Isolation and expression analysis of CsCML genes in response to abiotic stresses in the tea plant (Camellia sinensis)

Calmodulin-like (CML) proteins are a class of important Ca2+ sensors in plants, which play vital roles in regulating plant growth and development and response to abiotic stress. Tea plant (Camellia sinensis L.) is the most popular non-alcoholic economic beverage crop around the world. However, the potential functions of CMLs in either tea plants growth or in the response to environmental stresses are still unclear. In the present study, five CsCML genes (CsCML16, CsCML18-1, CsCML18-2, CsCML38, and CsCML42) were isolated from tea plant, and functionally characterized. The CsCML genes showed diverse expression patterns in leaves, roots, old stems, immature stems and flowers of tea plants. To investigate the expression changes of the genes under various abiotic stresses and ABA treatment, time-course experiments were also performed, the results indicated that the expression levels of CsCML16, 18-2 and 42 were significantly induced under low temperature and salt condition, while CsCML38 was induced distinctly under drought stress and ABA treatment. Overall, CsCML genes showed diverse function in tea plant under various stimuli. These results will increase our knowledge of the significance of CsCML genes in tea plant in response to abiotic stresses and hormone treatments.

Multiple sequence alignment and phylogenetic analysis. The deduced amino acid sequences of five CsCML were analyzed by multiple sequence alignments. As shown in Fig. 2, CsCML16 and EgCML16 (Erythranthe guttate) clustered in the same branch and showed the highest similarity, followed by ZjCML16 (Ziziphus jujube), PmCML16 (Prunus mume), MdCML16 (Malus domestica) and VvCML16 (Vitis vinifera). CsCML18-1 and CsCML18-2 were classified into the same cluster, indicating that they were paralogous genes. Both CsCML18-1 and CsCML18-2 showed high similarity to CML18 from other plant species. CsCML38 clustered with DcCML38 (Daucus carota subsp. Sativus), followed by RcCML38 (Ricinus communis), PeCML38 (Populus euphratica) and GaCML38 (Gossypium arboretum). For CsCML42, this gene shares highly similarity with ThCML42 (Tarenaya hassleriana), AtCML42 and NcCML42 (Noccaea caerulescens). Phylogenetic tree Name Gene ID in tea tree genome  www.nature.com/scientificreports www.nature.com/scientificreports/ analysis revealed that the CsCMLs amino acid sequences showed highly homologous to those of other plant species. expression analysis of CsCMLs genes in different tissues. Tissue-specific gene expression profile might be associated with the specific physiological and developmental functions in plants. The expression levels of CsCML family gene in different tissues, including leaves, roots, old stems, immature stems and flowers, were examined by qRT-PCR. As shown in Fig. 3, all CsCMLs were expressed in different tissues of tea plant with the expression level of tea roots as control. The expression levels of CsCML16 and CsCML18-1 were remarkably higher in flowers than in other tissues. CsCML16 also had a higher expression level in leaves than as expressed by other genes. However, their expression levels were induced slightly in other tissues, indicating that CsCMLs display tissue-specific expression in tea plant. The expression levels of CsCMLs in different tissues suggest that CsCML genes may have different functional variation in tea plant and this remains to be further investigated.

expression analysis of CsCMLs genes in tea plant under various abiotic stresses.
To explore the expression profiles of the CsCMLs genes under various abiotic stresses, time-course experiments of 5 CsCML genes in two-year-old seedlings treated with low temperature (10 °C), drought (PEG 6000), salinity (NaCl), and hormone (ABA) we performed. Generally, the 5 CsCML genes displayed distinctively expression patterns at different time points after stress treatments (Fig. 4). The expression levels of CsCML16, CsCML18-2 and CsCML42 were induced significantly by cold stress, and these genes showed highest expression levels at 24 h after cold stress. In contrast, the expression level of CsCML18-1 was suppressed by cold stress. The reverse expression patterns between CsCML18-1 and CsCML18-2 revealed a diverse function in paralogs of CML genes in plants. In addition, CsCML38 showed relative stable expressions under cold stress, which indicate that CsCML38 is insensitive to cold stress.
Under drought stress caused by 20% PEG 6000 treatment, the expression of CsCML38 was induced distinctly. However, expressions of CsCML16 and CsCML18-1 were reduced significantly after drought stress. The expression level of CsCML18-2 had a little fluctuation between 0-12 h, but decreased significantly at 24 h, and then showed highest expression levels at 36 h. CsCML42 was obviously inhibited at 4 h, followed by an increase until 24 h, and a decline at 36 h. The results indicate that CsCML genes showed different expression patterns under drought stress. In addition, the expression patterns of CsCMLs under drought stress were significantly different www.nature.com/scientificreports www.nature.com/scientificreports/  www.nature.com/scientificreports www.nature.com/scientificreports/ from that under cold and salt stress, suggesting that different response mechanisms were activated under different abiotic stresses (Fig. 5).
ABA, an important plant hormone, plays vital roles in the regulation of plant adaptation to surrounding environment. With the induction of ABA treatment, the expression of CsCML38 showed the highest change, indicating that CsCML38 was very sensitive to ABA treatment. The expression level of CsCML16 significantly increased and reached maximum at 12 h, followed by an acute decrease at 24 h. The expression level of CsCML18-1 rapidly decreased at 4 h, and the induction of CsCML18-1 steadily increased until 24 h. The expression level of CsCML18-2 increased significantly at 4 h, then decreased gradually. The expression level of CsCML42 declined at 2 h, followed by an increase at 12 h, and a decrease at 24 h (Fig. 6).
Salt stress is a common threat which restricts growth and development of plants. After NaCl treatment, the expression levels of CsCML16, CsCML18-2 and CsCML42 were increased significantly with the highest expression levels during 4-24 h after stress. However, the expression of CsCML38 decreased after salt stress, indicating that salt stress could suppress the expression of CsCML38. The expression patterns of CsCML16, CsCML18-2 and CsCML42 under salt stress were similar to that under cold stress, suggesting that these genes may be involved in similar signal pathway in response to salt and cold stresses (Fig. 7).

Discussion
Ca 2+ ion is a critical second messenger and could induce various physiological responses in plants in response to diverse stimuli. CML, as a Ca 2+ sensor, mediates interpretation of Ca 2+ signals in plant cell and also plays an important role in plants respond to various environmental stresses. In the present study, five CML genes namely CsCML16, CsCML18-1, CsCML18-2, CsCML38 and CsCML42 were identified and cloned from tea plant leaves. These genes encode small proteins containing 136-201 amino acids. Apart from CML18-1 protein which contains only one EF-hand motif, four proteins were found to have two EF-hand motifs. The CsCML gene family in tea plants contains EF-hand motifs with no other identifiable functional domains. These findings are in accordance with previously reports in Arabidopsis, rice and tomato 10,17,24 . Phylogenetic analysis revealed that amino acid  www.nature.com/scientificreports www.nature.com/scientificreports/ sequences of putative CsCMLs from tea plant have high similarity with CMLs from other plant species. These results indicated that the close evolutionary relationship existed in plant CML proteins.
Furthermore, the expression profiles of CsCML in different tissues indicated that CsCML genes were differentially expressed in tea plant. Expression levels of CsCML16 and CsCML18-1 in flowers were significantly higher than in other tissues. Interestingly, the expression levels of CsCML16 and CsCML18 were lower in flowers of Arabidopsis 24 , whereas AtCML24 and AtCML25 strongly affected the pollen germination and and pollen tube growth 25,26 . It can; therefore, be suggested that CsCML16 and CsCML18-1 expressed differently to adapt different species. Conversely, expression levels of CsCML18-2, CsCML38 and CsCML42 in roots were significantly higher than in leaves, stem and flower organs, suggesting that different CML gene members from different species have distinct expression levels in various tissues, and may function in different physiological processes. The diversified expression of these CsCML genes revealed that they might play significant role at different plant developmental stages.
It is known that CML genes are involved in response to different environmental stresses and hormones. ABA plays an important role in cellular signal transduction in response to abiotic stresses. It could regulate the expression of several ABA-responsive genes through cellular Ca 2+ ion changes 27 . Over expression of Oryza sativa multi-stress-responsive gene 2 (OsMSR2), which was a novel CML gene, improved the drought and salt tolerance of plants via ABA-mediated pathways 28 . Subsequently, another novel CML gene named OsDSR1 (Oryza sativa drought stress response-1) showed greater sensitivity to ABA during the development of plant 29 . OsCML4 could improve drought tolerance by effectively scavenging of ROS in rice 17 . In Arabidopsis, some studies have reported that AtCML24 was induced by cold and drought stress, suggest that AtCML24 might be involved in cold-related Ca 2+ signals transduction 30 . AtCML37, AtCML39 and AtCML38 were also in response to several stimulus-induced and developmental signalling pathways, including salt, drought, and ABA 31 , while AtCML42 may function to increase resistance to pathogens 32 . MtCML40 in Medicago truncatula was reported to be involved in salt, cold tolerance as well as ABA treatment 33 . Altogether, accumulating evidence has demonstrated that CML proteins play important roles in Ca 2+ signal transduction during plant growth and adaptation to abiotic stress.
The transcriptional regulation analysis under abiotic stress and hormones showed that the expression level of CsCMLs was affected in tea plants. The results revealed that the expression levels of CsCML16, CsCML18-2 and CsCML42 were induced significantly under low temperature condition, suggesting that these genes were involved in cold tolerance. Previous investigations have confirmed that CMLs are involved in plant responses to cold stress. For example, cold treatment could induce the expression levels of AtCML24 and OsMSR2, which might participate in cold-induced Ca 2+ signal transduction 28,30 .
Under drought stress, the expression levels of CsCML16 and CsCML18-1 decreased. This result is in contrast with the study by Jung et al. 34 who reported that OsCML16 was involved in drought tolerance through enhanced root growth. CML38 is a likely homolog of an endogenous suppressor of antiviral silencing 35,36 . In the present study, CsCML38 was responsive to all treatments except cold stress. However, the molecular mechanism of CsCML38 in response to abiotic stresses remains unclear. Whether it is similar to other CsCML homologs needs to be verified in the future study. The result reveals that CsCML genes have diverse functions in response to different stimuli. Meanwhile, the role of CsCML genes may be activated by different signals.
Both CsCML18-2 and CsCML42 were up-regulated under low temperature and ABA treatments. This result indicates that CMLs are links of signal pathways which involved in different stimuli. It is in accordance with the role of a CDPK in ABA-dependent cold acclimation 37 . The up-regulated CsCML16, CsCML18-2 and CsCML42 in the present study suggest that these genes are involved in high salinity tolerance. The function of these salt responsive CML genes in the regulation of salt tolerance may be due to targeting high affinity K + transporter-dependent Na + accumulation 38 .
In summary, our results indicated that CsCMLs possibly function as stress-responsive genes to improve stress tolerance in tea plant. Although the study of CsCML family genes is still largely unexplored; our results, to some Amplification and cloning of CsCML genes from tea plant. In order to find the CsCML family genes of tea plants, CsCML21 nucleic acid sequence (GenBank accession: JQ999983) and the transcriptome data (SRA accession number: SRR5075641) were used for homogenous alignment by using BLAST program. The ORF regions of CsCML genes were generated by RT-PCR using primers in Table 2. PCR amplification was conducted by the following program: 94 °C for 2 min, 35 cycles of amplification at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 2 min, followed by final extension at 72 °C for 10 min. The PCR products were loaded on 1.2% agarose gels for electrophoretic analysis and purified by a DNA purification kit (Qiagen, Valencia, CA). The purified DNA products was ligated into TOPO-TA vector and transformed into component cells (E. coli DH5α). Three independent clones from each isolate were sequenced by GenScript company (Nanjing, China).
Nucleotide and amino acid sequences analysis. Phylogenetic analysis of the CML gene sequences was carried out by using BLAST program in the National Center for Biotechnology Information website (http:// www.ncbi.nlm.nih.gov/BLAST). The phylogenetic tree was carried out by MEGA 7.0 using the neighbor-joining method. The predictions of possible open reading frame (ORF), theoretical isoelectric point (pI), and the molecular mass (Mw) were performed using online software (https://web.expasy.org/protparam/). Multiple sequence alignments of selected amino acids were conducted using ClustalW software. The MEME Suite software (http:// Name Primer sequences 5′-3′   Table 3. Primers used for the qRT-PCR analysis in this study.