ARTICLE   Open Access    

iTRAQ-based quantitative proteomic analysis of tea plant (Camellia sinensis (L.) O. Kuntze) during cold acclimation and de-acclimation procedures

  • # These authors contributed equally: Changqing Ding, Xinyuan Hao

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  • To gain a better understanding on the mechanism of cold acclimation in tea plant [Camellia sinensis (L.) O. Kuntze] at the proteome level, an iTRAQ based quantitative proteome analysis was carried out to identify differentially accumulated proteins in the mature leaves which were collected at non-acclimated (NA), fully acclimated (CA) and de-acclimated (DA) stages. 407 and 477 proteins identified from CA and DA showed significant abundance changes (at 95% confidence) compared with NA, respectively. Moreover, 251 protein species changed their abundance in DA compared with CA. Those differential abundance protein species were mainly involved in metabolism, cell wall, photosynthesis, energy, protein synthesis, antioxidation, carbohydrate metabolic process and binding, and mapped to the pathways of biosynthesis of secondary metabolites, microbial metabolism in diverse environment, ribosome, metabolic pathway, sugar metabolism, protein processing, photosynthesis, and plant-pathogen interaction pathway. However, no significant correlation was detected between the identified proteins and cognate gene transcript levels by correlation analysis and qRT-PCR analysis. This study presents a comprehensive proteome in mature leaves at different cold acclimation status and provides new insights into cold acclimation mechanisms in tea plants.
  • Salvia rosmarinus L. (old name Rosmarinus officinalis), common name Rosemary thrives well in dry regions, hills and low mountains, calcareous, shale, clay, and rocky substrates[1]. Salvia rosmarinus used since ancient times in traditional medicine is justified by its antiseptic, antimicrobial, anti-inflammatory, antioxidant, and antitumorigenic activity[1,2]. The main objective of the study is to evaluate the antimicrobial activity of different extracts of Salvia rosmarinus in vitro, and its compounds related to in silico targeting of enzymes involved in cervical cancer. Since the start of the 20th century, some studies have shown that microbial infections can cause cervical cancers worldwide, infections are linked to about 15% to 20% of cancers[3]. More recently, infections with certain viruses like Human papillomaviruses (HPV) and Human immunodeficiency virus (HIV), bacteria like Chlamydia trachomatis, and parasites like schistosomiasis have been recognized as risk factors for cancer in humans[3]. Then again, cancer cells are a group of diseases characterized by uncontrolled growth and spread of abnormal cells. Many things are known to increase the risk of cancer, including dietary factors, certain infections, lack of physical activity, obesity, and environmental pollutants[4]. Some studies have found that unbalanced common flora Lactobacillus bacteria around the reproductive organ of females increases the growth of yeast species (like Candida albicans) and some studies have found that women whose blood tests showed past or current Chlamydia trachomatis infection may be at greater risk of cervical cancer. It could therefore be that human papillomavirus (HPV) promotes cervical cancer growth[3]. Salvia rosmarinus is traditionally a healer chosen as a muscle relaxant and treatment for cutaneous allergy, tumors, increases digestion, and the ability to treat depressive behavior; mothers wash their bodies to remove bacterial and fungal infections, promote hair growth, and fight bad smells[5] .

    The study of plant-based chemicals, known as phytochemicals, in medicinal plants is gaining popularity due to their numerous pharmacological effects[6] against drug resistance pathogens and cancers. The causes of drug resistance to bacteria, fungi, and cancer are diverse, complex, and only partially understood. The factors may act together to initiate or promote infections and carcinogenesis in the human body is the leading cause of death[7]. Antimicrobial medicines are the cornerstone of modern medicine. The emergence and spread of drug-resistant pathogens like bacteria and fungi threaten our ability to treat common infections and to perform life-saving procedures including cancer chemotherapy and cesarean sections, hip replacements, organ transplantation, and other surgeries[7]. On the other hand, information about the current magnitude of the burden of bacterial and fungal drug resistance, trends in different parts of the world, and the leading pathogen–drug combinations contributing to the microbial burden is crucial. If left unchecked, the spread of drug resistance could make many microbial pathogens much more lethal in the future than they are today. In addition to these, cancers can affect almost any part of the body and have many anatomies and molecular subtypes that each require specific management strategies to avoid or inhibit them. There are more than 200 different types of cancer that have been detected. The world's most common cancers affecting men are lung, prostate, colorectal, stomach, and liver cancers[8]. While breast, cervix, colorectal, lung, and stomach cancers are the most commonly diagnosed among women[8]. Although some cancers said to be preventable they seem to still be one of the causes of death to humans, for example cervical cancer. The need to fill the gap to overcome the problem of searching for antimicrobials and anticancers from one source of Salvia rosmarinus is of importance.

    Cervical cancer is a common cancer in women and a prominent cause of death[9]. In Ethiopia, cervical cancer is a big deal for women aged 15 to 44, coming in as the second most common cancer[9]. Globally, it's the fourth most common prevalent disease for women[10]. Aberrant methylation of tumor-suppressor genes' promoters can shut down their important functions and play a big role in causing cervical tumors[10]. There are various cervical cancer repressor genes (proteins turn off or reduce gene expression from the affected gene), such as CCNA1, CHF, HIT, PAX1, PTEN, SFRP4, and TSC1. The genes play a crucial role in causing cervical cancer by regulating transcription and expression through promoter hypermethylation, leading to precursor lesions during cervical development and malignant transformation[11]. The process of DNA methylation is primarily carried out by a group of enzymes known as DNA methyltransferases (DNMT1). It has been reported that DNMT1 (PDB ID: 4WXX), a protein responsible for DNA methylation can contribute to the development of cervical cancer. DNMT1 inhibits the transcription of tumor suppressor genes, facilitating tumorigenesis, which finally develops into cervical cancer. Tumor suppressor gene transcription is inhibited by DNMT1, which helps cancer grow and eventually leads to cervical cancer. Repressive genes' hypermethylation may be decreased, their expression can be increased, and the phenotype of malignant tumors can be reversed by inhibiting the DNMT1 enzyme.

    On the other hand, infection by the human papilloma virus (HPV) phenotype 16, enzyme 6 (PDB ID: 4XR8) has been correlated with a greatly increased risk of cervical cancer worldwide[12]. Based on variations in the nucleotide sequences of the virus genome, over 100 distinct varieties of the human papilloma virus (HPV) have been identified (e.g. type 1, 2 etc.). Genital warts can result from certain types 6 and 11 of sexually transmitted HPVs. Other HPV strains, still, that can infect the genitalia, do not show any symptoms of infection[8]. Persistent infection with a subset of approximately 13 so-called 'high-risk' sexually transmitted HPVs, including such as types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 different from the ones that cause warts may lead to the development of cervical intraepithelial neoplasia (CIN), vulvar intraepithelial neoplasia (VIN), penile intraepithelial neoplasia (PIN), and/or anal intraepithelial neoplasia (AIN). These are precancerous lesions and can progress to invasive cancer. Almost all occurrences of cervical cancer have HPV infection as a required component[13]. Superfluous infection by HPV type 16 E6 (PDB ID: 4XR8) has been correlated with a greatly increased genital risk of precursor cervical cancer worldwide[11]. Scholars more defined in major biochemical and biological activities of HPV type 16 E6 (PDB ID: 4XR8) in high-risk HPV oncogenes and how they may work together in the development of cervical disease and cancer[13].

    One potential approach to treat cervical cancer is to inhibit the activity of the DNMT1 and HPV type 16 E6 enzymes specifically[1316]. Over 50% of clinical drug forms worldwide originate from plant compounds[17]. In the past, developing new drugs was a lengthy and costly process. However, with the emergence of bioinformatics, the use of computer-based tools and methods have become increasingly important in drug discovery. One such method is molecular docking and ADMET profiling which involves using the structure of a drug to screen for potential candidates. This approach is known as structure-based drug design and can save both time and resources during the research process[15]. Structural-based drug designing addresses ligand binding sites with a known protein structure[15]. Using free binding energies, a computational method known as docking examines a large number of molecules and suggests structural theories for impeding the target molecule[17]. Nowadays, due to increasing antibiotic resistance like bacteria, fungi, and cancer cells, natural products remain an important source for discovering antimicrobial compounds and novel drugs for anti-cancers like cervical cancers. Therefore, the purpose of this research is to assess the antimicrobial activity of extracts, molecular docking, ADMET profiling in anticancer properties of compounds isolated from Salvia rosmarinus, on a targeting DNMT1 and HPV type 16 E6 in human cervical cancer. In the present study, various solvent crude extracts obtained from Salvia rosmarinus were used for antimicrobial activity and the isolated compounds 1 and 2 were submitted for in silico study to target the DNMT1 and HPV type 16 E6 enzymes to inhibit the growth of human cervical cancer cells.

    Healthy Salvia rosmarinus leaves were collected in Bacho district, Southwest Showa, Oromia, Ethiopia, during the dry season of November 2022. The plant materials were authenticated by Melaku Wondafrish, Natural Science Department, Addis Ababa University and deposited with a voucher number 3/2-2/MD003-80/8060/15 in Addis Ababa University's National Herbarium.

    The most common organic solvent used in extractions of medicinal plants is 2.5 L of petroleum ether, chloroform/methanol (1:1), and methanol. The test culture medium for microbes was used and performed in sterile Petri dishes (100 mm diameter) containing sterile Muller–Hinton Agar medium (25 mL, pH 7) and Sabouraud Dextrose Agar (SDA) for bacteria and fungi, respectively. A sterile Whatman filter paper (No. 1) disc of 6 mm diameter was used to determine which antibiotics an infective organism is sensitive to prescribed by a minimum zone of inhibition (MZI). Ciprofloxacin antibiotic reference (manufactured by Wellona Pharma Ciprofloxacin tablet made in India) and Ketoconazole 2% (made in Bangladesh) were used as a positive controls for antibacterial and antifungal, respectively and Dimethyl sulfoxide (DMSO) 98.9% was used as a negative control for antimicrobial tests. In the present study, the height of the column was 650 mm and the width was 80 mm. Several studies by previous researchers showed the acceptable efficiency of column chromatography (up to 43.0% w/w recovery) in the fractionation and separation of phenolic compounds from plant samples[18]. In column chromatography, the ideal stationary phase used silica gel 60 (0.200 mm) particles. The 1H-NMR spectrums of the compounds were analyzed using a 600 MHz NMR machine and 150 MHz for 13C NMR. The compounds were dissolved in MeOD for compound 1 and in DMSO for compound 2 for NMR analysis. On the other hand, UV spectroscopy (made in China) used 570 nm ultraviolet light to determine the absorbency of flavonoids (mg·g−1) phytochemicals.

    The samples (extracts) were analyzed to detect the presence of certain chemical compounds such as alkaloids (tested using Wagner's reagents), saponins (tested using the froth test), steroids (tested with Liebermann Burchard's tests), terpenoids (tested with Lidaebermann Burchard's tests), quinones, and flavonoids (tested using Shinoda tests)[19].

    The leaves of Salvia rosmarinus (500 g) were successively extracted using maceration using petroleum ether, chloroform/methanol (1:1), and methanol, every one 2.5 L for 72 h to afford 3.6, 6, and 53 g crude extracts, respectively. The methanol/chloroform (1:1) extract (6 g) was loaded to silica gel (150 g) column chromatography using the increasing polarity of petroleum ether, methanol/chloroform (1:1) solvent system to afford 80 fractions (100 mL each). The fraction obtained from chloroform/methanol 1:1 (3:2) after repeated column chromatography yielded compound 1 (18 mg). Fractions 56-65, eluted with chloroform/methanol (1:1) were combined and purified with column chromatography to give compound 2 (10 mg).

    The microorganisms were obtained from the Ethiopia Biodiversity Institution (EBI). Two gram-positive bacteria namely Staphylococcus aureus serotype (ATCC 25923) and Streptococcus epidermidis (ATCC14990); and three gram-negative bacteria, namely Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 5702), and Klebsiella pneumonia (ATCC e13883) were inoculated overnight at 37 °C in Muller–Hinton Agar/MHA culture medium and two fungus strains of Candida albicans (ATCC 16404) and Aspergillus niger (ATCC 11414) were inoculated overnight at 27−30 °C in Sabouraud Dextrose Agar/SDA culture medium[20].

    The antibacterial and antifungal activities of different crude extracts obtained from Salvia rosmarinus plant leaves were evaluated by the disk diffusion method (in accordance with the 13th edition of the CLSI M02 document on hardydiagnostics.com/disk-diffusion). Briefly, the test was performed in sterile Petri dishes (100 mm diameter) containing solid and sterile Muller–Hinton Agar medium (25 mL, pH 7) and Sabouraud Dextrose Agar (SDA) for bacteria and fungi, respectively. The extracts were placed on the surface of the media that had previously been injected with a sterile microbial suspension (one microbe per petri dish) after being adsorbed on sterile paper discs (5 μL per Whatman disc of 6 mm diameter). To prevent test samples from eventually evaporating, all Petri dishes were sealed with sterile laboratory films. They were then incubated at 37 °C for 24 h, and the zone diameter of the inhibition was measured and represented in millimeters. Ciprofloxacin antibiotic reference (manufactured by Wellona Pharma Ciprofloxacin tablet, India) was used as a positive control and DMSO was used as a negative control for antibacterial activity test while Ketoconazole 2% (Bangladesh) was used as a positive control and 10 μL of 0.2% agar as a negative control for antifungal activity tests[20]. The term 'inhibitory concentration' refers to the minimum sample concentration required to kill 99.9% of the microorganisms present[21]. Three repetitions of the crude extract sample were used to precisely measure the inhibitory halo diameter (in mm), which was then expressed as mean ± standard deviation to assess the anti-microbial activity.

    Cervical cancer-causing protein was identified through relevant literature. The protein molecule structure of DNA (cytosine-5)-methyltransferase 1 (DNMT1) (PDB ID: 4WXX)[21] and HPV type 16 E6 (PDB ID: 4XR8)[21] - a protein known to cause cervical cancer - were downloaded from the Protein Data Bank[22]. The stability of the protein molecule was assessed using Rampage[23].

    Phytochemical constituents of Salvia rosmarinus plant leaves were used to select a source of secondary metabolites (ligands). Ligand molecules were obtained through plant extraction, and isolation, and realized with PubChem (https://pubchem.ncbi.nlm.nih.gov/). The ligands were downloaded in Silver diamine fluoride format (SDF) and then converted to PDB format using an online SMILES translator (https://cactus.nci.nih.gov/translate/). The downloaded files were in PDB format, which was utilized for running various tools and software[24].

    The Biovia Discovery Studio Visualizer software was used to analyze the protein molecule. The protein molecule was converted into PDB format and its hierarchy was analyzed by selecting ligands and water molecules. Both the protein molecule and the water molecules lost their attached ligands during the analysis. Finally, the protein's crystal structure was saved in a PDB file[25].

    PyRx software was utilized to screen secondary metabolites and identify those ligands with the lowest binding energy to the protein target. The ligands with the lowest binding energy were further screened for their drug-likeliness property through analysis. It is worth noting that PyRx runs on PDBQT format. To begin using PyRx, it needs to load a protein molecule. This molecule should be converted from PDB to the protein data bank, partial charge (Q), and Atom Type (PDBQT) format. Once the protein molecule is loaded, it can import ligands from a specific folder in Silver diamine fluoride format. The ligand energy was minimized and changed to PDBQT format. The protein was docked with the ligand and screened based on minimum binding energy (https://cactus.nci.nih.gov/translate/).

    The optimal ligand was selected for final docking using AutoDock Vina and Biovia by modifying the reference of Discovery Studio Client 2021 (https://cactus.nci.nih.gov/translate/).

    The protein target from the Protein Data Bank (PDB) was loaded onto the graphical interface of AutoDock Vina. To prepare the protein for docking, water molecules were removed, hydrogen polar atoms were added, and Kollman charges were assigned to the protein molecule. Ultimately, PDBQT format was used to store the protein. After being imported in PDB format, the Ligand molecule was transformed to PDBQT format. Next, a grid box was chosen to represent the docked region. The command prompt was used to run AutoDock Vina and the outcomes were examined (https://cactus.nci.nih.gov/translate/).

    Docking the ligand with the protein target DNMT1(PDB ID: 4WXX)[22] and HPV type 16 E6 (PDB ID: 4XR8)[21] enzymes were performed using Biovia Discovery Studio Client 2021 by loading the protein target first followed by the ligand in PDB format. The charges were attached to the protein molecule, and the energy was minimized for the ligands. Both the protein and ligand molecules were prepared for docking. Once the docking process was complete, the results were analyzed based on several parameters, including absolute energy, clean energy, conf number, mol number, relative energy, and pose number. The interaction between the protein and ligand was analyzed using structure visualization tools, such as Biovia Discovery Studio Visualizer and PyMol (https://cactus.nci.nih.gov/translate/).

    The process of visualizing the structure was carried out using the PyMol tool. PyMol is a freely available software. Firstly, the protein molecule in PDBQT form was loaded on the PyMol graphical screen. Then, the output PDBQT file was added. The docked structure was visualized and the 'molecule' option was changed to 'molecular surface' under the 'shown as' menu (https://cactus.nci.nih.gov/translate/).

    Drug likeliness properties of the screened ligands were evaluated using the SwissADME online server. SMILE notations were obtained from PubChem and submitted to the SwissADME web server for analysis. The drugs were subjected to Lipinski's rule of five[20] for analysis. Lipinski's rules of five were selected for final docking through AutoDock Vina and Biovia Discovery Studio Client 2021. Ligands 1 and 2 were analyzed using Lipinski's rule of five for docking with AutoDock Vina and Biovia Discovery Studio Client 2021.

    The antimicrobial analysis data generated by triplicate measurements reported as mean ± standard deviation, and a bar graph also generated by GraphPad Prism version 8.0.1 (244) for Windows were used to perform the analysis. GraphPad Prism was used and combined with scientific graphing, comprehensive bar graph fitting (nonlinear regression), understandable statistics, and data organization. Prism allows the performance and modification of basic statistical tests commonly used and determined through the statistical applications in microbiology labs (https://graphpad-prism.software.informer.com/8.0/).

    Phytochemical screening of the different extracts for the presence (+) and absence (−) of alkaloids, steroids, glycosides, coumarins, terpenoids, flavonoids, carbohydrates, tannins, and saponins were done. The present study showed that alkaloids, terpenoids, flavonoids, and tannins tests in S. rosmarinus leaves of petroleum ether, chloroform/methanol (1:1), and methanol extracts were high whereas glycoside, coumarins, and carbohydrates had a moderate presence. The extract of S. rosmarinus leaves contain commonly bioactive constituents such as alkaloids, steroids, terpenoids, flavonoids, tannins, and saponins. These bioactive chemicals have active medicinal properties. Phytochemical compounds found in S. rosmarinus leaves have the potential to treat cancer cells and pathogens. The study also found that these flavonoids are related to natural phenolic compounds with anticancer and antimicrobial properties in the human diet (Table 1).

    Table 1.  Phytochemical screening tests result of petroleum ether, chloroform/methanol (1:1) and methanol extracts of Salvia rosmarinus leaves.
    Botanical name Phytochemicals Phytochemical screening tests Different extracts
    Petroleum ether Chloroform/methanol (1:1) Mehanol
    Salvia rosmarinus Alkaloids Wagner's test ++ ++ ++
    Steroids Libermann Burchard test ++ + ++
    Glycoside Keller-Killiani test +
    Coumarins Appirade test + +
    Terpenoids Libermann Burchard test ++ ++ ++
    Flavonoids Shinoda test ++ ++ ++
    Carbohydrate Fehling's test ++ ++
    Tannins Lead acetate test ++ ++ ++
    Saponins Foam test + + +
    + indicates moderate presence, ++ indicates highly present, − indicates absence.
     | Show Table
    DownLoad: CSV

    Two compounds were isolated and characterized using NMR spectroscopic methods (Fig. 1 & Supplementary Fig. S1ac). Compound 1 (10 mg) was isolated as yellow crystals from the methanol/chloroform (1:1) leaf extract of Salvia rosmarinus. The TLC profile showed a spot at Rf 0.42 with methanol/chloroform (3:2) as a mobile phase. The 1H-NMR spectrum (600 MHz, MeOD, Table 2, Supplementary Fig. S1a) of compound 1 showed the presence of one olefinic proton signal at δ 5.3 (t, J = 3.7 Hz, 1H), two deshielded protons at δ 4.7 (m, 1H), and 4.1 (m, 1H) associated with the C-30 exocyclic methylene group, and one O-bearing methine proton at δH 3.2 (m, 1H), and six methyl protons at δ 1.14 (s, 3H), 1.03 (d, J = 6.3 Hz, 3H), 1.00 (s, 3H), 0.98 (s, 3H), 0.87 (s, 3H), and 0.80 (s, 3H). A proton signal at δ 2.22 (d, J = 13.5 Hz, 1H) was attributed to methine proton for H-18. Other proton signals integrate for 20 protons were observed in the range δ 2.2 to 1.2. The proton decoupled 13C-NMR and DEPT-135 spectra (151 MHz, MeOD, Supplementary Fig. S1b & c) of compound 1 revealed the presence of 30 well-resolved carbon signals, suggesting a triterpene skeleton. The analysis of the 13C NMR spectrum displayed signals corresponding to six methyl, nine methylene, seven methine, and eight quaternary carbons. Among them, the signal observed at δ 125.5 (C-12) belongs to olefinic carbons. The methylene carbon showed signals at δC 39.9, 28.5, 18.1, 36.7, 23.9, 30.4, 26.5, 32.9, and 38.6. The quaternary carbons showed a signal at δC 39.4, 41.9, 38.4, 138.2, 41.8, and 47.8. The signals of exocyclic methylene carbon signals appeared at δ 153.1 and 103.9. The spectrum also showed sp3 oxygenated methine carbon at δ 78.3 and carboxyl carbon at δ 180.2. The spectrum revealed signals due to methyl groups at δC 27.4, 16.3, 15.0, 20.2, 22.7, and 16.4. The remaining carbon signals for aliphatic methines were shown at δC 55.3, 55.2, 53.0, and 37.1. The NMR spectral data of compound 1 is in good agreement with data reported for micromeric acid, previously reported from the same species by Abdel-Monem et al.[26]. (Fig. 1, Table 2).

    Figure 1.  Structure of isolated compounds from the leaves of Salvia rosmarinus.
    Table 2.  Comparison of the 13C-NMR spectral data of compound 1 and micromeric acid (MeOD, δ in ppm).
    Position NMR data of compound 1 Abdel-Monem
    et al.[26]
    1H-NMR 13C-NMR 13C-NMR
    1 38.60 39.9
    2 27.8 28.5
    3 3.2 (m, 1H) 78.3 80.3
    4 39.4 39.9
    5 55.3 56.7
    6 18.1 18.3
    7 36.7 34.2
    8 41.9 40.7
    9 53 48.8
    10 38.4 38.2
    11 23.9 24.6
    12 5.3 (t, J = 3.7 Hz, 1H) 125.5 127.7
    13 138.2 138
    14 41.8 43.3
    15 30.4 29.1
    16 26.5 25.6
    17 47.8 48
    18 δ 2.22 (d, J = 13.5 Hz, 1H) 55.2 56.1
    19 37.1 38.7
    20 153.1 152.8
    21 32.9 33.5
    22 39.0 40.1
    23 27.4 29.4
    24 16.3 16.9
    25 15.0 16.6
    26 20.2 18.3
    27 22.7 24.6
    28 180.2 177.8
    29 16.4 17.3
    30 4.7 (m, 1H), and 4.1 (m, 1H) 103.9 106.5
     | Show Table
    DownLoad: CSV

    Compound 2 (18 mg) was obtained as a white amorphous isolated from 40% methanol/chloroform (1:1) in petroleum ether fraction with an Rf value of 0.49. The 1H NMR (600 MHz, DMSO, Supplementary Fig. S2a) spectral-data showed two doublets at 7.79 (d, J = 8.7 Hz, 2H), and 6.90 (d, J = 8.7 Hz, 2H) which are evident for the presence of 1,4-disubstituted aromatic group. The oxygenated methylene and terminal methyl protons were shown at δ 4.25 (q, J = 7.1 Hz, 2H) and 1.29 (t, J = 7.1 Hz, 3H), respectively. The13C-NMR spectrum, with the aid of DEPT-135 (151 MHz, DMSO, Table 3, Supplementary Fig. S2b & c) spectra of compound 2 confirmed the presence of well-resolved seven carbon peaks corresponding to nine carbons including threee quaternary carbons, one oxygenated methylene carbon, one terminal methyl carbon, and two symmetrical aromatic methine carbons. The presence of quaternary carbon signals was shown at δ 120.9 (C-1), 148.2 (C-4), and ester carbonyl at δ 166.0 (C-7). The symmetry aromatic carbons signal was observed at δ 131.4 (C-2, 6), and 116.8 (C-3, 5). The oxygenated methylene and terminal methyl carbons appeared at δC 60.4 (C-8) and 14.7 (C-9), respectively. The spectral results provided above were in good agreement with those for benzocaine in the study by Alotaibi et al.[27]. Accordingly, compound 2 was elucidated to be benzocaine (4-Aminobenzoic acid-ethyl ester) (Table 3, Fig. 1, Supplementary Fig. S2ac), this compound has never been reported before from the leaves of Salvia rosmarinus.

    Table 3.  Comparison of the 1H-NMR, and 13C-NMR spectral data of compound 2 and benzocaine (DMSO, δ in ppm).
    Position NMR data of compound 2 Alotaibi et al.[27]
    1H-NMR 13C-NMR 1H-NMR 13C-NMR
    1 120.9 119
    2 7.79 (d, J = 8.7 Hz, 2H) 131.4 7.86 (d, J = 7.6 Hz) 132
    3 6.90 (d, J = 8.7 Hz, 2H) 116.8 6.83 (d, J = 7.6 Hz) 114
    4 148.2 151
    5 6.90 (d, J = 8.7 Hz, 2H) 116.8 6.83 (d, J = 7.6 Hz) 114
    6 7.79 (d, J = 8.7 Hz, 2H) 131.4 7.86 (d, J = 7.6 Hz) 132
    7 166.0 169
    8 4.3 (q, J = 7.1 Hz, 2H) 60.4 4.3 (q, J = 7.0 Hz) 61
    9 1.3 (t, J = 7.1 Hz, 3H) 14.7 1.36 (t, J = 7.0 Hz) 15
     | Show Table
    DownLoad: CSV

    The extracts and isolated compounds from Salvia rosmarinus were evaluated in vitro against microbes from gram-positive bacteria (S. aureus and S. epidermidis), gram-negative bacteria (E. coli, P. aeruginosa, and K. pneumoniae) and fungi (C. albicans and A. Niger) (Table 4). The petroleum ether extracts exhibited significant activity against all the present study-tested microbes at 100 μg·mL−1, resulting in an inhibition zone ranging from 7 to 21 mm. Chloroform/methanol (1:1) and methanol extracts demonstrated significant activity against all the present study-tested microbes at 100 μg·mL−1 exhibiting inhibition zones from 6 to 14 mm and 6 to 13 mm, respectively (Table 4). The chloroform/methanol (1:1) extracts were significantly active against bacteria of E. coli and K. pneumonia, and A. Niger fungi at 100 μg·mL−1. On the other hand, chloroform/methanol (1:1) extracts were significantly inactive against the S. rosmarinus and P. aeruginosa of bacteria and C. albicans of fungi, and again chloroform/methanol (1:1) extracts overall significantly active produced an inhibition zone of 12 to 14 mm (Table 4). Methanol extracts exhibited significant activity against S. aureus, E. coli bacteria, and A. Niger fungi at 100 μg·mL−1. The inhibition zone was recorded to be 11 to 13 mm. However, methanol extracts exhibited significant inactivity against K. pneumoniae (Table 4). The overall result of our studies shows that Salvia rosmarinus was extracted and evaluated in vitro, exhibiting significant antibacterial and antifungal activity, with inhibition zones recorded between 6 to 21 mm for bacteria and 5 to 21 mm for fungi. In our study, the positive control for ciprofloxacin exhibited antibacterial activity measured at 21.33 ± 1.15 mm, 15.00 ± 0.00 mm, and 14.20 ± 0.50 mm for petroleum ether, chloroform/methanol (1:1), and methanol extracts, respectively. Similarly, the positive control for ketoconazole demonstrated antifungal activity of 22.00 ± 1.00 mm, 13.67 ± 0.58 mm, and 15.00 ± 0.58 mm for petroleum ether, chloroform/methanol (1:1), and methanol extracts, respectively. Additionally, our findings indicated that the mean values of flavonoids (mg/g) tested were 92.2%, 90.4%, and 94.0% for petroleum ether, chloroform/methanol (1:1), and methanol extracts, respectively. This suggests that the groups of phenolic compounds evaluated play a significant role in antimicrobial activities, particularly against antibiotic-resistant strains.

    Table 4.  Comparison of mean zone of inhibition (MZI) leaf extracts of Salvia rosmarinus.
    Type of specimen, and standard antibiotics for
    each sample
    Concentration (μg·mL−1) of extract
    in 99.8% DMSO
    Average values of the zone of inhibition (mm)
    Gram-positive (+) bacteria Gram-negative (−) bacteria Fungai
    S. aurous S. epidermidis E. coli P. aeruginosa K. pneumoniae C. albicans A. niger
    Petroleum ether extracts
    S. rosmarinus 50 18.50 ± 0.50 15.33 ± 0.58 0.00 ± 0.00 0.00 ± 0.00 10.00 ± 0.00 15.93 ± 0.12 4.47 ± 0.50
    75 19.87 ± 0.06 17.00 ± 0.00 9.33 ± 0.29 10.53 ± 0.50 10.93 ± 0.12 18.87 ± 0.23 5.47 ± 0.50
    100 21.37 ± 0.78 17.50 ± 0.50 11.47 ± 0.50 13.17 ± 0.29 12.43 ± 0.51 20.83 ± 0.76 6.70 ± 0.10
    Standard antibiotics Cipro. 21.33 ± 1.15 18.33 ± 0.58 9.33 ± 0.58 12.30 ± 0.52 15.00 ± 0.00
    Ketocon. 22.00 ± 1.00 10.67 ± 0.58
    Chloroform/methanol (1:1) extracts
    50 5.47 ± 0.42 0.00 ± 0.00 10.33 ± 0.00 0.00 ± 0.00 9.70 ± 0.00 0.00 ± 0.12 8.47 ± 0.50
    S. rosmarinus
    75 5.93 ± 0.06 0.00 ± 0.00 11.33 ± 0.29 0.00 ± 0.50 12.50 ± 0.12 0.00 ± 0.23 10.67 ± 0.50
    100 6.47 ± 0.06 0.00 ± 0.00 14.17 ± 0.50 7.33 ± 0.29 14.17 ± 0.51 0.00 ± 0.76 12.67 ± 0.10
    Standard antibiotics Cipro. 15.00 ± 0.00 11.00 ± 1.00 11.33 ± 0.58 10.00 ± 0.52 12.67 ± 0.00
    Ketocon. 7.00 ± 1.00 13.67 ± 0.58
    Methanol extracts
    50 9.17 ± 0.29 5.50 ± 0.50 0.00 ± 0.00 7.50 ± 0.00 0.00 ± 0.00 6.57 ± 0.12 0.00 ± 0.50
    S. rosmarinus
    75 9.90 ± 0.10 6.93 ± 0.12 9.33 ± 0.29 8.50 ± 0.50 0.00 ± 0.00 8.70 ± 0.23 0.00 ± 0.50
    100 11.63 ± 0.55 7.97 ± 0.06 11.47 ± 0.50 9.90 ± 0.10 0.00 ± 0.00 10.83 ± 0.76 13.13 ± 0.10
    Standard antibiotics Cipro. 13.00 ± 0.00 11.50 ± 0.50 14.20 ± 0.58 13.33 ± 0.29 10.00 ± 0.00
    Ketocon. 12.00 ± 1.00 15.00 ± 0.58
    Mean values of flavonoids (mg·g−1) by 570 nm
    S. rosmarinus
    Petroleum ether extracts Chloroform/methanol (1:1) extracts Methanol extracts
    50 0.736 0.797 0.862
    75 0.902 0.881 0.890
    100 0.922 0.904 0.940
    Samples: Antibiotics: Cipro., Ciprofloxacin; Ketocon., ketoconazole (Nizoral); DMSO 99.8%, Dimethyl sulfoxide.
     | Show Table
    DownLoad: CSV

    Determining the three solvent extracts in S. rosmarinus plants resulted in relatively high comparable with positive (+) control. Especially, the S. rosmarinus petroleum ether leaf extracts against drug resistance human pathogenic bacteria S. aureus, S. epidermidis, E. coli, P. aeruginosa, and K. pneumoniae were minimum zone of inhibition (MZI) recorded that 21.37 ± 0.78, 17.50 ± 0.50, 11.47 ± 0.50, 13.17 ± 0.29, and 12.43 ± 0.51 mm, respectively and against human pathogenic fungi C. albicans and A. niger were minimum zone of inhibition (MZI) recorded that 20.83 ± 0.76 and 6.70 ± 0.10 mm, respectively which was used from bacteria against S. aureus MZI recorded that 21.37 ± 0.78 mm higher than the positive control (21.33 ± 1.15 mm). The S. rosmarinus of chloroform/methanol (1:1) extracts were found to be against E. coli (14.17 ± 0.50 mm) and K. pneumoniae (14.17 ± 0.51 mm) higher than the positive control 11.33 ± 0.58 and 12.67 ± 0.00 mm, respectively. The methanol extracts of leaves in the present study plants were found to have overall MZI recorded less than the positive control. The Salvia rosmarinus crude extracts showed better antifungal activities than the gram-negative (−) bacteria (Table 4, Fig 2, Supplementary Fig. S3). Therefore, the three extracts, using various solvents of different polarity indexes, have been attributed to specific biological activities. For example, the antimicrobial activities of Salvia rosmarinus extracts may be due to the presence of alkaloids, terpenoids, flavonoids, tannins, and saponins in natural products (Table 1).

    Figure 2.  Microbes' resistance with drugs relative to standard antibiotics in extracts of Salvia rosmarinus. The figures represent understudy of three extracts derived from Salvia rosmarinus. (a) Petroleum ether, (b) chloroform/methanol (1:1), and (c) methanol extracts tested in Salvia rosmarinus.

    Compounds 1 and 2 were isolated from chloroform/methanol (1:1) extract of Salvia rosmarinus (Fig. 1, Tables 2 & 3). The plant extract exhibited highest antibacterial results recorded a mean inhibition with diameters of 21 and 14 mm at a concentration of 100 mg·mL−1 against S. aureus and E. coli/K. pneumoniae, respectively. After testing, overall it was found that the highly active petroleum ether extract of Salvia rosmarinus was able to inhibit the growth of S. aureus and C. albicans, with inhibition zones of 21 and 20 mm, respectively. The petroleum ether extracts showed good efficacy against all tested microbes, particularly gram-positive bacteria and fungi (Table 4). This is noteworthy because gram-negative bacteria generally exhibit greater resistance to antimicrobial agents. Petroleum ether and chloroform/methanol (1:1) extracts of the leaves were used at a concentration of 100 mg·mL−1, resulting in impressive inhibition zone diameters of 11 and 14 mm for E. coli, 13 and 7 mm for P. aeruginosa, and 12 and 14 mm for K. pneumoniae, respectively.

    The present study found that at a concentration of 50 μg·mL−1, petroleum ether, chloroform/methanol (1:1), and MeOH extracts did not display any significant inhibition zone effects against the tested microbes. This implies that the samples have a dose-dependent inhibitory effect on the pathogens. The leaves of Salvia rosmarinus have been found to possess remarkable antimicrobial properties against gram-negative bacteria in different extracts such as E. coli, P. aeruginosa, and K. pneumoniae with 14.17 ± 0.50 in chloroform/methanol (1:1), 13.17 ± 0.29 in petroleum ether and 14.17 ± 0.51 in chloroform/methanol (1:1), respectively. However, in the present study, Salvia rosmarinus was found to possess remarkable high zones of inhibition with diameters of 21.37 ± 0.78 and 17.50 ± 0.50 mm antimicrobial properties against S. aureus, and S. epidermidis of gram-positive bacteria, respectively (Supplementary Fig. S3). The results are summarized in Fig. 2ac.

    The crystal structure of human DNMT1 (351-1600), classification transferase, resolution: 2.62 Å, PDB ID: 4WXX. Active site dimensions were set as grid size of center X = −12.800500 Å, center Y = 34.654981 Å, center Z = −24.870231 Å (XYZ axis) and radius 59.081291. A study was conducted to investigate the binding interaction of the isolated compounds 1 and 2 of the leaves of Salvia rosmarinus with the binding sites of the DNMT1 enzyme in human cervical cancer (PDB ID: 4WXX), using molecular docking analysis.

    The study also compared the results with those of standard anti-cancer agents Jaceosidin (Table 5 & Fig. 3). The compounds isolated had a final fixing energy extending from −5.3 to −8.4 kcal·mol−1, as shown in Table 4. It was compared to jaceosidin (–7.8 kcal·mol−1). The results of the molecular docking analysis showed that, compound 1 (−8.4 kcal·mol−1) showed the highest binding energy values compared with the standard drugs jaceosidin (–7.8 kcal·mol−1). Compound 2 has shown lower docking affinity (–5.3 kcal·mol−1) but good matching amino acid residue interactions compared to jaceosidin. After analyzing the results, it was found that the isolated compounds had similar residual interactions and docking scores with jaceosidin.

    Table 5.  Molecular docking results of ligand compounds 1 and 2 against DNMT1 enzyme (PDB ID: 4WXX).
    Ligands Binding affinity

    ( kcal·mol−1)
    H-bond Residual interactions
    Hydrophobic/electrostatic Van der Waals
    1 −8.4 ARG778 (2.85249), ARG778 (2.97417), VAL894 (2.42832) Lys-889, Pro-879, Tyr-865, His-795, Cys-893, Gly-760, Val-759, Phe-892, Phe-890, Pro-884, Lys-749
    2 −5.3 ARG596 (2.73996), ALA597 (1.84126), ILE422 (2.99493), THR424 (2.1965), ILE422 (2.93653) Electrostatic Pi-Cation-ARG595 (3.56619), Hydrophobic Alkyl-ARG595 (4.15839), Hydrophobic Pi-Alkyl-ARG595 (5.14967) Asp-423, Glu-428, Gly-425, Ile-427, Trp-464, Phe-556, Gln-560, Gln-594, Glu-559, Gln-598, Ser-563
    Jaceosidin −7.8 ASP571 (2.93566), GLN573 (2.02126), GLU562 (2.42376), GLN573 (3.49555), GLU562 (3.46629) Hydrophobic Alkyl-PRO574 (4.59409), Hydrophobic Alkyl-ARG690 (5.09748), Hydrophobic Pi-Alkyl-PHE576 (5.1314), Hydrophobic Pi-Alkyl-PRO574 (4.97072), Hydrophobic Pi-Alkyl-ARG690 (5.07356) Glu-698, Cys-691, Ala-695, Pro-692, Val-658, Glu-566, Asp-565
     | Show Table
    DownLoad: CSV
    Figure 3.  The 2D and 3D binding interactions of compounds against DNMT1 enzyme (PDB ID: 4WXX). The 2D and 3D binding interactions of compound 1 and 2 represent against DNMT1 enzyme, and jaceosidin (standard) against DNMT1 enzyme.

    Hence, compound 1 might have potential anti-cancer agents. However, anti-cancer in vitro analysis has not yet been performed. Promising in silico results indicate that further research could be beneficial. The 2D and 3D binding interactions of compounds 1 and 2 against human cervical cancer of DNMT1 enzyme (PDB ID: 4WXX) are presented in Fig. 3. The binding interactions between the DNMT1 enzyme (PDB ID: 4WXX), and compound 1 (Fig. 3) and compound 2 (Fig. 3) were displayed in 3D. Compounds and amino acids are connected by hydrogen bonds (green dash lines) and hydrophobic interactions (non-green lines).

    Crystal structure of the HPV16 E6/E6AP/p53 ternary complex at 2.25 Å resolution, classification viral protein, PDB ID: 4XR8. Active site dimensions were set as grid size of center X = −43.202782 Å, center Y = −39.085513 Å, center Z = −29.194115 Å (XYZ axis), R-value observed 0.196, and Radius 65.584122. A study was conducted to investigate the binding interaction of the isolated compounds 1 and 2 of the leaves of Salvia rosmarinus with the binding sites of the enzyme of human papilloma virus (HPV) type 16 E6 (PDB ID: 4XR8), using molecular docking analysis software. The study also compared the results with those of standard anti-cancer agents jaceosidin (Table 6 & Fig. 4). The compounds isolated had a bottom most fixing energy extending from −6.3 to −10.1 kcal·mol−1, as shown in Table 6. It was compared to jaceosidin (–8.8 kcal·mol−1). The results of the molecular docking analysis showed that, compound 1 (−10.1 kcal·mol−1) showed the highest binding energy values compared with the standard drugs jaceosidin (–8.8 kcal·mol−1). Compound 2 has shown lower docking affinity (–6.3 kcal·mol−1) but good matching amino acid residue interactions compared to jaceosidin. After analyzing the results, it was found that the isolated compounds had similar residual interactions and docking scores with jaceosidin.

    Table 6.  Molecular docking results of ligand compounds 1 and 2 against HPV type 16 E6 (PDB ID: 4XR8).
    Ligands Binding affinity
    (kcal·mol−1)
    H-bond Residual interactions
    Hydrophobic/electrostatic Van der Waals
    1 −10.1 ASN101 (2.25622), ASP228 (2.88341) Asp-148, Lys-176, Lys-180, Asp-178, Ile-179, Tyr-177, Ile-334, Glu-382, Gln-336, Pro-335, Gln-73, Arg-383, Tyr-100
    2 −6.5 TRP63 (1.90011), ARG67 (2.16075), ARG67 (2.8181) Hydrophobic Pi-Sigma-TRP341 (3.76182), Hydrophobic Pi-Pi Stacked-TYR156 (4.36581), Hydrophobic Pi-Pi T-shaped-TRP63 (5.16561), Hydrophobic Pi-Pi T-shaped-TRP63 (5.44632), Hydrophobic Alkyl-PRO155 (4.34691), Hydrophobic Pi-Alkyl-TRP341 (4.11391), Hydrophobic Pi-Alkyl-ALA64 (4.61525) Glu-154, Arg-345, Asp-66, Met-331, Glu-112, Lys-16, Trp-231
    Jaceosidin −8.8 ARG146 (2.06941), GLY70 (3.49991), GLN73 (3.38801) Electrostatic Pi-Cation-ARG67 (3.93442), Hydrophobic Pi-Alkyl-PRO49 (5.40012) Tyr-342, Tyr-79, Ser-338, Arg-129, Pro-335, Leu-76, Tyr-81, Ser-74, Tyr-71, Ser-80, Glu-46
     | Show Table
    DownLoad: CSV
    Figure 4.  The 2D and 3D binding interactions of compounds against HPV type 16 E6 (PDB ID: 4XR8). The 2D and 3D binding interactions of compound 1 and 2 represent against HPV type 16 E6 enzyme, and jaceosidin (standard) against HPV type 16 E6 enzyme.

    Hence, compounds 1 and 2 might have potential anti-cancer agents of HPV as good inhibitors. However, anti-cancer in vitro analysis has not been performed yet on HPV that causes cervical cancer agents. Promising in silico results indicate that further research could be beneficial. The 2D and 3D binding interactions of compounds 1 and 2 against human papilloma virus (HPV) type 16 E6 enzyme (PDB ID: 4XR8) are presented in Fig. 4. The binding interactions between the HPV type 16 E6 enzyme (PDB ID: 4XR8) and compound 1 (Fig. 4) and compound 2 (Fig. 4) were displayed in 3D. Compounds and amino acids are connected by hydrogen bonds (magenta lines) and hydrophobic interactions (non-green lines).

    In silico bioactivities of a drug, including drug-likeness and toxicity, predict its oral activity based on the document of Lipinski's Rule[25] was stated and the results of the current study showed that the compounds displayed conform to Lipinski's rule of five (Table 7). Therefore, both compounds 1 and 2 should undergo further investigation as potential anti-cancer agents. Table 8 shows the acute toxicity predictions, such as LD50 values and toxicity class classification (ranging from 1 for toxic, to 6 for non-toxic), for each ligand, revealing that none of them were acutely toxic. Furthermore, they were found to be similar to standard drugs. Isolated compound 1 has shown toxicity class classification 4 (harmful if swallowed), while 2 showed even better toxicity prediction giving results of endpoints such as hepatotoxicity, mutagenicity, cytotoxicity, and irritant (Table 8). All the isolated compounds were predicted to be non-hepatotoxic, non-irritant, and non-cytotoxic. However, compound 1 has shown carcinogenicity and immunotoxicity (Table 9). Hence, based on ADMET prediction analysis, none of the compounds have shown acute toxicity, so they might be proven as good drug candidates.

    Table 7.  Drug-likeness predictions of compounds computed by Swiss ADME.
    Ligands Formula Mol. Wt. (g·mol−1) NRB NHA NHD TPSA (A°2) Log P (iLOGP) Log S (ESOL) Lipinski's rule of five
    1 C30H46O3 454.68 1 3 2 57.53 3.56 −6.21 1
    2 C 9H11NO2 165.19 3 2 1 52.32 1.89 −2.21 0
    Jaceosidin C17H14O7 330.3 3 7 3 105 1.7 1 0
    NHD, number of hydrogen donors; NHA, number of hydrogen acceptors; NRB, number of rotatable bonds; TPSA, total polar surface area; and log P, octanol-water partition coefficients; Log S, turbid metric of solubility.
     | Show Table
    DownLoad: CSV
    Table 8.  Pre ADMET predictions of compounds, computed by Swiss ADME.
    Ligands Formula Skin permeation value
    (logKp - cm·s−1)
    GI
    absorption
    Inhibitor interaction
    BBB permeability Pgp substrate CYP1A2 inhibitor CYP2C19 inhibitor CYP2C9 inhibitor CYP2D6 inhibitor
    1 C30H46O3 −4.44 Low No No No No No No
    2 C 9H11NO2 −5.99 High Yes No No No No No
    Jaceosidin C17H14O7 −6.13 High No No Yes No Yes Yes
    GI, gastrointestinal; BBB, blood brain barrier; Pgp, P-glycoprotein; and CYP, cytochrome-P.
     | Show Table
    DownLoad: CSV
    Table 9.  Toxicity prediction of compounds, computed by ProTox-II and OSIRIS property explorer.
    Ligands Formula LD50
    (mg·kg−1)
    Toxicity
    class
    Organ toxicity
    Hepatotoxicity Carcinogenicity Immunotoxicity Mutagenicity Cytotoxicity Irritant
    1 C30H46O3 2,000 4 Inactive Active Active Inactive Inactive Inactive
    2 C 9H11NO2 NA NA Inactive Inactive Inactive Inactive Inactive Inactive
    Jaceosidin C17H14O7 69 3 Inactive Inactive Inactive Inactive Inactive Inactive
    NA, not available.
     | Show Table
    DownLoad: CSV

    Rosemary is an evergreen perennial plant that belongs to the family Lamiaceae, previously known as Rosmarinus officinalis. Recently, the genus Rosmarinus was combined with the genus Salvia in a phylogenetic study and became known as Salvia rosmarinus[28,29] and it has been used since ancient times for various medicinal, culinary, and ornamental purposes. In the field of food science, rosemary is well known as its essential oil is used as a food preservative, thanks to its antimicrobial and antioxidant properties, rosemary has many other food applications such as cooking, medicinal, and pharmacology uses[30]. According to the study, certain phytochemical compounds found in Salvia rosmarinus leaves have the potential to halt the growth of cancer cells, and pathogens or even kill them[31]. In literature, alkaloids are found mostly in fungi and are known for their strong antimicrobial properties, which make them valuable in traditional medicine[32,33]. However, in the present study, S. rosmarinus species have been shown to possess alkaloids. Most alkaloids have a bitter taste and are used to protect against antimalarial, antiasthma, anticancer, antiarrhythmic, analgesic, and antibacterial[33] also some alkaloids containing nitrogen such as vincristine, are used to treat cancer.

    Steroids occur naturally in the human body. They are hormones that help regulate our body's reaction to infection or injury, the speed of metabolism, and more. On the other hand, steroids are reported to have various biological activities such as chronic obstructive pulmonary disease (COPD), multiple sclerosis, and imitate male sex hormones[34]. It is a natural steroid compound occurring both in plants and animals[35]. Thus, were found in the present study. Terpenoids are derived from mevalonic acid (MVA) which is composed of a plurality of isoprene (C5) structural units. Terpenoids, like mono-terpenes and sesquiterpenes, are widely found in nature and more than 50,000 have been found in plants that reduce tumors and cancers. Many volatile terpenoids, such as menthol and perillyl alcohol, are used as raw materials for spices, flavorings, and cosmetics[36]. In the present study, high levels of these compounds were found in Salvia rosmarinus leaves.

    Flavonoids are a class of phenolic compounds commonly found in fruits and vegetables and are considered excellent antioxidants[37]. Similarly, the results of this study revealed that S. rosmarinus contain flavonoids. According to the literature, these flavonoids, terpenoids, and steroid activities include anti-diabetic, anti-inflammatory, anti-cancer, anti-bacterial, hepatic-protective, and antioxidant effects[36]. Tannins are commonly found in most terrestrial plants[38] and have the potential to treat cancer, and HIV/AIDS as well as to treat inflamed or ulcerated tissues. Similarly, in the present study, tannins were highly found in the presented plant. On the other hand, due to a sudden rise in the number of contagious diseases and the development of antimicrobial resistance against current drugs, drug development studies are vital to discovering novel medicinal compounds[30] and add to these cancer is a complex multi-gene disease[39] as in various cervical cancer repressor genes[11] that by proteins turn off or reduce gene expression from the affected gene to cause cervical cancer by regulating transcription and expression through promoter hypermethylation (DNMT1), leading to precursor lesions during cervical development and malignant transformation.

    In a previous study[40], a good antibacterial result was recorded at a median concentration (65 μg·mL−1). Methanol extract showed a maximum and minimum zone antibacterial result against negative bacteria E. coli 14 + 0.71 and most of the petroleum ether tests show null zone of inhibition. However, in the present study at a concentration of 100 μg·mL−1, the methanol extract demonstrated both maximum and minimum antibacterial zones against E. coli 11.47 ± 0.50. Conversely, the test conducted with petroleum ether exhibited a good zone of inhibition by increasing concentration. Further research may be necessary to determine the optimal concentration for this extract to maximize its efficacy. The results obtained in gram-negative bacteria such as E. coli, P. aeruginosa, and K. pneumoniae are consistent with previous research findings[41]. However, in the present study, Salvia rosmarinus has been found to possess high zones of inhibition with diameters of 21.37 ± 0.78 and 17.50 ± 0.50 mm antimicrobial properties against S. aureus, and S.epidermidis of gram-positive bacteria, respectively (Table 4 & Fig. 2, Supplementary Fig. S3). According to a previous study[42], the ethanolic leaf extract of Salvia rosmarinus did exhibit activity against C. albicans strains. In the present study, the antifungal activity of petroleum ether extracts from Salvia rosmarinus were evaluated against two human pathogenic fungi, namely C. albicans and A. niger. The findings showed that at a concentration of 100 μg·mL−1, the extracts were able to inhibit the growth of C. albicans 20.83 ± 0.76 resulting in a minimum zone of inhibition.

    Antimicrobial agents can be divided into groups based on the mechanism of antimicrobial activity. The main groups are: agents that inhibit cell wall synthesis, depolarize the cell membrane, inhibit protein synthesis, inhibit nucleic acid synthesis, and inhibit metabolic pathways in bacteria. On the other hand, antimicrobial resistance mechanisms fall into four main categories: limiting the uptake of a drug; modifying a drug target; inactivating a drug; and active drug efflux. Because of differences in structure, etc., there is a variation in the types of mechanisms used by gram-negative bacteria vs gram-positive bacteria. Gram-negative bacteria make use of all four main mechanisms, whereas gram-positive bacteria less commonly use limiting the uptake of a drug[43]. The present findings showed similar activity in chloroform/methanol (1:1) and methanol extracts of leaves of Salvia rosmarinus than gram-negative bacteria like P. aeruginosa and Klebsiella pneumoniae. However, Staphylococcus epidermidis of gram-positive bacteria under chloroform/methanol (1:1) extracts have similarly shown antimicrobial résistance. This occurred due to intrinsic resistance that may make use of limiting uptake, drug inactivation, and drug efflux that need further study. The structure of the cell wall thickness and thinners of gram-negative and gram-positive bacteria cells, respectively when exposed to an antimicrobial agent, there happen two main scenarios may occur regarding resistance and persistence. In the first scenario, resistant cells survive after non-resistant ones are killed. When these resistant cells regrow, the culture consists entirely of resistant bacteria. In the second scenario, dormant persistent cells survive. While the non-persistent cells are killed, the persistent cells remain. When regrown, any active cells from this group will still be susceptible to the antimicrobial agent.

    Ferreira et al.[44] explained that with molecular docking, the interaction energy of small molecular weight compounds with macromolecules such as target protein (enzymes), and hydrophobic interactions and hydrogen bonds at the atomic level can be calculated as energy. Several studies have been conducted showing natural products such as epigallocatechin-3-gallate-3-gallate (EGCG), curcumin, and genistein can be used as an inhibitor of DNMT1[4547] . In the literature micromenic (1) is used for antimicrobial activities and for antibiotic-resistance like methicillin-resistant Staphylococcus aureus (MRSA)[48], and benzocaine (2) is used to relieve pain and itching caused by conditions such as sunburn or other minor burns, insect bites or stings, poison ivy, poison oak, poison sumac, minor cuts, or scratches[49]. However, in the present study, Salvia rosmarinus was used as a source of secondary metabolites (ligands) by using chloroform/methanol (1:1) extract of the plant leaves yielded to isolate micromeric (1) and benzocaine (2) in design structure as a candidate for drugs as inhibitors of the DNMT1 enzyme by inhibiting the activity of DNMT1 that prevent the formation of cervical cancer cells.

    Cervical cancer is one of the most dangerous and deadly cancers in women caused by Human papillomaviruses (HPV). Some sexually transmitted HPVs (type 6 owner of E6) may cause genital warts. There are several options for the treatment of early-stage cervical cancer such as surgery, nonspecific chemotherapy, radiation therapy, laser therapy, hormonal therapy, targeted therapy, and immunotherapy, but there is no effective cure for an ongoing HPV infection. In the present study, Salvia rosmarinus leaves extracted and isolated compounds 1 and 2 are one of the therapeutic drugs design structure as a candidate drug for inhibiting HPV type 16 E6 enzyme. Similarly, numerous researchers have conducted studies on the impact of plant metabolites on the treatment of cervical cancer. Their research has demonstrated that several compounds such as jaceosidin, resveratrol, berberin, gingerol, and silymarin may be active in treating the growth of cells[47].

    Small-molecule drugs are still most commonly used in the treatment of cancer[50]. Molecular docking in in silico looks for novel small-molecule (ligands) interacting with genes or DNA or protein structure agents which are still in demand, newly designed compounds are required to have a specific even multi-targeted mechanism of action to anticancer and good selectivity over normal cells. In addition to these, in the literature, anti-cancer drugs are not easily classified into different groups[51]. Thus, drugs have been grouped according to their chemical structure, presumed mechanism of action, and cytotoxic activity related to cell cycle arrest, transcription regulation, modulating autophagy, inhibition of signaling pathways, suppression of metabolic enzymes, and membrane disruption[52]. Another problem for grouping anticancers often encountered is the resistance that may emerge after a brief period of a positive reaction to the therapy or may even occur in drug-naïve patients[50]. In recent years, many studies have investigated the molecular mechanism of compounds affecting cancer cells and results suggest that compounds exert their anticancer effects by providing free electron charge inhibiting some of the signaling pathways that are effective in the progression of cancer cells[53] and numerous studies have shown that plant-based compounds such as phenolic acids and sesquiterpene act as anticancer agents by affecting a wide range of molecular mechanisms related to cancer[53]. The present investigations may similarly support molecular mechanisms provided for the suppression of metabolic enzymes of cervical cancer.

    The main aim of the study was to evaluate the antimicrobial activity of different extracts of Salvia rosmarinus in vitro, and its compounds related to in silico targeting of enzymes involved in cervical cancer. The phytochemical screening tests indicated the presence of phytochemicals such as alkaloids, terpenoids, flavonoids, and tannins in its extracts. The plant also exhibited high antimicrobial activity, with varying efficacy in inhibiting pathogens in a dose-dependent manner (50−100 μg·mL−1). However, this extract exhibited a comparatively high inhibition zone in gram-positive and gram-negative bacteria had lower inhibition zones against E. coli, P. aeruginosa, and K. pneumoniae, respectively, and stronger antifungal activity 20.83 ± 0.76 mm inhibition zone against C. albicans fungi. Molecular docking is a promising approach to developing effective drugs through a structure-based drug design process. Based on the docking results, the in silico study predicts the best interaction between the ligand molecule and the protein target DNMT1 and HPV type 16 E6. Compound 1 (–8.3 kcal·mol−1) and 2 (–5.3 kcal·mol−1) interacted with DNMT1 (PDB ID: 4WXX) and the same compound 1 (–10.1 kcal·mol−1) and 2 (–6.5 kcal·mol−1) interacted with HPV type 16 E6 (PDB ID: 4XR8). Compounds 1 and 2 may have potential as a medicine for treating agents of cancer by inhibiting enzymes DNMT1 and HPV type 16 E6 sites, as well as for antimicrobial activities. None of the compounds exhibited acute toxicity in ADMET prediction analysis, indicating their potential as drug candidates. Further studies are required using the in silico approach to generate a potential drug through a structure-based drug-designing approach.

  • The authors confirm contribution to the paper as follows: all authors designed and comprehended the research work; plant materials collection, experiments performing, data evaluation and manuscript draft: Dejene M; research supervision and manuscript revision: Dekebo A, Jemal K; NMR results generation: Tufa LT; NMR data analysis: Dekebo A, Tegegn G; molecular docking analysis: Aliye M. All authors reviewed the results and approved the final version of the manuscript.

  • All data generated or analyzed during this study are included in this published article.

  • This work was partially supported by Adama Science and Technology University under Grant (ASTU/SP-R/171/2022). We are grateful for the fellowship support from Adama Science and Technology University (ASTU), the identification of plants by Mr. Melaku Wendafrash, and pathogenic strain support from the Ethiopian Biodiversity Institute (EBI). We also thank the technical assistants of the Applied Biology and Chemistry departments of Haramaya University (HU) for their help.

  • The authors declare that they have no conflict of interest.

  • Supplemental Fig. S1 Experimental design and wokeflow of the iTRAQ analysis on tea plant during different cold acclimation stages.
    Supplemental Fig. S2 Protein abundance distribution between the three different sample stages (CA vs NA, CA vs DA and DA vs NA).
    Supplemental Fig. S3 Venn charts for correlation between proteome and transcriptome database.
    Supplemental Fig. S4 Clustering analyses of expression patterns between identified proteins and its corresponding associated gene (A. CA vs NA; B. DA vs CA; C. DA vs NA).
    Supplemental Table S1 Primers used for quantitative RT-PCR.
    Supplemental Table S2 Raw determination data in proteome analysis (sheet "raw determination data"), raw data of proteomic accumalation analyses comparing with transcriptome data (sheet "expression data analysis"), and KEGG and GO term annotation for detected proteins (sheet "KEGG and GO term annotation").
    Supplemental Table S3 Total pathway analysis results of total and enriched protein species in the comparisons among different samples.
    Supplemental Table S4 Information of the total identified and differentially accumulated protein species mapped in KEGG pathway.
    Supplemental Table S5 Differentially accumulated protein species among the three comparisons (CA vs NA, DA vs NA and DA vs CA) (sheet "differentially accumulated proteins") and Gene Ontology (GO) enrichment analysis on the basis of clustering analysis (sheet "GO analyses of large clusters").
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  • Cite this article

    Ding C, Hao X, Wang L, Li N, Huang J, et al. 2023. iTRAQ-based quantitative proteomic analysis of tea plant (Camellia sinensis (L.) O. Kuntze) during cold acclimation and de-acclimation procedures. Beverage Plant Research 3:16 doi: 10.48130/BPR-2023-0016
    Ding C, Hao X, Wang L, Li N, Huang J, et al. 2023. iTRAQ-based quantitative proteomic analysis of tea plant (Camellia sinensis (L.) O. Kuntze) during cold acclimation and de-acclimation procedures. Beverage Plant Research 3:16 doi: 10.48130/BPR-2023-0016

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iTRAQ-based quantitative proteomic analysis of tea plant (Camellia sinensis (L.) O. Kuntze) during cold acclimation and de-acclimation procedures

Beverage Plant Research  3 Article number: 16  (2023)  |  Cite this article

Abstract: To gain a better understanding on the mechanism of cold acclimation in tea plant [Camellia sinensis (L.) O. Kuntze] at the proteome level, an iTRAQ based quantitative proteome analysis was carried out to identify differentially accumulated proteins in the mature leaves which were collected at non-acclimated (NA), fully acclimated (CA) and de-acclimated (DA) stages. 407 and 477 proteins identified from CA and DA showed significant abundance changes (at 95% confidence) compared with NA, respectively. Moreover, 251 protein species changed their abundance in DA compared with CA. Those differential abundance protein species were mainly involved in metabolism, cell wall, photosynthesis, energy, protein synthesis, antioxidation, carbohydrate metabolic process and binding, and mapped to the pathways of biosynthesis of secondary metabolites, microbial metabolism in diverse environment, ribosome, metabolic pathway, sugar metabolism, protein processing, photosynthesis, and plant-pathogen interaction pathway. However, no significant correlation was detected between the identified proteins and cognate gene transcript levels by correlation analysis and qRT-PCR analysis. This study presents a comprehensive proteome in mature leaves at different cold acclimation status and provides new insights into cold acclimation mechanisms in tea plants.

    • During the life cycles of plants, high salinity, desiccation and low temperatures are the major environmental stresses. These stresses impair the development and productivity of crops, horticultural plants and wild species, and even threaten their survival. For the perennial plants grown in frigid and temperate zones, low temperature in winter is a serious stress to overcome. Based on long term evolution, plants have developed complicated mechanisms to perceive and respond to low temperature stresses. In higher plants, cold acclimation (CA) is an important mechanism to defend against cold temperatures in winter[1]. Many physiological, biochemical, and structural changes happen during plant CA process[2, 3]. The increasing number of transcriptome analyses of gene expression at the RNA transcript level have helped us to better understand the molecular basis of CA[4, 5]. However, the transcript level of a given RNA does not always strictly correlate with the corresponding protein level in plant cells. And little has been done to elucidate the protein abundance changes during CA and de-acclimation (DA) processes[6, 7]. As a valuable approach to study the stress responses at the level of protein abundance, proteome analysis has been implemented in many studies. In Arabidopsis, putative plasma membrane proteins associated with CA were identified using a mass spectrometric approach[8]. Based on the two-dimensional difference gel electrophoresis (2-D DIGE) analysis, 26 differently expressed proteins were identified in rice, and the cellular phospholipase Dα1 protein was proven as a key candidate involved in the CA signaling pathway[9]. Balbuena et al.[10] found that the tolerant lines of sunflower showed a higher number of differentially expressed proteins in leaves, compared with freezing susceptible lines. As an important semi-permeable cellular membrane, plasma membrane plays vital roles in response to abiotic stress such as low temperature stress in plant. And plasma membrane proteins may change during CA[11]. Although many cold-stress-related proteins have been identified and analyzed, and much knowledge about CA has been added recently, the understanding of the CA mechanism is still limited, especially in woody and evergreen plants.

      Tea plant [Camellia sinensis (L.) O. Kuntze] is a woody, perennial, evergreen plant and is widely planted in developing counties of the tropics and sub-tropics as an important cash crop[12]. Low temperature in winter is a key environmental factor restricting the growth of tea plants, which could lead to damage to tea plantations, decline of production, and even plant death. Therefore, understanding the tea plant CA mechanism and functional genes, and application in tea plant breeding is a crucial way to improve tea plant cold tolerance. Similar to many other plants, huge changes happen at cellular, physiological and metabolic levels during the tea plant CA process, such as the relative electrical conductivity, concentration of malondialdehyde and relative water content decrease[13, 14]. Oppositely, the palisade tissue thickness increased and the plasma membrane stability enhanced through the increasing of total proteins and unsaturated fatty acids[15]. The content of soluble sugars also increased in winter[14]. Additionally, cold induced or related genes were identified and functions were validated by different technologies in tea plant[1620]. To further highlight the mechanisms of CA, we performed a transcriptome analysis based on RNA-seq. Many differentially expressed genes were identified and confirmed using quantitative RT-PCR analysis. These genes were grouped into signal transduction genes, cold-responsive transcription factor genes, plasma membrane stabilization related genes, osmosensing-responsive genes and detoxification genes, etc[4]. The transcriptome analysis provided a valuable chance to look into global gene expression changes at RNA level during the CA process in tea plant. Transcriptomic data are not often consistent with protein or metabolism data due to post-transcriptional modifications. So to provide new ideas towards the CA mechanism in tea plant, we examined the proteome during CA and DA processes in tea plant. Tea plant, being a broad leaved woody evergreen, may provide novel information on cold resistance mechanisms of other broad leaved evergreen plants in winter.

      In the present study, leaf samples at the non-acclimated (NA), fully acclimated (CA) and de-acclimated (DA) stages were collected according to our previous work[4] and analyzed using isobaric tags for relative and absolute quantitation (iTRAQ) quantitative proteomic approach following the workflow shown in Supplemental Fig. S1. Finally, over 1,300 differentially expressed and functioning in varied biological processes proteins were identified.

    • The tea plants of C. sinensis (L.) O. Kuntze 'Longjing 43' grown in the field of the Tea Research Institute, Chinese Academy of Agricultural Sciences (TRI, CAAS) (N 30°10', E 120°5'), were used in this study. Intact mature leaves at NA, CA and DA stages were collected for further study according to Wang et al.[4]. Each biological replicate contained ten intact leaves collected from ten individual plant and three biological replicates of each stage were collected. Those collected samples were frozen in liquid N2, and stored at −80 °C for protein extraction and iTRAQ assay and qRT-PCR analysis.

    • Protein extraction was performed according to the method of Yang et al.[21] with minor revision. Frozen leaves were ground to a fine power and weighted 1.0 g, and then 5 ml lysis buffer (7 M Urea, 2 M Thiourea, 4% CHAPS, 40 mM Tris-HCl, pH 8.5) was added. After that, 1 mM and 2 mM PMSF and EDTA were added respectively. Five minutes later, DTT was added with a final concentration of 10 mM. Then the above suspension was sonicated (200 Watts) for 15 min and centrifuged (30,000× g) at 4 °C for 15 min. Then those new supernatant was transferred into another tube and five-fold 10% chilled TCA acetone was added and incubated overnight at −20 °C. The precipitate was washed three times with chilled acetone for at least 30 min, and harvested by a centrifugation at 4 °C, 30,000× g for 15 min after each washing. The pellet was air dried before being dissolved in 500 μl 0.5 M TEAB, followed by sonication at 200 Watts for 15 min. Sonicated solution was centrifuged at 4 °C, 30,000× g for 15 min. Finally, supernatant was transferred to a new tube and quantified using BSA as standard protein with GE Healthcare's 2-D Quant Kit (Code No. 80-6483-56) following the instructions. After the quantification, SDS-PAGE and standard colloidal staining were performed to measure the quality of the protein sample.

    • One hundred micrograms of protein were digested using Trypsin Gold (Promega, Madison, WI, USA) with a ration of protein : trypsin = 30 : 1, and incubated at 37 °C for 16 h. Then, the digested proteins were dried using vacuum centrifugation and reconstituted in 0.5 M TEAB (Triethylammonium bicarbonate buffer). The reconstituted NA, CA and DA samples were labeled with different isobaric tags according to the munufacturer's protocol for 8-plex iTRAQ (Applied Biosystems).

      The labeled peptides were separated using Strong Cation Exchange Choematography (SCX), and analyzed using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)[10]. Shimadzu LC-20AB HPLC Pump system was used for SCX chromatography as follows: the digested peptides were dissolved with 4 mL buffer A (25 mM NaH2PO4 in 25% ACN, pH 2.7) and then were loaded onto a Ultremex SCX column (4.6 mm × 250 mm) containing 5 μm particles (Phenomenex). Parameters of peptide separating procedures were setted as follows: first buffer A for 10 min, then 5%−35% buffer B (25 mM NaH2PO4, 1 M KCl in 25% ACN, PH 2.7) for 40 min, and thirdly 35%−80% buffer B for 1 min, with a flow rate 1 mL/min. The absorbance at 214 nm was monitored and separated samples were collected every 1 min. Finally, each sample were separated into 20 fractions, then each fraction was desalted using Strata XC18 column (Phenomenex) and dried by vacuum.

      The desalted and dried fractions were re-suspended using buffer A (2% ACN, 0.1% FA) and centrifuged (20,000× g) for 10 min. Then, 8 μl of 0.5 μg·μl−1 supernatant was injected into a Shimadzu LC-20AD nano HPLC system with an analytical C18 column (inner diameter 75 μm). The flow rate was as follows: 8 μL·min−1 for 4 min, 300 μL·min−1 for 40 min with 2% to 35% B (95% ACN, 0.1% FA), followed by 5 min linear gradient to 80%, and mainted for 4 min with 80% B, finally returned to 5% within 1 min.

      After that, the samples were nanoelectrospray ionized (1.6 kV) and put into an Q EXACTIVE (Thermo Fisher Scientific, San Jose, CA, USA) coupled online with HPLC system. And the high-energy collision dissociation (HCD) operating mode with a normalized collision energy setting of 27.0 was applied for peptides selected for MS/MS. An Orbitrap with a resolution of 70,000 automatic gain control (AGC) was applied for detection and spectra optimization. For AGC target, the parameters of MS and MS 2 were setted as 3e6 and 1e5 respectively. While for MS and MS 2 scans, the m/z parameters were setted as 350 to 2,000 Da and 100−1,800 Da respectively. The 15 most abundant precursor ions which have a threshold ion count above 20,000 and 15 s dynamic exclusion duration time were identified based on a data-dependent procedure.

    • Proteome Discoverer 1.2 (PD 1.2, Thermo Fisher Scientific) was used for the conversion of raw data files acquired from the Orbitrap and the converted MGF file was used for protein identification using Mascot 2.3.02 (www.matrixscience.com) software (Matrix Science, London, UK). Tea plant unigene (from NCBI) translation database, theaceae_txid27065 database in NCBInr, and sequences from the tea plant transcriptome studies implemented by Wang et al.[4] were chosen as databases. The mass tolerance was set as '± 0.05 Da' and '± 0.1 Da' for intact peptide and fragmented ion identification respectively and one missed cleavage was allowed in trypsin digests. The charge states of peptides were set to +2 and +3. Deamidated (NQ), Gln->pyro-Glu (N-term Q) and Oxidation (M) were deemed as potential variable modifications. Moreover, Carbamidomethyl (C), iTRAQ8plex (K) and iTRAQ8plex (N-term) were defined as fixed modifications. A random sequence of database and the real database were used for raw spectra test in the decoy checkbox. An automatic decoy database search was conducted based on the decoy checkbox in Mascot. To improve the accuracy of peptide identification, only those peptides with significance scores (≥ 20) at the 95% confidence interval and detected as greater than 'identity' by Mascot probability analysis could be counted as identified. At least one unique peptide should be identified for each confident protein[22].

      Protein amount was estimated by spectral counts according to Balbuena et al.[10] A pairwise comparison was performed for NA, CA and DA and 1.5 fold cutoff wit p-value < 0.05 was defined as up-accumulated or down-accumulated proteins.

    • All identified proteins were classified using standard Gene Ontology (GO) online tool (www.geneontology.org) analysis. The enriched GO terms among the comparisons were identified using the statistical method described by Zheng & Wang[23]. The KEGG pathways analysis was carried out by sequence alignment against the Kyoto Encyclopedia of Genes and Genomes database[24] using BLASTP algorithm (E-value threshold 10−5). Differentially accumulated protein species among the different samples were grouped by Cluster 3.0 software and the output files were read by javaTreeview.

      To detect the correlation between protein level and corresponding transcript level at different CA stages, all the identified protein species were matched to the corresponding transcripts in the transcriptome database[4], and then correlation analyses were implemented according to Zheng et al.[25].

    • RNAprep pure Plant Kit (Tiangen, Beijing, China) was used for total RNA extraction from the samples that were used for protein extraction according to the manufacturer's instructions. RNA concentration and integrity estimation, cDNA synthesis and real-time PCR were performed according to previous descriptions[4], and the polypyrimidine tract-binding protein gene of the tea plant (CsPTB) was used as an internal reference[26]. The gene-specific primers for qRT-PCR were designed according to the corresponding coding sequences in the genome database[4], and all the primer sequences were provided in Supplemental Table S1.

    • During the tea plant CA procedure, its relative electrical conductivity following subsequent freezing decreased, then its cold tolerance could be improved. When tea plant was de-acclimated, its relative electrical conductivity increased and its cold tolerance became weaker[4]. We collected tea plant leaves from three different stages, NA, CA and DA for iTRAQ assaies.

      In total, 2,573 unique peptides out of 2,751 peptides were harvested. Based on Mascot searching in tea plant unigene translation database, NCBInr, theaceae_txid27065 database, and 51,940 sequences database 1,331 proteins were identified. Peptide length was generally 7 to 16 amino acids, and protein mass distribution was concentrated mainly at 10 to 40 kDa. Approximately 25% proteins had 5%−10% coverage by peptides, and 712 of 1,331 identified proteins were only represented by a single peptide (Supplemental Table S2). Above analyses suggested that high quality protein abundance libraries with low redundancy were constructed successfully.

      Protein quantification revealed 407 differential accumulated proteins between CA compared with NA, and among those proteins 202 were up-accumulated, while 205 were down-accumulated. Compared with CA, 115 up-accumulated and 136 down-accumulated proteins were detected in DA. In addition, compared with NA, 477 differentially accumulated proteins, including 253 up-accumulated proteins and 224 down-accumulated proteins, were identified in DA (Fig. 1). The distribution analysis of differential abundance proteins indicated that, when comparing CA with NA samples, generally down-accumulated proteins had greater abundance differences. When comparing DA with NA samples, both up-accumulated proteins and down-accumulated proteins had large abundance difference (Supplemental Fig. S2).

      Figure 1. 

      Number of differentially accumulated proteins among different samples.

    • Based on the GO enrichment analysis, those differently enriched GO terms (P-value < 0.05) among the comparisons between NA, CA and DA were listed in Table 1. Most of the significant differently enriched GO terms were grouped in biological process in the comparisons. Moreover, in cellular component category, more enriched GO terms were detected in DA vs CA and DA vs NA than in CA vs NA. Similar numbers of enriched GO term were grouped in molecular function category among the three different comparisons. Briefly, in the comparison of CA vs NA, extracellular region, plastid stroma, ammonia ligase activity, acid-ammonia (or amide) ligase activity, ribosome biogenesis and glutamine metabolic process were the most highly enriched; in DA vs CA, cell wall, plastid part, adenylyltransferase activity, racemase and epimerase activity, electron transport chain, and oxidation reduction were primarily enriched; in DA vs NA, extracellular region, cell wall, binding, protein kinase activity, electron transport chain and oxidation reduction were mainly enriched. Furthermore, comparing all the enriched GO terms in CA vs NA with DA vs CA, only one common enriched GO term, plastid stroma, was found. Six terms were found when comparing CA vs NA and DA vs NA, namely acid-ammonia (or amide) ligase activity, extracellular region, ammonia ligase activity, ribosome biogenesis, binding, glutamine metabolic process. Fourteen terms were found in the comparison of DA vs CA and DA vs NA, including cell wall, oxidation reduction, nucleoside phosphate metabolic process, electron transport chain, photosynthetic electron transport chain, nucleobase, nucleoside and nucleotide metabolic process, nucleotide metabolic process, photosynthesis, light reaction, purine nucleotide metabolic process, oxidoreduction coenzyme metabolic process, pyridine nucleotide metabolic process, nicotinamide nucleotide metabolic process, photosynthesis, and generation of precursor metabolites and energy.

      Table 1.  Gene Ontology (GO) enrichment analysis of differentially accumulated protein species among the comparisons between sample NA, CA and DA ( p -value < 0.05).

      GO termNA vs CACA vs DADA vs NA
      Cellular componentExtracellular regionCell wallExtracellular region
      Plastid stromaPlastid partCell wall
      Cytoplasmic vesiclePlastid stromaPlastid envelope
      VesicleThylakoid light-harvesting complexExternal encapsulating structure
      Chloroplast thylakoid membraneOrganelle envelope
      Light-harvesting complexEnvelope
      Plastid thylakoid membraneMicrobody
      Chromosome
      Membrane part
      Molecular functionAmmonia ligase activityAdenylyltransferase activityBinding
      Acid-ammonia (or amide) ligase activityRacemase and epimerase activityProtein kinase activity
      Oxidoreductase activity, acting on the
      CH-NH2 group of donors, disulfide as
      acceptor
      O-acyltransferase activityAmmonia ligase activity
      Ligase activity, forming carbon-nitrogen
      bonds
      Racemase and epimerase activity, acting
      on carbohydrates and derivatives
      Acid-ammonia (or amide) ligase activity
      Binding
      Biological processRibosome biogenesisElectron transport chainElectron transport chain
      Glutamine metabolic processOxidation reductionOxidation reduction
      Cellular carbohydrate metabolic processNucleoside phosphate metabolic processPhotosynthetic electron transport chain
      Reproductive developmental processWater-soluble vitamin metabolic processNucleotide metabolic process
      Reproductive processNucleobase, nucleoside and nucleotide metabolic processGeneration of precursor metabolites and energy
      Glycine metabolic processPhotosynthetic electron transport chainRibosome biogenesis
      Cellular amino acid metabolic processNucleotide metabolic processNucleoside phosphate metabolic process
      Cellular amine metabolic processSeed germinationGlutamine metabolic process
      Sulfur amino acid metabolic processMucilage metabolic processNucleobase, nucleoside and nucleotide metabolic process
      Reproductive structure developmentGlucan metabolic processNegative regulation of molecular function
      Carbohydrate metabolic processVitamin metabolic processProtein complex assembly
      ReproductionNADP metabolic processOxidoreduction coenzyme metabolic process
      Amine metabolic processNADPH regenerationTissue development
      Respiratory electron transport chainNicotinamide metabolic processPyridine nucleotide metabolic process
      Flower developmentAlkaloid metabolic processCellular macromolecular complex assembly
      Organic acid catabolic processNucleobase, nucleoside, nucleotide and nucleic acid metabolic processCellular protein complex assembly
      Carboxylic acid catabolic processCellular glucan metabolic processNicotinamide nucleotide metabolic process
      Response to chemical stimulusPhotosynthesis, light reactionProtein complex biogenesis
      Response to inorganic substancePurine nucleotide metabolic processCellular component biogenesis
      Energy derivation by oxidation of organic compoundsOxidoreduction coenzyme metabolic
      process
      Cellular component organization
      Organic acid metabolic processPyridine nucleotide metabolic processSulfur metabolic process
      Carboxylic acid metabolic processNicotinamide nucleotide metabolic processPrimary metabolic process
      Cellular ketone metabolic processCellular polysaccharide metabolic processPhotosynthesis
      Oxoacid metabolic processGlycogen metabolic processPurine nucleotide metabolic process
      Sulfur amino acid biosynthetic processEnergy reserve metabolic processFatty acid metabolic process
      Serine family amino acid metabolic processPhotosynthesisPhotosynthesis, light reaction
      Generation of precursor metabolites and energyPlastid membrane organization
      Stomatal movement
      Membrane organization
    • Pathway analysis is an important approach to expose the crucial biochemical metabolism and signal transduction pathways including given proteins[27]. The identified protein species in this study were annotated based on KEGG database. Generally, more differential abundance protein species were annotated and assigned to larger number of pathways in CA vs NA and DA vs CA, compared with DA vs CA. Moreover, the differential abundance protein species in three different comparisons were mainly mapped onto carbon fixation in photosynthetic organisms, metabolic pathway, ribosome, starch and sucrose metabolism, biosynthesis of secondary metabolites and microbial metabolism in diverse environments, protein processing in endoplasmic reticulum, photosynthesis, plant-pathogen interaction and oxidative phosphorylation. Interestingly, the pathways of glycolysis/gluconeogenesis, starch and sucrose metabolism and pyruvate metabolism related to glycometabolism were the largest proportion of differential abundance protein species in CA vs NA and DA vs NA. In addition, lysosome, glutathione metabolism, peroxisome and ascorbate and aldarate metabolism and phagosome pathways had dramatic difference in the number of differential abundance protein species in the three comparisons. As listed in Table 2 and Supplemental Tables S3 & S4, many pathways only had detectable changes in one or two specific samples, for example, proteasome, fatty acid metabolism, streptomycin biosynthesis, biosynthesis of unsaturated fatty acids and so on.

      Table 2.  Pathway analysis of total proteins and enriched proteins in the comparisons among different samples.

      NumberPathwayCountPathway ID
      Total
      (1,015)
      CA vs NA
      (319)
      CA vs DA
      (196)
      DA vs NA
      (362)
      1Metabolic pathways45814991168ko01100
      2Biosynthesis of secondary metabolites247753374ko01110
      3Microbial metabolism in diverse environments190703273ko01120
      4Ribosome83241727ko03010
      5Carbon fixation in photosynthetic organisms71331331ko00710
      6Glycolysis / Gluconeogenesis6522525ko00010
      7Methane metabolism5317821ko00680
      8Starch and sucrose metabolism52191024ko00500
      9Protein processing in endoplasmic reticulum49171115ko04141
      10Lysosome49111016ko04142
      11Pyruvate metabolism4818720ko00620
      12Amino sugar and nucleotide sugar metabolism4311814ko00520
      13Photosynthesis43131317ko00195
      14Glutathione metabolism367714ko00480
      15Phenylpropanoid biosynthesis3411713ko00940
      16Alanine, aspartate and glutamate metabolism3112711ko00250
      17Citrate cycle (TCA cycle)3110310ko00020
      18Plant-pathogen interaction3012715ko04626
      19Pentose phosphate pathway3013612ko00030
      20Glycine, serine and threonine metabolism2913613ko00260
      21Antigen processing and presentation291058ko04612
      22Oxidative phosphorylation2811812ko00190
      23Glyoxylate and dicarboxylate metabolism2612511ko00630
      24Purine metabolism25747ko00230
      25Huntington's disease25945ko05016
      26Phenylalanine metabolism2510810ko00360
      27Fructose and mannose metabolism25887ko00051
      28Peroxisome23749ko04146
      29Galactose metabolism22458ko00052
      30Arginine and proline metabolism22735ko00330
      31Phagosome213511ko04145
      32Nitrogen metabolism21131013ko00910
      33Cysteine and methionine metabolism19716ko00270
      34Spliceosome19636ko03040
      35Other glycan degradation19637ko00511
      36Alzheimer's disease18838ko05010
      37Valine, leucine and isoleucine degradation17417ko00280
      38Parkinson's disease16533ko05012
      39Butanoate metabolism16525ko00650
      40Ascorbate and aldarate metabolism16734ko00053
      41RNA degradation16526ko03018
      42Pentose and glucuronate interconversions15747ko00040
      43Glycerolipid metabolism15637ko00561
      44Endocytosis13543ko04144
      45Toxoplasmosis13533ko05145
      46Propanoate metabolism13523ko00640
      47Phenylalanine, tyrosine and tryptophan biosynthesis13332ko00400
      48Aminoacyl-tRNA biosynthesis13414ko00970
      49Cyanoamino acid metabolism13826ko00460
      50Renin-angiotensin system13356ko04614
      51Aminobenzoate degradation12446ko00627
      52Inositol phosphate metabolism12424ko00562
      53RNA transport12727ko03013
      54Valine, leucine and isoleucine biosynthesis12312ko00290
      55Selenoamino acid metabolism11334ko00450
      56Tyrosine metabolism11634ko00350
      57Tropane, piperidine and pyridine alkaloid biosynthesis11351ko00960
      58Proteasome1122ko03050
      59Reductive carboxylate cycle (CO2 fixation)1113ko00720
      60Tryptophan metabolism10315ko00380
      61Two-component system10658ko02020
      62MAPK signaling pathway10432ko04010
      63Fatty acid metabolism1046ko00071
      64Porphyrin and chlorophyll metabolism922ko00860
      65beta-Alanine metabolism9312ko00410
      66Glycosphingolipid biosynthesis - globo series9515ko00603
      67alpha-Linolenic acid metabolism9114ko00592
      68Terpenoid backbone biosynthesis9112ko00900
      69Prion diseases9723ko05020
      70Type I diabetes mellitus932ko04940
      71Pyrimidine metabolism911ko00240
      72One carbon pool by folate9534ko00670
      73Flavonoid biosynthesis8412ko00941
      74Chagas disease834ko05142
      75Lysine biosynthesis8112ko00300
      76Insulin signaling pathway8212ko04910
      77Chloroalkane and chloroalkene degradation8212ko00625
      78Carotenoid biosynthesis8213ko00906
      79Pathogenic Escherichia coli infection8146ko05130
      80Proximal tubule bicarbonate reclamation8122ko04964
      81Glycosphingolipid biosynthesis - ganglio series7223ko00604
      82PPAR signaling pathway723ko03320
      83Limonene and pinene degradation733ko00903
      84Glycosaminoglycan degradation7223ko00531
      85Metabolism of xenobiotics by cytochrome P4507343ko00980
      86Sphingolipid metabolism7334ko00600
      87Glycerophospholipid metabolism7313ko00564
      88Vibrio cholerae infection724ko05110
      89Drug metabolism - cytochrome P4507343ko00982
      90Lysine degradation733ko00310
      91Amyotrophic lateral sclerosis (ALS)7424ko05014
      92Calcium signaling pathway611ko04020
      93NOD-like receptor signaling pathway6323ko04621
      94Protein digestion and absorption642ko04974
      95Isoquinoline alkaloid biosynthesis6321ko00950
      96Pathways in cancer6323ko05200
      97Prostate cancer6323ko05215
      98Neurotrophin signaling pathway624ko04722
      99Folate biosynthesis6133ko00790
      100Ubiquinone and other terpenoid-quinone biosynthesis623ko00130
      101Protein export522ko03060
      102Photosynthesis - antenna proteins5222ko00196
      103Histidine metabolism522ko00340
      104Riboflavin metabolism5422ko00740
      105Sulfur metabolism5121ko00920
      106Streptomycin biosynthesis52ko00521
      107Benzoate degradation523ko00362
      108Bisphenol degradation5111ko00363
      109MAPK signaling pathway - yeast5122ko04011
      110Arachidonic acid metabolism5111ko00590
      111Progesterone-mediated oocyte maturation5312ko04914
      112Type II diabetes mellitus511ko04930
      113Biosynthesis of ansamycins5424ko01051
      114Chlorocyclohexane and chlorobenzene degradation522ko00361
      115Novobiocin biosynthesis5211ko00401
      116Fatty acid biosynthesis412ko00061
      117Polycyclic aromatic hydrocarbon degradation4311ko00624
      118Linoleic acid metabolism41ko00591
      119Systemic lupus erythematosus412ko05322
      120Bacterial invasion of epithelial cells421ko05100
      121Regulation of actin cytoskeleton4223ko04810
      122Biosynthesis of unsaturated fatty acids42ko01040
      123Geraniol degradation412ko00281
      124Focal adhesion4121ko04510
      125Collecting duct acid secretion43ko04966
      126Pantothenate and CoA biosynthesis41ko00770
      127Epithelial cell signaling in Helicobacter pylori infection43ko05120
      128Oocyte meiosis413ko04114
      129Cell cycle412ko04110
      130Fluorobenzoate degradation31ko00364
      131Gap junction323ko04540
      132Toll-like receptor signaling pathway311ko04620
      133N-Glycan biosynthesis31ko00510
      134Taurine and hypotaurine metabolism3111ko00430
      135Amoebiasis31ko05146
      136mRNA surveillance pathway322ko03015
      137Caprolactam degradation3111ko00930
      138Carbohydrate digestion and absorption322ko04973
      139Ether lipid metabolism3111ko00565
      140Toluene degradation31ko00623
      141Shigellosis3122ko05131
      142Tight junction321ko04530
      143Phototransduction - fly322ko04745
      144Vitamin B6 metabolism311ko00750
      145Drug metabolism - other enzymes2ko00983
      146Leukocyte transendothelial migration221ko04670
      147Leishmaniasis211ko05140
      148D-Glutamine and D-glutamate metabolism2ko00471
      149Phosphatidylinositol signaling system21ko04070
      150Cell cycle - Caulobacter2111ko04112
      151Naphthalene degradation211ko00626
      152Bacterial secretion system21ko03070
      153Ethylbenzene degradation21ko00642
      154C5-Branched dibasic acid metabolism2ko00660
      155Base excision repair211ko03410
      156Fc gamma R-mediated phagocytosis2212ko04666
      157Viral myocarditis221ko05416
      158GnRH signaling pathway2112ko04912
      159Flavone and flavonol biosynthesis222ko00944
      160Arrhythmogenic right ventricular cardiomyopathy (ARVC)221ko05412
      161Dilated cardiomyopathy221ko05414
      162Apoptosis211ko04210
      163ECM-receptor interaction21ko04512
      164Hypertrophic cardiomyopathy (HCM)221ko05410
      165DDT degradation221ko00351
      166Ubiquitin mediated proteolysis211ko04120
      167Adherens junction221ko04520
      168Melanogenesis11ko04916
      169Vascular smooth muscle contraction11ko04270
      170Basal transcription factors111ko03022
      171Lipopolysaccharide biosynthesis1ko00540
      172Nucleotide excision repair1ko03420
      173Stilbenoid, diarylheptanoid and gingerol biosynthesis111ko00945
      174Steroid biosynthesis1ko00100
      175RIG-I-like receptor signaling pathway1ko04622
      176Cardiac muscle contraction111ko04260
      177Phototransduction11ko04744
      178Sulfur relay system111ko04122
      179Gastric acid secretion11ko04971
      180Retinol metabolism111ko00830
      181Circadian rhythm - plant1ko04712
      182Mismatch repair1ko03430
      183Salivary secretion11ko04970
      184Benzoxazinoid biosynthesis1ko00402
      185Fatty acid elongation in mitochondria1ko00062
      186Notch signaling pathway1111ko04330
      187Thiamine metabolism111ko00730
      188DNA replication1ko03030
      189Indole alkaloid biosynthesis1ko00901
      190Synthesis and degradation of ketone bodies111ko00072
      191Long-term potentiation11ko04720
      192Diterpenoid biosynthesis1ko00904
      193SNARE interactions in vesicular transport1ko04130
      194Glioma11ko05214
      195Olfactory transduction11ko04740
    • The differential abundance protein species in the three different comparisons were sorted on the basis of the abundance patterns during different CA stages (Fig. 2). The patterns of differential abundance protein species varied widely and were clustered roughly into 11 groups. Among these clusters, cluster C only contained three protein species, namely pertin acetylesterase family protein, macrophage migration inhibitory factor family protein and chloroplast nucleoid DNA binding protein. These protein species were down-accumulated in three comparisons, especially in DA vs NA and DA vs CA. Furthermore, the cluster G and H were formed only by FERONIA receptor-like kinase and ribosomal protein respectively. They were dramatically down-accumulated and up-accumulated respectively in DA vs NA and CA vs NA. However, their abundance had very small change in DA vs CA. Except above minor clusters, the other eight clusters constituted at least six protein species. The biological functions of the protein species involved in these large clusters were classified based on GO enrichment analysis (Supplemental Table S5). Interestingly, most of differential abundance protein species functioned in or composed cell part, cell, organelle, catalytic activity, metabolic process, binding, cellular process, organelle part and response to stimulus. Cluster A and cluster D were formed by a small number of protein species. The protein species in cluster A were up-accumulated in DA vs NA and DA vs CA, were down-accumulated in CA vs NA, while the protein species in cluster D had an opposite profile. Similarly, cluster F and I also had an opposite abundance profile. The protein species abundances in cluster F were increased in DA vs CA and decreased in both CA vs NA and DA vs NA. Cluster B and E were respectively consisted of 36 and 49 protein species that showed reduced abundance in three comparisons, especially for the protein species in cluster E among the comparisons of CA vs NA and DA vs NA. Cluster J and neighboring cluster K had similar abundance profiles because the protein species involved in these clusters were increased at different levels among the three comparisons.

      Figure 2. 

      Hierachical clustering of proteins showing different abundance profiles across different samples. The data were transferred using log2.

      Among these differential abundance protein species, some stress-related or responding protein species were identified. These protein species included ribosomal proteins (RPs), photosynthesis related proteins, energy metabolism proteins, osmosensing-responsiveness proteins, antioxidation-related proteins, and some signal transduction, transporter and post-translationally modified proteins, etc. These protein species were grouped into different clusters. Most of these protein species were up-accumulated in CA and/or DA stage compared to NA, indicated that complex proteomics changes were happened during CA procedure in tea plant.

    • To illuminate the potential correlation between proteome and the corresponding transcriptome, all the identified proteins were correlated to corresponding transcriptome first, and thereafter association analyses were carried out between identified proteins and corresponding differentially expressed genes among the comparisons between different CA stages. Results showed that 1310 proteins out of total 1331 identified proteins were successfully associated with transcripts (Supplemental Fig. S3). Unexpectedly, the association analysis results showed that the identified proteins had low correlation coefficients (r) with the cognate genes level in the comparisons of CA vs NA, DA vs CA and DA vs NA in transcriptome analyses, with r values of 0.0023, 0.1519 and 0.1120 respectively (Fig. 3). Because the value of correlation coefficient was close to zero, the protein level was poorly correlated with transcript levels. To further show the details about the expression patterns of identified proteins and its corresponding associated gene, clustering analyses of expression patterns was implemented (Supplemental Fig. S4). The clustering results showed that the identified protein species and differentially expressed genes were mainly grouped into three kinds of cluster. First is positive correlation, such as the cluster F in CA vs NA, cluster C in CA vs DA and cluster D in DA vs NA. Second is confused correlation, which includes both week negative and positive correlation, such as cluster G in CA vs NA, cluster D in DA vs CA and cluster E in DA vs NA. These correlation relationships took a large share. Third is clear negative correlation, such as the cluster B, C, D, E and I in CA vs NA, cluster E in DA vs CA, cluster C, F, G in DA vs NA. Main proteins included in these groups have functions that include beta-primeverosidase, glycyl-tRNA synthetase, alpha-glucan water dikinase, AT-HF, phosphoenolpyruvate carboxylase, carbonic anhydrase, hydrolase family protein and plastocyanin-like domain-containing protein.

      Figure 3. 

      Correlation analysis of transcript (log2 FPKM value) and protein (log2 iTRAQ value) among different samples. (a) CA vs NA; (b) CA vs DA; (c) DA vs NA.

    • In order to validate the correlationship between proteome and the corresponding transcriptome, 20 protein species were chosen for qRT-PCR analysis, including 17 differential abundance protein species and three unchanged abundance protein species. The expression pattern analysis showed that only seven genes had similar patterns with iTRAQ results (Fig. 4). The results were consistent with the association analysis results between proteome and transcriptome. These results may be due to various post translational modifications and other complex regulatory networks in tea plant response to cold stress.

      Figure 4. 

      Analysis of transcript levels of the selected proteins among NA, CA and DA stages by qRT-PCR. Those genes which showed similar patterns are marked using black boxes. All data are the mean ± SD (n = 3).

    • As a perennial, evergreen and originating from tropical regions, low temperature is widely accepted as the most critical factor limiting tea plant growth and geographical distribution. An understanding of the adaptive mechanisms under low temperature stress of tea plant is necessary to enhance its cold tolerance. Although some studies have focused on the cellular, physiological, metabolic and transcrioptomics changes in tea plant during CA procedures[4, 13, 14, 16, 2830], but the molecular mechanism at the proteome level remains unclear. In the present study, we conducted comparative proteomic analysis and iTRAQ labeling method to examine the whole protein profile changes at the different CA states in tea plant leaves. A large number of differential abundance proteins, putatively related to cold stress, were identified. Comparisons of the differentially accumulated proteins revealed that more differentially accumulated proteins were detected among the comparisons of CA vs NA and DA vs NA. The result indicates that many changes which resulted from CA remained unchanged in DA. This may indicate that de-acclimated plants might have greater cold resistance than NA plants, and might be more amenable to re-acclimation than un-acclimated plants. A similar response was observed in Arabidopsis with regards to transcript changes of those genes involved in photosynthesis, calcium signaling and general stress responses maintaining acclimated expression patterns[31]. However, more work needed to be done to test this hypothesis. GO enrichment analysis is a commonly used tool to determine the potential function of differentially accumulated proteins in different data set comparisons[23]. In this study, the significant different GO terms among different comparisons between NA, CA and DA were obtained using GO enrichment analysis. As has been observed previously in other systems[21, 3235], our results showed that the differentially accumulated protein species during CA and DA mainly involve in cell wall, photosynthesis, energy, protein synthesis, metabolism, antioxidation, carbohydrate metabolic process, and binding. Recent studies showed that these biological processes or cellular components were common to CA. Degand et al. reported that the differentially accumulated proteins identified from chicory root after CA were mainly classified into the functional categories of protein synthesis, metabolism, energy or cell structure[36]. Kosmala et al. found that proteins related to photosynthetic machinery, cell energy and cell metabolic pathways played important roles in the CA procedure of Festuca pratensis[32]. The proteomic analysis of Thellungiella rosette leaves under cold stress revealed that most identified proteins were involved in photosynthesis, defense response, cell wall and cytoskeleton, RNA metabolism, energy pathway, protein metabolism and signal transduction pathways[37]. The proteomic studies of the CA mechanism in sunflower found that those cold-responsive proteins from three different cold tolerant lines were mostly involved in metabolism, protein synthesis, energy, and defense processes[10]. Moreover, the proteomic results in plantain also indicated that the majority of differentially accumulated proteins were involved in oxidation-reduction, photosynthesis, and several primary metabolic processes[21]. Our results also showed that 20 pathways were significantly enriched in tea plant during CA process at transcriptome level, and the metabolism was the largest category, which included 'carbohydrate metabolism pathway', 'energy metabolism pathway', 'xenobiotics biodegradation and metabolism' and 'lipid metabolism', etc[4]. Therefore, it can be assumed that the CA of tea plant is characteristic of many previously identified integral metabolic changes and suggest that many of the previous regulatory mechanisms controlling CA and DA can be used to direct future research into improving cold tolerance of tea plant.

      Interestingly, according to the significantly different GO terms listed in Table 1, cell wall is a significantly over-represented ontology that is specifically associated with DA. Cell wall is the first physical barrier and it plays a vital role in plant responses to abiotic stress[38]. Previous research suggested that many changes happen in the cell wall when plants were placed under cold stress, such as the increase in weight of cell walls, cell wall composition changes, and expression of cell wall-related gene changes and those changes showed close relationships with plant cold resistance[3941]. Our results were consistant with previous studies and provided some novel findings for tea plant CA mechanism research.

      Tea plant is an evergreen woody plant, and thus adjusting photosynthetic processes to deal with alterations in membrane fluidity and structure are important[37, 42]. We observed the terms of plastid part, thylakoid light-harvesting complex, chloroplast thylakoid membrane, light-harvesting complex, plastid thylakoid membrane, NADP metabolic process and DANPH regeneration being over-represented among differentially accumulating proteins in DA vs CA, plastid envelope in DA vs NA, plastid stroma in both CA vs NA and DA vs CA, and photosynthetic electron transport chain and photosynthesis in both DA vs CA and DA vs NA in concurrence with the hypothesis that modifications in photosynthesis are required for CA and DA in tea plant. These observations are consistent with the fact that photosynthetic processes can lead to greater production of damaging oxidative radicles when thylakoid membranes undergo changes of state during chilling[43]. Furthermore, reactive oxygen species scavenging is an important mechanism needed for coping with oxidative stress under cold stress in plants[44]. Consequently, over-representation of the terms of oxidation reduction and oxidoreduction coenzyme metabolic process suggest involvement of antioxidative mechanism. The terms of electron transport chain was significantly over-represented in both DA vs CA and DA vs NA, while in DA vs CA was energy reserve metabolic process. Moreover, the alterations in protein synthesis were indicated by over-representation of the terms of ammonia ligase activity, acid-ammonia (or amide) ligase activity and ribosome biogenesis in both DA vs CA and DA vs NA comparisons. Energy production related proteins were significantly up-regulated in rice leaf blades and were down-regulated in poplar leaves under a chilling environment[45, 46]. The terms of metabolic process and other terms involved in metabolism were over-represented among protein annotations in DA vs NA. The terms of carbohydrate metabolic process in CA vs NA and racemase and epimerase activity, acting on carbohydrates and derivatives in DA vs CA are involved in carbohydrate metabolism. The term of binding has significant difference in both CA vs NA and DA vs NA. Significant different terms of membrane part and fatty acid process in DA vs NA were also detected. Membrane modifications – particularly regarding alterations in fatty acid composition, have long been associated with CA processes[47]. Likewise, it is well known that carbohydrate accumulation can be protective against cold stress[48]. And these results were consistent with our previous study at the transcriptome level[4].

      CA is an important mechanism for perennial plants to obtain or enhance freezing tolerance[1]. Extended freezing temperatures in winter pose a great challenge for the survival of evergreen perennials such as tea plant, and, along with dormancy formation, such plants develop physiological and molecular changes to successfully archieve overwintering[49]. Because freezing of extra-cellular water pose significant challenges in regards to dehydration, cellular changes in water content, water status and osmotic potentials are essential events[50]. Consequently, cells decrease water content during fall or early winter and accumulate osmoprotectants such as specific storage proteins, sugars, starch and alter membrane chemistries to better deal with dehydration[51]. These adaptive mechanisms rely in part on gene induction and regulation, resulting in related protein enrichment in the fully acclimated stage. Therefore, the functional category analysis revealed that cell wall, photosynthesis, energy, protein synthesis, metabolism, antioxidation, carbohydrate metabolic process, and binding take crucial roles in tea plant CA.

      Pathway enrichment analysis was conducted to determine the main signal transduction and biochemical metabolic pathways involved by those differentially accumulated proteins in a previous study. The results showed that microbial metabolism in diverse environments, metabolic pathways and biosynthesis of secondary metabolites were the top three enriched pathways. In concordance with GO analyses above, carbon fixation in photosynthetic organisms, ribosome, carbon fixation in photosynthetic organisms, starch and sucrose metabolism, protein processing in endoplasmic reticulum, plant-pathogen interaction, oxidative phosphorylation and photosynthesis also take a large proportion of the differentially accumulated proteins. The changes of general metabolism and photosynthesis are the main responses of tea plant to low temperature. Accumulation of secondary metabolic products is an important character for tea plant. Many of the differentially accumulating proteins are involved in secondary metabolism related pathways. The role of these secondary metabolites in CA and DA are difficult to envision. For example, we observed increases in gallic acid and its derivatives which are known to have anti-oxidant properties, and thus might have protective roles in freezing stress[48]. However, increases in gallic acid production are not commonly observed during CA processes of other non-evergreen systems. It is well noted in the literature that protein content in plant cell can change within hours of low temperature exposure, and that protein metabolism play important roles in CA and freezing stress tolerance[52]. The enrichment of ribosome and protein processing in endoplasmic reticulum in this study also indicates the close correlation between protein synthesis and CA. Interestingly, a large number of proteins were involved in the pathways of microbial metabolism in diverse environments and plant-pathogen interaction. This was consistent with the results found in the plasma membrane of oat and rye after CA[53]. Recent studies report that some of the pathogenesis-related proteins are induced during winter months and have been shown to have antifreeze activity, cryoprotective activity, or antifungal activity[49].

      Furthermore, the signal transduction pathway plays a pivotal role in the response to the stress of low temperatures[54]. Plasma membrane play an important role in response to low temperature stress[47]. Plasma membrane can sense and transduce cold signals and then signal responses that alter its structure, chemical composition and function to improve cold resistance[11, 53, 55]. Li et al.[56] and Takahashi et al.[53] had identified hundreds of differentially abundant plasma membrane proteins in Arabidopsis, oat and rye using shotgun proteomic technology suggesting that these proteins may have a role in CA and freezing tolerance development. These proteins included signal transduction, disease/defense-related, energy-related, transporter and post-translationally modified proteins, etc. Phosphatidic acid is one of the major membranous second-messenger molecules and is produced by phospholipase D. During CA, plants generally increase their phospholipase D levels and phosphatidic acid content which is correlated with enhanced low temperature resistance[8,57]. Heat shock proteins are a kind of membrane protein that act as molecular chaperones[58], and usually play crucial roles in response to cold stress by re-establishing normal protein conformation and thus cellular homeostasis[53,59]. In tea plant, we also found phospholipase D (comp1149_c0_seq1) and heat shock proteins (comp671_c0_seq1, comp1412_c0_seq1 and comp16542_c0_seq2) were up-accumulated in CA and DA compared with NA. The plasma membrane contains many proteins, and some of those involved in cold-responsive process in tea plant were also reported in other plants[53,56]. These include, but are not limited to, proteins such as early response to dehydration proteins (ERDs), fasciclin-like arabinogalactan proteins (FLAs), aquaporins, ATPases, clathrins weren't differentially accumulated in our results (Supplemental Table S1). Studies using isolated plasma membrane should be conducted to elucidate the accurate changes of plasma membrane-specific proteins during CA in the future.

    • In the present study, 1,331 proteins were identified from NA, CA and DA tea leaves using iTRAQ analysis. 407 and 477 proteins were differently accumulated in comparison NA vs CA and DA Vs CA respectively. Function and KEGG pathway analysis revealed that those differently accumulated proteins were mainly mapped onto the metabolic, biosynthesis of secondary metabolites, microbial metabolism in diverse environment, ribosome, sugar metabolism, protein processing, photosynthesis and plant-pathogen interaction pathways. Further GO enrichment analysis indicated that those proteins were mainly involved in protein synthesis, photosynthesis, energy, sugar metabolism, antioxidation and stress defense. Correlation analysis showed that the proteome changes were not well-correlated with corresponding gene transcription changes. Overall, our study revealed general information about the proteome changes in tea plant leaf during NA, CA and DA procedures and provided some new insights on cold tolerance mechanism in tea plant.

      • This work was supported by the National Natural Science Foundation of China (U22A20499), the China Agriculture Research System of MOF and MARA (CARS-19), the Chinese Academy of Agricultural Sciences through an Innovation Project for Agricultural Sciences and Technology (CAAS-ASTIP-2021-TRICAAS) and the special project of Zhejiang province (2020R52036).

      • The authors declare that they have no conflict of interest. Xinchao Wang is the Editorial Board member of Beverage Plant Research who was blinded from reviewing or making decisions on the manuscript. The article was subject to the journal's standard procedures, with peer-review handled independently of this Editorial Board members and his research groups.

      • # These authors contributed equally: Changqing Ding, Xinyuan Hao

      • Supplemental Fig. S1 Experimental design and wokeflow of the iTRAQ analysis on tea plant during different cold acclimation stages.
      • Supplemental Fig. S2 Protein abundance distribution between the three different sample stages (CA vs NA, CA vs DA and DA vs NA).
      • Supplemental Fig. S3 Venn charts for correlation between proteome and transcriptome database.
      • Supplemental Fig. S4 Clustering analyses of expression patterns between identified proteins and its corresponding associated gene (A. CA vs NA; B. DA vs CA; C. DA vs NA).
      • Supplemental Table S1 Primers used for quantitative RT-PCR.
      • Supplemental Table S2 Raw determination data in proteome analysis (sheet "raw determination data"), raw data of proteomic accumalation analyses comparing with transcriptome data (sheet "expression data analysis"), and KEGG and GO term annotation for detected proteins (sheet "KEGG and GO term annotation").
      • Supplemental Table S3 Total pathway analysis results of total and enriched protein species in the comparisons among different samples.
      • Supplemental Table S4 Information of the total identified and differentially accumulated protein species mapped in KEGG pathway.
      • Supplemental Table S5 Differentially accumulated protein species among the three comparisons (CA vs NA, DA vs NA and DA vs CA) (sheet "differentially accumulated proteins") and Gene Ontology (GO) enrichment analysis on the basis of clustering analysis (sheet "GO analyses of large clusters").
      • Copyright: © 2023 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (4)  Table (2) References (59)
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    Ding C, Hao X, Wang L, Li N, Huang J, et al. 2023. iTRAQ-based quantitative proteomic analysis of tea plant (Camellia sinensis (L.) O. Kuntze) during cold acclimation and de-acclimation procedures. Beverage Plant Research 3:16 doi: 10.48130/BPR-2023-0016
    Ding C, Hao X, Wang L, Li N, Huang J, et al. 2023. iTRAQ-based quantitative proteomic analysis of tea plant (Camellia sinensis (L.) O. Kuntze) during cold acclimation and de-acclimation procedures. Beverage Plant Research 3:16 doi: 10.48130/BPR-2023-0016

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