-tailed and sequencing ligated were performed after cDNA purification using AMPure XP beads. AMPure XP system (Beckman Coulter, Beverly, United States) was used to purify library fragments for preferential selection of cDNA fragments with a length of 240 bp. The PCR products were amplified and purified, and the quality of the libraries was assessed using an Agilent Bioanalyzer 2,100. Clusters were generated using the TruSeq PE Cluster Kit version 4-cBot-HS (Illumina, USA). The library was paired-end sequenced on an Illumina 2,500 sequencer to obtain raw reads.

Sequence quality assessment and identification of differentially expressed genes

Adapter-containing readings, ploy-N-containing readings, and low-quality readings were removed from the raw data to obtain clean reads. Q30 was used to measure the clean reads quality. The clean reads were mapped to the tea reference genome (http://tpia.teaplant.org/) via the HISAT2 (http://ccb.jhu.edu/software/hisat2/index.shtml) software [18]. The gene expression levels were assessed using fragments per kilobase of transcript per million fragments mapped (FPKM). Genes with fold change ≥ 2 and false discovery rate (FDR) < 0.01 were categorised as differentially expressed genes by DESeq2 software [19]. Genes were functionally annotated based on the following databases: NCBI non-redundant protein sequences (Nr; ftp://ncbi.nih.gov/blast/db/), Swiss-Prot (http://www.uniprot.org/), Gene ontology (GO; http://www.geneontology.org/), Clusters of Orthologous Groups of proteins (COG; http://www.ncbi.nlm.nih.gov/COG/), protein family (Pfam; http://pfam.xfam.org/), and Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/).

Sequence alignment and phylogenetic Tree Construction

To understand the structure of gibberellin 2-oxidase (GA2ox), the protein sequences of GA2ox from tea plant and 10 other different plants were compared to each other using BLAST (http://www.ncbi.nlm.nih.gov/BLAST) with default parameters. GA2ox putative proteins from tea plant and 29 other species were used to construct a phylogenetic tree using the neighbour-joining method via Mega X with 500 bootstrap replications [20]. The motifs were identified using the MEME (http://meme-suite.org/tools/meme) website.

Quantitative real-time PCR analysis

Quantitative real-time PCR (qRT-PCR) was performed on the Bio-Rad CFX96 system to verify the accuracy of the transcriptomic data. And 8 different genes were selected randomly to validate the expression levels. Primers were designed by AlleleID 6.0 software (Supplementary Table 2). The CsGAPDH was used as a reference gene. The procedure was as follows: 95 °C for 30 s; 95 °C for 5 s; 60 °C for 30 s for a total of 40 cycles. 0.8 µL primer (10 µM), 50 ng cDNA and 12.5 µL 2X SYBR Green Fast qPCR Mix (Biomarkers, China) were included in the total volume of the 25 µL reaction volume. The 2−ΔΔCt method was used for the relative expression of genes [21].

Statistical analysis

The SPSS 23.0 (https://www.ibm.com/support/pages/spss-statistics-230-fix-pack-3) and Microsoft Excel 2016 were used for statistical analysis. Differences between groups were evaluated using t test and P < 0.05 was considered to be significantly different.

Results

Quantitative analysis of hormonal compounds

In tea plants, ABA accounted for 1.03–1.57 µg/g, followed by 1-Aminocyclopropanecarboxylic acid (ACC) of 159–210 ng/g and salicylic acid (SA) of 108–166 ng/g. Among the GAs, GA20 was present at the highest level of 8–21 ng/g; GA1, GA4, GA7, GA9, GA19, GA24, GA15 and GA53 were not detected in tea plants. For auxins, IAA showed the highest level of 28–43 ng/g, but 3-Indolebutyric acid (IBA) was not detected. For CK, trans-Zeatin (tZ) showed the highest content of 0.35–0.64 ng/g, but cis-Zeatin (cZ) and Dihydrozeatin (DZ) were not detected. In addition, jasmonic acid (JA) accounted for 20–106 ng/g in tea plants (Fig. 1).

In comparison, only the hormonal compounds ACC, SA, GA3 and GA20 were higher in ‘Yuchunzao’ compared to ‘Echa 1’. All other hormonal compounds were lower in ‘Yuchunzao’ compared to ‘Echa 1’. Among them, GA3, GA20 and Indole-3-carboxylic acid (ICA) levels were significantly different between two tea varieties. Moreover, the ratio of GA/ABA was higher in ‘Yuchunzao’.

Fig. 1 Phenotypes of ‘Yuchunzao’ (left) and ‘Echa 1’ (right) and the content of selected endogenous hormone compounds in ‘Yuchunzao’ and ‘Echa 1’. IAA: Indole-3-acetic acid, ME_IAA: Methyl indole-3-acetate, ICAld: Indole-3-carboxaldehyde, ICA: Indole-3-carboxylic acid, ACC: 1-Aminocyclopropanecarboxylic acid, IP: N6-Isopentenyladenine, tZ: trans-Zeatin, MEJA: Methyl jasmonate, JA: Jasmonic acid, JA_ILE: Jasmonoyl-L-Isoleucine, SA: Salicylic acid, ABA: Abscisic acid, GA3: Gibberellin A3, GA20: Gibberellin A20. The * indicates significant difference, ns indicates non-significant difference

Quality assessment of transcriptomic raw data

A total of 42.94 Gb of clean data was obtained after removing the reads containing adaptors and low-quality sequences (NCBI SRA accession: PRJNA1005819). Clean data reached 6.42Gb for each sample. The GC percentage was over 45.03% for all samples in clean data. The Q30 for all samples was more than 94.15%, which represents high quality. Over 87.10% the clean reads were mapped to the tea genome, with 74.73–75.15% unique mapped readings (Table 1). In conclusion, these data indicate that the quality of sequences is reliable and can be used for further analysis.

Table 1 Quality of the sequencing data

Samples	Total reads	Clean reads	% ≥ Q30	Mapped reads	Uniq mapped reads	
Yuchunzao-1	45,617,018	22,808,509	94.15%	88.07%	75.13%	
Yuchunzao-2	49,605,490	24,802,745	94.21%	87.95%	75.05%	
Yuchunzao-3	48,833,214	24,416,607	94.33%	88.15%	75.15%	
Echa 1–1	52,716,790	26,358,395	94.58%	87.54%	74.73%	
Echa 1–2	47,396,402	23,698,201	94.20%	87.54%	75.00%	
Echa 1–3	42,902,610	21,451,305	94.74%	87.10%	75.12%	

Identification and functional annotation of differential expressed genes

A total of 5,395 DEGs were identified between the buds of ‘Yuchunzao’ and ‘Echa 1’, including 2,950 down-regulated genes and 2,445 up-regulated genes. Functional annotation analysis revealed 1,815, 3,227, 1,972, 3,762 and 4,828 DEGs in the COG, GO, KEGG, Swiss-Prot and NR databases, respectively. A total of 8 DEGs (CSS0017652, CSS0050504, CSS0039293, CSS0011049, CSS0030210, CSS0017383, CSS0023205 and CSS0037997) were randomly selected for qRT-PCR analysis in order to verify the reliability of DEGs RNA-seq data. The results showed that expression of most of DEGs showed consistency between RNA-seq analysis and qRT-PCR verification. RNA-seq analysis was proved to be reliable (Fig. 2).

Fig. 2 Quantitative real-time (qRT-PCR) verification for randomly selected differentially expressed genes (DEGs). Fold change of FPKM represents RNA-seq data. 2–ΔΔCt represents qRT-PCR data. The * indicates significant difference, ns indicates non-significant difference

Based on KEGG analysis, Metabolism related DEGs were mostly enriched, especially for amino acid biosynthesis, carbon metabolism, starch and sucrose metabolism associated DEGs. For Plant hormone signal transduction, a total of 40 DEGs were identified (Fig. 3). Of them, only one GA synthesis related gene was identified, which was gibberellin 2-oxidase 1 (CsGA2ox1, CSS0002801). It showed lower expression in ‘Yuchunzao’ than in ‘Echa 1’ (Log2FC -3.42).

Fig. 3 Enrichment of DEGs in different KEGG pathways

In the COG functional classification, DEGs were divided into 26 clusters, and CsGA2ox1 was annotated in “Secondary metabolites biosynthesis, transport and catabolism” class (Supplementary Fig. 1A). In GO classification analysis, CsGA2ox1 was simultaneously annotated in iron ion binding, GA catabolic process and oxidation-reduction process (Supplementary Fig. 1B).

Participation of the CsGA2ox1 gene as a component of the GA synthesis in the diterpenoid biosynthesis pathway

In the present study, the CsGA2ox1 was found to be down-regulated in buds of ‘Yuchunzao’, compared to ‘Echa 1’. The predicted CsGA2ox1 protein contained the specific structural domain 2OG-FeII_Oxy which was conserved in the 2OG-FeII_Oxy superfamily (Fig. 4A). CsGA2ox1 was found to be have three conserved motifs, and location of these motifs were consist with 2OG-FeII_Oxy domain (Fig. 4B).GA2ox catalyzes bioactive GA and direct precursors of GA to be inactive GA [22]. CsGA2ox1 enzymes catalyze the formation of GA8, GA34, GA51 and GA29 from GA1, GA4, GA9 and GA20, respectively. In the present study, GA3 and GA20 were accumulated but the expression of CsGA2ox1 was down regulated in ‘Yuchunzao’ (Fig. 4C). In addition, phylogenetic tree showed that CsGA2ox1 was more closely related to the orthologs from Coffea arabica, Datura stramonium, Quercus suber, Quercus robur and Actinidia chinensis var. chinensis (Supplementary Fig. 2).

Fig. 4 Protein structure analysis of GA2ox. (A) Conserved structure of CsGA2ox1. (B) Modal analysis of GA2ox. Camellia sinensis (CsGA2ox1), Actinidia chinensis var. chinensis (AcGA2ox), Diospyros kaki (DkGA2ox2), Nerium oleander (NoGA2ox2), Coffea arabica (CaGA2ox), Olea europaea subsp. Europaea (OeGA2ox), Salix suchowensis (SsGAox), Populus alba (PaGA2ox), Paeonia suffruticosa (PsGA2ox), Jasminum sambac (JsGA2ox), Sesamum indicum (SiGA2ox). (C) Involvement of CsGA2ox1 in GA synthesis in the diterpenoid synthesis pathway. Green indicates down-regulated genes. Red indicates up-regulated metabolites

Discussion

In this study, transcriptome analysis and hormonal compounds levels of the early sprouting tea cultivar ‘Yuchunzao’ and the control cultivar ‘Echa 1’ were performed to identify the key factor regulating sprouting time of tea plants in spring. Quantitative analysis of hormonal compounds showed that ICA was significantly lower in ‘Yuchunzao’, and GA3 and GA20 were significantly higher in ‘Yuchunzao’. Transcriptome analysis indicated a diterpenoid biosynthesis related gene CsGA2ox1, which was involved in GA metabolism. It was downregulated in ‘Yuchunzao’ and showed reverse trend with the content of GA20 and GA3.

The content of major hormonal compounds in the new shoots varies in plants. In Castanea mollisima, the dominant hormonal compounds were JA-ILE, SA and JA [23]. In blueberry flower buds, the major hormonal compounds were JA, ABA and ACC [24]. In Phalaenopsis, ABA, IAA and SA accounted for the highest levels [25]. In tea buds, the major endogenous hormonal compounds were SA, JA, ACC and ABA. This result was similar to the previous studies [26, 27]. However, the most significant hormonal compounds such as ABA, ACC and SA were not significantly different between ‘Yuchunzao’ and ‘Echa 1’. Notably, ABA levels were usually considered to be negatively correlated with bud sprouting, and ABA level of ‘Yuchunzao’ was lower than that in ‘Echa 1’ in this study, which may be interpret the reason of early sprouting of ‘Yuchunzao’ to some extent. The mechanism of ABA regulating sprouting needs to be further determined. It is certain that the increase in the ratio of GA/ABA could promote the germination of cucumber and soybean seeds, and vice versa [28, 29]. In this study, the ratio of GA/ABA was higher during the tea buds of ‘Yuchunzao’ (Fig. 1), while ABA levels did not differ significantly between the two varieties, so the increase of the GA/ABA ratio was mainly attributed to the rise in GA rather than the fall in ABA. We hypothesised that GA is more closely involved in regulating sprouting than ABA. In addition, although the content of GA20 and GA3 was significantly lower than the major hormonal compounds in tea shoots, they were different significantly between two cultivars. Thus, we suggested that GA20 and GA3 might be the major hormonal compounds contributing to the sprouting of tea plants.

GA is a class of diterpenoid phytohormones that are associated with plant growth and development [22]. Furthermore, GA has been pointed out as an important factor in the release of dormancy of plant and promoting growth of shoots after the release of dormancy [30]. The low temperature was required for dormant release, and the GA partially replaced the need for low temperature resulting in the promotion of sprouting [31]. GA has more than 100 types, only GA1, GA3, GA4, and GA7 were biologically active [30]. GA3 is the most active component of the sprout-promoting GAs [32, 33]. In addition, exogenous GA3 could accelerate tea bud sprout and the growth of tea plant [34, 35]. Bioactive GA3 is synthesized from GA20, and GA3 level may increase by elevated GA20 (Fig. 4C). GA20 content showed significant positive correlation with plant height, petiole length, and leaf development indicators such as leaf length width and area [36]. The level of GA20 in tea buds was about four times higher after dormant release than at mid-dormancy [37], suggesting that GA20 is highly correlated with tea bud release. In this study, the content of both GA20 and GA3 were higher in ‘Yuchunzao’ than in ‘Echa 1’, which indicating that GA20 and GA3 play important roles in promoting geminating of tea shoots.

CsGA2ox1 is an enzyme involved in GA metabolism, it can catalyze GA1,4,9,20 to GA8,34,51,29, respectively [38]. GA20 is also the precursor of bioactive GA3. Therefore, there is a substrate competition between GA3 and GA8,34,51,29 (Fig. 4C). In this study, CsGA2ox1 showed lower expression in ‘Yuchunzao’ than in ‘Echa 1’, the GA20 and GA3 were accumulated in ‘Yuchunzao’, which indicating that the down regulation of CsGA2ox1 induced the accumulation of GA20 and GA3. GA2ox catabolism of GA is a key mechanism for maintaining bud dormancy [39, 40]. Low expression of GA2ox could increase rice and pear seed germination, and vice versa [41, 42]. In addition, expression of GA2ox inhibits the early bud break phenotype in SVLRNAi transgenic hybrid aspen plants [43]. In the present study, the CsGA2ox1 gene was found to be down-regulated in ‘Yuchunzao’. We could suggest that low expression of CsGA2ox1 resulted in high levels of GA20, which led to early sprout phenotypes in ‘Yuchunzao’. Except for GA, one of auxin ICA was significantly lower in ‘Yuchunzao’. ICA has been proved to promote plant growth [44, 45]. However, auxin is not the direct trigger for bud release, it may induce GA synthesis to promote sprouting and sustained bud growth [46]. And in this study, ICA levels were significantly different between two varieties, but their absolute contents were too low to be detected. Thus, the relationship between ICA level and sprouting of tea plants need further verification.

In addition, in KEGG metabolism-related pathways, DEGs are enriched for amino acid biosynthesis, carbon metabolism, and starch and sucrose metabolism. Since ‘Yuchunzao’ germinated earlier than ‘Echa 1’, the tenderness of the buds sampled at the same time was different, leading to differences in the intrinsic quality components of the two varieties. Sugars were stored in the buds to replenish energy during bud growth and development, and the pathways of sugar metabolism in the buds were constantly changing to meet the different energy requirements of the buds at various stages of growth [1]. Furthermore, plant hormones may also cause metabolite differences. Phytohormones and sugar crosstalk affect bud sprout and growth [1, 31]. Therefore, further research is needed to verify their relationship with sprouting.

Conclusion

In conclusion, GA20 and GA3 were accumulated in ‘Yuchunzao’, which showed reverse trend with the expression of GA metabolism related gene CsGA2ox1. It suggested that low expression of CsGA2ox1 led to the accumulation of its substrate GA20 and GA3, which causes early tea bud sprouting. However, the direct relationship between CsGA2ox1 and GA in tea remains to be investigated. This study provides theoretical basis of tea plants sprout and guides the tea breeding in practice.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Acknowledgements

Not applicable.

Author contributions

Ziyuan Qiu prepared samples for analysis, determined and analyzed the data, interpreted the results, and wrote the original draft paper. Wenhui Guo and Qian Yu determined and analyzed the data and interpreted the results. Mengjie Zhao, Han Lv, and Xuewen Hua measured supplementary experimental data. Yu Wang collected samples and assisted. Qingping Ma and Zhaotang Ding designed the study, analyzed the data, interpreted the results, and revised the manuscript. Dongxue Li made revisions to the first draft of the article. All authors reviewed the manuscript.

Funding

This work was supported by Natural Science Fund of Shandong Province (ZR2021QC159), National Natural Science Foundation of China (32302607), Youth innovation team project of Shandong Province (2023KJ208), the Open Project of Liaocheng University Landscape Architecture Discipline (319462212) and Innovation Training Program for College Students (CXCY2023253).

Data availability

All transcriptomic original sequencing data associated with this study have been submitted to the NCBI SRA under the accession number PRJNA1005819.

Declarations

Ethics approval guidelines and consent to participate

Our research did not involve any human or animal subjects, material, or data. We declare that the plant material used in the experiment was collected and studied by relevant institutional, national, and international guidelines and legislation. Plant material was cultivated and planted in our own greenhouse. This study did not involve any herbariums.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Publisher’s note

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