Document Type : Research Paper

Authors

• Hossein Askari ¹
• Amineh Zeinali ²
• Mitra Parsa ³
• mona kashanchi ⁴
• Reza Azadi Gonbad ⁵
• Aghdas Banaei ⁶
• Sanam Safaei-Chaeikar ⁷
• Ehsan Kahneh ⁷
• Ali Seraji ⁸

¹ Associate Professor, Department of Cellular and Molecular Biology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran.

² Industrial and Environmental Biotechnology Department, Research Institute of Applied Science, ACECR, Shahid Beheshti University, Tehran, Iran,

³ Industrial and Environmental Biotechnology Department, Research Institute of Applied Science, ACECR, Shahid Beheshti University, Tehran, Iran, Po box: 198396411

⁴ 3. Industrial and Environmental Biotechnology Department, Research Institute of Applied Science, ACECR, Shahid Beheshti University, Tehran, Iran, Po box: 198396411

⁵ Assist. Professor, Tea Research Center, Horticultural Sciences Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Lahijan, Iran

⁶ Department of Nano and Biophysics, Research Institute of Applied Sciences , Academic Center of Education, Culture and Research (ACECR), Tehran, Iran

⁷ Tea Research Center, Horticultural Science Research Institute, AREEO, Lahijan, Iran.

⁸ Assistant Professor of Tea Research Center, Horticultural Science Research Institute, AREEO, Lahijan, Guilan, Iran.

Abstract

Background and objectives: Elicitors are used more frequently to promote plant growth and secondary metabolites. One of the main challenges for tea producers is the slow growth and poor quality of green tea leaves (Camellia sinensis) in tea-growing regions of Iran. In many plants, foliar application of chitosan or nano-chitosan (NC) enhances secondary metabolite production. It has a positive impact on plants' physiological and biochemical indicators. This study investigated the impact of nano-chitosan on some biochemical and physiological parameters with and without mineral nutrition (NPK), as well as assessing the quality of green leaves by comparing the relative expression levels of three enzymes involved in the flavonoids pathway in the Kashef cv. tea plant in northern Iran.
Methodology: For this, two scenarios were used: four different concentrations of nano chitosan solution (0, 25, 50, and 100 mgL^-1) were prepared and combined with NPK (1%-1%-0.5%), and without NPK were prepared. At the Lahijan Tea Research Center in Iran, two experiments were conducted as foliar treatments applied twice at two-week intervals, after the first season's harvest in 2021. Twenty days following the initial foliar treatment, plant sampling was conducted to examine physiological, biochemical, metabolic, and molecular characteristics. The first and second leaves, as well as the buds, were gathered for metabolic and molecular testing. The third and fourth leaves were collected for physiological and biochemical analyses. Chlorophyll content and relative water content were measured in physiological experiments. Protein content and the antioxidant enzymes CAT, SOD, and PPO were investigated biochemically. Additionally, metabolic properties were determined using the folin-sio-catheo method as well as HPLC to determine catechins, epigallocatechins, and gallocatechins. Molecular analysis was also performed by examining the relative expression of three critical enzymes in the flavonoid biosynthesis pathway, F3H, DFR, and LAR.
Results: The results showed that utilizing NC along with NPK significantly increased the content of total polyphenols in tea compared to the control (without NC and NPK). All treatments reduced catechin content 4- to 6-fold. With an increase in NC concentration, epigallocatechin content increased. Gallocatechin content also revealed a slight increase in 100 mg.L^-1 NC concentration. Chlorophyll content indicated a significant difference with a falling trend in treatments with low concentrations of NC; however, a significant difference with a growing trend was seen in treatments with 100 mgL^-1 of nano chitosan. In comparison to the control, various NC treatments had similar protein content. Except for the 50 mg.L^-1 NC+NPK treatment, there was an apparent significant difference in the SOD enzyme activity in each NC treatment, with a positive trend. With increasing NC concentrations, CAT enzyme activity also rose in various treatments. In treatments with insignificant NC concentrations, PPO enzyme activity significantly decreased. In different treatments, leaf water content rose. Only at a dose of 100 mg.L^-1 NC+NPK did the relative expression of the F3H enzyme rise nearly three times compared to the control; in contrast, other treatments had no meaningful effect on relative expression. Different NC+NPK treatments raised DFR relative expression, and 100 mg.L^-1 NC demonstrated the highest expression (4 times). Compared to the control, LAR relative expression increased at 0, 50, and 100 mg.L-1 NC and NPK. The treatment without NC and with NPK displayed the highest level of LAR expression, with an expression almost 2.5 times higher than the control.
Conclusion: In Kashef cultivar tea plants, chitosan nanoparticles in various concentrations coupled with NPK increased the production of catechin compounds. This effectively reduced oxidative stress and enhanced green tea leaf quality. In addition to addressing oxidative stress, NC may play a practical role in green tea quality. Due to its biodegradable properties, nano chitosan can be used instead of chemicals to improve tea plants' green leaves quality and lower environmental pollution.

Keywords

• Tea plant
• Camellia sinensis (L.) O. Kuntze
• NPK
• nano chitosan
• flavonoid
• Dihydro flavonol 4 reductase
• Flavonone 3 hyroxilasr
• Leuoco anthocyanin reductase

Main Subjects

• Biotechnology