CsLHY positively regulates cold tolerance by activating CsSWEET17 in tea plants.

Low temperature is one of the most important environmental factors limiting tea plants' geographic distribution and severely affects spring tea's yield and quality. Circadian components contribute to plant responses to low temperatures; however, comparatively little is known about these components in tea plants. In this study, we identified a core clock component the LATE ELONGATED HYPOCOTYL, CsLHY, which is mainly expressed in tea plants' mature leaves, flowers, and roots. Notably, CsLHY maintained its circadian rhythmicity of expression in summer, but was disrupted in winter and held a high expression level. Meanwhile, we found that CsLHY expression rhythm was not affected by different photoperiods but was quickly broken by cold, and the low temperature induced and kept CsLHY expression at a relatively high level. Yeast one-hybrid and dual-luciferase assays confirmed that CsLHY can bind to the promoter of Sugars Will Eventually be Exported Transporters 17 (CsSWEET17) and function as a transcriptional activator. Furthermore, suppression of CsLHY expression in tea leaves not only reduced CsSWEET17 expression but also impaired the freezing tolerance of leaves compared to the control. Our results demonstrate that CsLHY plays a positive role in the low-temperature response of tea plants by regulating CsSWEET17 when considered together.

Integrative transcriptome and whole-genome bisulfite sequencing analyses of a temperature-sensitive albino tea plant cultivar.

Green tea made from albino buds and leaves has a strong umami taste and aroma. The cultivar 'Zhonghuang 2' (ZH2, Camellia sinensis) is a natural mutant with young shoots that are yellow in spring and green or yellow-green in summer. However, the mechanism of leaf color change remains unclear. Here, we found that young shoots of ZH2 were yellow at low temperature (LT) and green at high temperature (HT), indicating that ZH2 is a temperature-sensitive cultivar. Transmission electron microscopy analysis showed that the grana in the chloroplasts of young shoots grown at LT were poorly stacked, which caused a lack of photoreactions and chlorophyll. RNA-seq results showed 1279 genes differentially expressed in the young shoots grown at LT compared with those at HT, including genes related to cytochrome synthesis, chloroplast development, photosynthesis, and DNA methylation. A whole-genome bisulfite sequencing assay revealed that the dynamics of DNA methylation levels in the CG, CHG, and CHH contexts decreased under LT, and the change was most obvious in the CHH context. Furthermore, 72 genes showed significant changes in both expression and DNA methylation levels, and most of them were related to cytochrome synthesis, chloroplast development, photosynthesis, transcription factors, and signaling pathways. These results demonstrate that DNA methylation is involved in the LT-regulated albino processes of ZH2. Changes in DNA methylation levels were associated with changes in gene expression levels, affecting the structure and function of chloroplasts, which may have a phenotypic impact on shoot and leaf color.

Transcriptome Analysis Reveals That Ascorbic Acid Treatment Enhances the Cold Tolerance of Tea Plants through Cell Wall Remodeling.

Cold stress is a major environmental factor that adversely affects the growth and productivity of tea plants. Upon cold stress, tea plants accumulate multiple metabolites, including ascorbic acid. However, the role of ascorbic acid in the cold stress response of tea plants is not well understood. Here, we report that exogenous ascorbic acid treatment improves the cold tolerance of tea plants. We show that ascorbic acid treatment reduces lipid peroxidation and increases the Fv/Fm of tea plants under cold stress. Transcriptome analysis indicates that ascorbic acid treatment down-regulates the expression of ascorbic acid biosynthesis genes and ROS-scavenging-related genes, while modulating the expression of cell wall remodeling-related genes. Our findings suggest that ascorbic acid treatment negatively regulates the ROS-scavenging system to maintain ROS homeostasis in the cold stress response of tea plants and that ascorbic acid's protective role in minimizing the harmful effects of cold stress on tea plants may occur through cell wall remodeling. Ascorbic acid can be used as a potential agent to increase the cold tolerance of tea plants with no pesticide residual concerns in tea.

CsCIPK11-Regulated Metalloprotease CsFtsH5 Mediates the Cold Response of Tea Plants.

Photosystem II repair in chloroplasts is a critical process involved in maintaining a plant's photosynthetic activity under cold stress. FtsH (filamentation temperature-sensitive H) is an essential metalloprotease that is required for chloroplast photosystem II repair. However, the role of FtsH in tea plants and its regulatory mechanism under cold stress remains elusive. In this study, we cloned a FtsH homolog gene in tea plants, named CsFtsH5, and found that CsFtsH5 was located in the chloroplast and cytomembrane. RT-qPCR showed that the expression of CsFtsH5 was increased with leaf maturity and was significantly induced by light and cold stress. Transient knockdown CsFtsH5 expression in tea leaves using antisense oligonucleotides resulted in hypersensitivity to cold stress, along with higher relative electrolyte leakage and lower Fv/Fm values. To investigate the molecular mechanism underlying CsFtsH5 involvement in the cold stress, we focused on the calcineurin B-like-interacting protein kinase 11 (CsCIPK11), which had a tissue expression pattern similar to that of CsFtsH5 and was also upregulated by light and cold stress. Yeast two-hybrid and dual luciferase (Luc) complementation assays revealed that CsFtsH5 interacted with CsCIPK11. Furthermore, the Dual-Luc assay showed that CsCIPK11-CsFtsH5 interaction might enhance CsFtsH5 stability. Altogether, our study demonstrates that CsFtsH5 is associated with CsCIPK11 and plays a positive role in maintaining the photosynthetic activity of tea plants in response to low temperatures.

A Tea Plant (Camellia sinensis) FLOWERING LOCUS C-like Gene, CsFLC1, Is Correlated to Bud Dormancy and Triggers Early Flowering in Arabidopsis.

Flowering and bud dormancy are crucial stages in the life cycle of perennial angiosperms in temperate climates. MADS-box family genes are involved in many plant growth and development processes. Here, we identified three MADS-box genes in tea plant belonging to the FLOWERING LOCUS C (CsFLC) family. We monitored CsFLC1 transcription throughout the year and found that CsFLC1 was expressed at a higher level during the winter bud dormancy and flowering phases. To clarify the function of CsFLC1, we developed transgenic Arabidopsis thaliana plants heterologously expressing 35S::CsFLC1. These lines bolted and bloomed earlier than the WT (Col-0), and the seed germination rate was inversely proportional to the increased CsFLC1 expression level. The RNA-seq of 35S::CsFLC1 transgenic Arabidopsis showed that many genes responding to ageing, flower development and leaf senescence were affected, and phytohormone-related pathways were especially enriched. According to the results of hormone content detection and RNA transcript level analysis, CsFLC1 controls flowering time possibly by regulating SOC1, AGL42, SEP3 and AP3 and hormone signaling, accumulation and metabolism. This is the first time a study has identified FLC-like genes and characterized CsFLC1 in tea plant. Our results suggest that CsFLC1 might play dual roles in flowering and winter bud dormancy and provide new insight into the molecular mechanisms of FLC in tea plants as well as other plant species.

Integrative Transcriptome and Proteome Analysis Reveals the Absorption and Metabolism of Selenium in Tea Plants [Camellia sinensis (L.) O. Kuntze].

Certain tea plants (Camellia sinensis) have the ability to accumulate selenium. In plants, the predominant forms of bioavailable Se are selenite (SeO3 2-) and selenate (SeO4 2-). We applied transcriptomics and proteomics to hydroponically grown plants treated with selenite or selenate for 48 h in the attempt to elucidate the selenium absorption and assimilation mechanisms in tea. A total of 1,844 differentially expressed genes (DEGs) and 691 differentially expressed proteins (DEPs) were obtained by comparing the Na2SeO3 and Na2SeO4 treatments against the control. A GO analysis showed that the genes related to amino acid and protein metabolism and redox reaction were strongly upregulated in the plants under the Na2SeO3 treatment. A KEGG pathway analysis revealed that numerous genes involved in amino acid and glutathione metabolism were upregulated, genes and proteins associated with glutathione metabolism and ubiquinone and terpenoid-quinone biosynthesis were highly expressed. Genes participating in DNA and RNA metabolism were identified and proteins related to glutathione metabolism were detected in tea plants supplemented with Na2SeO4. ABC, nitrate and sugar transporter genes were differentially expressed in response to selenite and selenate. Phosphate transporter (PHT3;1a, PHT1;3b, and PHT1;8) and aquaporin (NIP2;1) genes were upregulated in the presence of selenite. Sulfate transporter (SULTR1;1 and SULTR2;1) expression increased in response to selenate exposure. The results of the present study have clarified Se absorption and metabolism in tea plants, and play an important theoretical reference significance for the breeding and cultivation of selenium-enriched tea varieties.

Genome-wide identification of SULTR genes in tea plant and analysis of their expression in response to sulfur and selenium.

Sulfur (S) is an essential macronutrient required by plants. Plants absorb and transport S through sulfate transporters (SULTRs). In this study, we cloned 8 SULTR genes (CsSULTR1;1/1;2/2;1/3;1/3;2/3;3/3;5/4;1) from tea plant (Camellia sinensis), all of which contain a typical sulfate transporter and antisigma factor antagonist (STAS) conserved domain. Phylogenetic tree analysis further divided the CsSULTRs into four main groups. Many cis-acting elements related to hormones and environmental stresses were found within the promoter sequence of CsSULTRs. Subcellular localization results showed that CsSULTR4;1 localized in the vacuolar membrane and that other CsSULTRs localized to the cellular membrane. The tissue-specific expression of the 8 CsSULTR genes showed different expression patterns during the active growing period and dormancy period. In particular, the expression of CsSULTR1;1 was highest in the roots, but that of CsSULTR1;2 was lowest in the dormancy period. The expression of CsSULTR1;1/1;2/2;1/3;2 was stimulated under different concentrations of selenium (Se) and S; moreover, CsSULTR1;2/2;1/3;3/3;5 was upregulated in response to different valences of Se.

Genome-wide identification, characterization, and expression analysis of tea plant autophagy-related genes (CsARGs) demonstrates that they play diverse roles during development and under abiotic stress.

BACKGROUND: Autophagy, meaning 'self-eating', is required for the degradation and recycling of cytoplasmic constituents under stressful and non-stressful conditions, which helps to maintain cellular homeostasis and delay aging and longevity in eukaryotes. To date, the functions of autophagy have been heavily studied in yeast, mammals and model plants, but few studies have focused on economically important crops, especially tea plants (Camellia sinensis). The roles played by autophagy in coping with various environmental stimuli have not been fully elucidated to date. Therefore, investigating the functions of autophagy-related genes in tea plants may help to elucidate the mechanism governing autophagy in response to stresses in woody plants. RESULTS: In this study, we identified 35 C. sinensis autophagy-related genes (CsARGs). Each CsARG is highly conserved with its homologues from other plant species, except for CsATG14. Tissue-specific expression analysis demonstrated that the abundances of CsARGs varied across different tissues, but CsATG8c/i showed a degree of tissue specificity. Under hormone and abiotic stress conditions, most CsARGs were upregulated at different time points during the treatment. In addition, the expression levels of 10 CsARGs were higher in the cold-resistant cultivar 'Longjing43' than in the cold-susceptible cultivar 'Damianbai' during the CA period; however, the expression of CsATG101 showed the opposite tendency. CONCLUSIONS: We performed a comprehensive bioinformatic and physiological analysis of CsARGs in tea plants, and these results may help to establish a foundation for further research investigating the molecular mechanisms governing autophagy in tea plant growth, development and response to stress. Meanwhile, some CsARGs could serve as putative molecular markers for the breeding of cold-resistant tea plants in future research.

Characterization of l-Theanine Hydrolase in Vitro and Subcellular Distribution of Its Specific Product Ethylamine in Tea (Camellia sinensis).

l-Theanine has a significant role in the taste of tea (Camellia sinensis) infusions. Our previous research indicated that the lower l-theanine metabolism in ethylamine and l-glutamate is a key factor that explains the higher content of l-theanine in albino tea with yellow or white leaves, compared with that of normal tea with green leaves. However, the specific genes encoding l-theanine hydrolase in tea remains unknown. In this study, CsPDX2.1 was cloned together with the homologous Arabidopsis PDX2 gene and the recombinant protein was shown to catalyze l-theanine hydrolysis into ethylamine and l-glutamate in vitro. There were higher CsPDX2.1 transcript levels in leaf tissue and lower transcripts in the types of albino (yellow leaf) teas compared with green controls. The subcellular location of ethylamine in tea leaves was shown to be in the mitochondria and peroxisome using a nonaqueous fractionation method. This study identified the l-theanine hydrolase gene and subcellular distribution of ethylamine in tea leaves, which improves our understanding of the l-theanine metabolism and the mechanism of differential accumulation of l-theanine among tea varieties.

Population sequencing enhances understanding of tea plant evolution.

Tea is an economically important plant characterized by a large genome, high heterozygosity, and high species diversity. In this study, we assemble a 3.26-Gb high-quality chromosome-scale genome for the 'Longjing 43' cultivar of Camellia sinensis var. sinensis. Genomic resequencing of 139 tea accessions from around the world is used to investigate the evolution and phylogenetic relationships of tea accessions. We find that hybridization has increased the heterozygosity and wide-ranging gene flow among tea populations with the spread of tea cultivation. Population genetic and transcriptomic analyses reveal that during domestication, selection for disease resistance and flavor in C. sinensis var. sinensis populations has been stronger than that in C. sinensis var. assamica populations. This study provides resources for marker-assisted breeding of tea and sets the foundation for further research on tea genetics and evolution.

Integrated transcriptomic and metabolomic analyses reveal the effects of callose deposition and multihormone signal transduction pathways on the tea plant-Colletotrichum camelliae interaction.

Colletotrichum infects diverse hosts, including tea plants, and can lead to crop failure. Numerous studies have reported that biological processes are involved in the resistance of tea plants to Colletotrichum spp. However, the molecular and biochemical responses in the host during this interaction are unclear. Cuttings of the tea cultivar Longjing 43 (LJ43) were inoculated with a conidial suspension of Colletotrichum camelliae, and water-sprayed cuttings were used as controls. In total, 10,592 differentially expressed genes (DEGs) were identified from the transcriptomic data of the tea plants and were significantly enriched in callose deposition and the biosynthesis of various phytohormones. Subsequently, 3,555 mass spectra peaks were obtained by LC-MS detection in the negative ion mode, and 27, 18 and 81 differentially expressed metabolites (DEMs) were identified in the tea leaves at 12 hpi, 24 hpi and 72 hpi, respectively. The metabolomic analysis also revealed that the levels of the precursors and intermediate products of jasmonic acid (JA) and indole-3-acetate (IAA) biosynthesis were significantly increased during the interaction, especially when the symptoms became apparent. In conclusion, we suggest that callose deposition and various phytohormone signaling systems play important roles in the tea plant-C. camelliae interaction.

CsSWEET1a and CsSWEET17 Mediate Growth and Freezing Tolerance by Promoting Sugar Transport across the Plasma Membrane.

Sugars Will Eventually be Exported Transporters (SWEETs) are important in plant biological processes. Expression levels of CsSWEET1a and CsSWEET17 are induced by cold acclimation (CA) and cold stress in Camellia sinensis. Here, we found that CsSWEET17 was alternatively spliced, and its exclusion (Ex) transcript was associated with the CA process. Both plasma membrane-localized CsSWEET1a and CsSWEET17 transport hexoses, but cytoplasm-localized CsSWEET17-Ex does not. These results indicate that alternative splicing may be involved in regulating the function of SWEET transporters in response to low temperature in plants. The extra C-terminal of CsSWEET17, which is not found in the tonoplast fructose transporter AtSWEET17, did not affect its plasma membrane localization but promoted its sugar transport activities. The overexpression (OE) of CsSWEET1a and CsSWEET17 genes resulted in an increased sugar uptake in Arabidopsis, affecting plant germination and growth. The leaf and seed sizes of the CsSWEET17-OE lines were significantly larger than those of the wild type. Moreover, the OE of CsSWEET1a and CsSWEET17 significantly reduced the relative electrolyte leakage levels under freezing stress. Compared with the wild type, the expression of AtCWINV genes was suppressed in both CsSWEET1a-OE and CsSWEET17-OE lines, indicating the alteration in sugar contents in the cell walls of the OE lines. Furthermore, the interaction between CsSWEET1a and CsSWEET17 was confirmed using yeast two-hybrid and bimolecular fluorescence complementation assays. We showed that CsSWEET1a and CsSWEET17 form homo-/heterodimers in the plasma membrane and mediate the partitioning of sugars between the cytoplasm and the apoplast, thereby regulating plant growth and freezing tolerance.

Characterization of CBL-CIPK signaling complexes and their involvement in cold response in tea plant.

Calcineurin B-like (CBL) proteins, a class of Ca2+-binding proteins, play vital roles in calcium signal transduction by interacting specifically with CBL-interacting protein kinases (CIPKs), and these two gene families and their interacting complexes are involved in regulating plant responses to various environmental stimuli. In the present study, eight CBL and 25 CIPK genes were identified in tea plant and divided into four and five subfamilies, respectively. Analysis of the expression of these genes in response to abiotic stresses (mature leaves treated with cold, salinity, and PEG and young shoots treated with cold) revealed that CsCBL1/3/5 and CsCIPK1/4/5/6a/7/8/10b/10c/12/14a/19/23a/24 could be induced by at least two stresses. Under cold stress, CsCBL9 and CsCIPK4/6a/6b/7/11/14b/19/20 were upregulated in both mature leaves and young shoots, CsCBL1/3/5 and CsCIPK1/8/10a/10b/10c/12/14a/23a/24 were induced only in mature leaves, and CsCIPK5/25 were induced only in young shoots. Yeast two-hybrid analysis showed that CsCBL1 could interact with CsCIPK1/10b/12 but not with CsCIPK6a/7/11/14b/20. CsCBL9 was found to interact with CsCIPK1/10b/12/14b but not with CsCIPK6a/7/11/20. These results suggest divergent responses to cold stress regulated by CBL-CIPK complexes between tea plant and Arabidopsis, as well as between mature leaves and young shoots in tea plant. A model of Ca2+-CsCBL-CsCIPK module-mediated abiotic stress signaling in tea plant is proposed.

Lifestyle Characteristics and Gene Expression Analysis of Colletotrichum camelliae Isolated from Tea Plant [Camellia sinensis (L.) O. Kuntze] Based on Transcriptome.

Colletotrichum camelliae is one of the most serious pathogens causing anthracnose in tea plants, but the interactive relationship between C. camelliae and tea plants has not been fully elucidated. This study investigated the gene expression changes in five different growth stages of C. camelliae based on transcriptome analysis to explain the lifestyle characteristics during the infection. On the basis of gene ontology (GO) enrichment analyses of differentially expressed genes (DEGs) in comparisons of germ tube (GT)/conidium (Con), appressoria (App)/Con, and cellophane infectious hyphae (CIH)/Con groups, the cellular process in the biological process category and intracellular, intracellular part, cell, and cell part in the cellular component category were significantly enriched. Hydrolase activity, catalytic activity, and molecular_function in the molecular function category were particularly enriched in the infection leaves (IL)/Con group. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that the DEGs were enriched in the genetic information processing pathway (ribosome) at the GT stage and the metabolism pathway (metabolic pathways and biosynthesis of secondary metabolism) in the rest of the stages. Interestingly, the genes associated with melanin biosynthesis and carbohydrate-active enzymes (CAZys), which are vital for penetration and cell wall degradation, were significantly upregulated at the App, CIH and IL stages. Subcellular localization results further showed that the selected non-annotated secreted proteins based on transcriptome data were majorly located in the cytoplasm and nucleus, predicted as new candidate effectors. The results of this study may establish a foundation and provide innovative ideas for subsequent research on C. camelliae.

ABA-dependent bZIP transcription factor, CsbZIP18, from Camellia sinensis negatively regulates freezing tolerance in Arabidopsis.

KEY MESSAGE: Overexpression of the tea plant gene CsbZIP18 in Arabidopsis impaired freezing tolerance, and CsbZIP18 is a negative regulator of ABA signaling and cold stress. Basic region/leucine zipper (bZIP) transcription factors play important roles in the abscisic acid (ABA) signaling pathway and abiotic stress response in plants. However, few bZIP transcription factors have been functionally characterized in tea plants (Camellia sinensis). In this study, a bZIP transcription factor, CsbZIP18, was found to be strongly induced by natural cold acclimation, and the expression level of CsbZIP18 was lower in cold-resistant cultivars than in cold-susceptible cultivars. Compared with wild-type (WT) plants, Arabidopsis plants constitutively overexpressing CsbZIP18 exhibited decreased sensitivity to ABA, increased levels of relative electrolyte leakage (REL) and reduced values of maximal quantum efficiency of photosystem II (Fv/Fm) under freezing conditions. The expression of ABA homeostasis- and signal transduction-related genes and abiotic stress-inducible genes, such as RD22, RD26 and RAB18, was suppressed in overexpression lines under freezing conditions. However, there was no significant change in the expression of genes involved in the C-repeat binding factor (CBF)-mediated ABA-independent pathway between WT and CsbZIP18 overexpression plants. These results indicate that CsbZIP18 is a negative regulator of freezing tolerance via an ABA-dependent pathway.

Genome-wide identification and expression analysis of flowering-related genes reveal putative floral induction and differentiation mechanisms in tea plant (Camellia sinensis).

The tea leaf is economically important, while reproductive growth reduce tea output. However, little is known about flowering mechanisms in tea plants. Here, we determined the approximate times of floral induction, floral transition and floral organ differentiation by morphological observation. We identified 401 and 356 flowering-related genes from the genomes of Camellia sinensis var. sinensis and Camellia sinensis var. assamica, respectively. Then, we compared the expression profiles of flowering-related genes in floriferous and oliganthous cultivars, the result showed that PRR7, GI, GID1B and GID1C expression is correlated with the floral induction; LFY, PNF and PNY expression was correlated with floral bud formation. Transcriptome analysis also showed that GI, PRR7 and GID1 were correlated with stress-induced flowering. Thus, we proposed putative mechanisms of flowering in tea plants. This study provides new insights into flowering and a theoretical basis for balancing vegetative and reproductive growth in tea plants and other economical plants.

Genome-wide identification, characterization, and expression analysis of nucleotide-binding leucine-rich repeats gene family under environmental stresses in tea (Camellia sinensis).

Plants often use nucleotide-binding leucine-rich repeats (NLRs) to recognize specific virulence proteins and activate the hypersensitive response thereby defending against invaders. However, data on NLRs and the resistance mechanism of NLR protein mediation in tea plant are extremely limited. In this study, 400 and 303 CsNLRs were identified from the genomes of C. sinensis var. sinensis (CSS) and C. sinensis var. assamica (CSA), respectively. Phylogenetic analysis revealed that the numbers in CNL groups are predominant in both CSS and CSA. RNA-Seq revealed that the expression of CsNLRs is induced by Colletotrichum fructicola, cold, drought, salt stress and exogenous methyl jasmonate. The 21 CsCNLs that are highly expressed in tea plant under biotic and abiotic stresses as well as during bud dormancy and in different tissues are identified. Gene structure analysis revealed several cis-regulatory elements associated with phytohormones and light responsiveness in the promoter regions of these 21 CsCNLs.

Pale green mutant analyses reveal the importance of CsGLKs in chloroplast developmental regulation and their effects on flavonoid biosynthesis in tea plant.

Tea cultivars with leaf color variation have attracted increasing attention in tea production and research due to their unusual appearances and appealing flavors. However, the molecular mechanism underlying this variation is little known due to the unavailability of genetic transformation and a highly complex genome. Here, a natural tea plant mutant producing pale green branches (pgb) was discovered and characterized. Ultrastructural and biochemical analyses showed that the leaves of the pgb mutant had defective chloroplast structure and significantly lower pigment content than the normal control. Comprehensive expression detection of chloroplast-development-related genes further indicated that a significant downregulation of CsGLKs in the pgb mutant likely caused the chloroplast defect. Transcriptome analyses and polyphenolic compound determination highlighted a tight correlation between photosynthesis and secondary metabolite biosynthesis in tea plant. These results provide useful information illuminating the mechanism of chloroplast development and leaf color variation in tea plant.

The involvements of calcium-dependent protein kinases and catechins in tea plant [Camellia sinensis (L.) O. Kuntze] cold responses.

Temperature is one of the most important environmental factors limiting tea plant growth and tea production. Previously we reported that both Ca2+ and ROS signals play important roles in tea plant cold acclimation. Here, we identified 26 CsCPK transcripts, analyzed their phylogenetic and sequence characters, and detected their transcriptions to monitor Ca2+ signaling status. Tissue-specific expression profiles indicated that most CsCPK genes were constitutively expressed in tested tissues, suggesting their possible roles in development. Cold along with calcium inhibitor assays suggested that CsCPKs are important cold regulators and CsCPK30/5/4/9 maybe the key members. Moreover, LaCl3 or EGTA pre-treatment could result in impaired Ca2+ signaling and compromised cold-responding network, but higher catechins accumulation revealed their potential positive roles in cold responses. Those findings indicated that catechins and other secondary metabolites in tea plant may form an alternative cold-responding network that closely correlated with Ca2+ signaling status.