Effects Of Copper And Cadmium Stress On The AsA-GSH Cycle In Tea Plants

As an important cash crop in hilly and mountainous areas,tea plants are often planted in areas with acid soil.Under acid soil conditions,heavy metal stress often occurs.Copper（Cu）and cadmium（Cd）stress are two types of heavy metal stress that tea plants often face.Cu and Cd stress affect the growth and content of tea plants,resulting in a decline in tea yield and quality.Therefore,it is very important to explore the pathways and genes related to heavy metal resistance.The rise of active oxygen under Cu and Cd stress has a great impact on the physiological and biochemical characteristics of tea plants.The synthesis and regeneration of ascorbic acid（AsA）and glutathione（GSH）are important ways for plants to remove reactive oxygen species and regulate oxidative steady-state imbalances caused by heavy metal stress.In this experiment,the synthesis and regeneration of AsA and GSH and the expression of related genes under the stress of Cu and Cd in Baiye 1 tea plants were studied.Our study laid the corresponding theoretical foundation for the tea plant to alleviate the response mechanism of Cu and Cd stress.In this paper,a CsGGP3 sequence was obtained by using the "Baiye 1" cDNA as a template,and the sequence characteristics and amino acid characteristics of the CsGGP3 gene were analyzed.The length of CsGGP3 is 1353bp,a total of 450 amino acids and the similarity to the CsGGP1 gene sequence（KC505203.1）and Shuchazao CsGGP2 gene sequence（XP₀28110023.1）registered in Genbank are 93.58%and 98.22%,respectively.The CsGGP gene is highly conserved among different tea varieties,and the CsGGP3 gene and CsGGP2 gene differ only by 7 amino acid positions.This may be the result of natural selection of different tea plants varieties during long-term evolution.Using ’Baiye 1’ as a hydroponic material,the expression level of CsGGP3 during 30 mM Cu and Cd stress was found to be consistent,and the expression of CsGGP3 under Cu and Cd stress showed consistency,which was significantly suppressed the expression level of CsGGP3 in ’Baiye 1’ tea plants.By spraying 30 mM Cu and Cd,the experiment explored the phenotypic changes of tea plants as well as the levels of active oxygen,AsA and GSH.Under the stress of Cu and Cd,the phenotypic state was affected in a short period of time.After 7 days of treatments,the leaves of the tea plants were easily shed,withered,and necrotic.The stress of Cd had an earlier effect on the phenotypic changes of tea plants.Spots and leaf color chlorosis appeared on the third day of treatment,while they appeared on the fifth day under Cu treatment.At the same time,Cu and Cd stress greatly changed the redox homeostasis in the tea plantation,and the active oxygen content increased sharply,resulting in an increase in cell membrane lipid peroxidation levels and changes in antioxidant substances.Reducing ascorbic acid increased significantly on the first day but its content decreased rapidly with the extension of stress time.The change of glutathione content was the opposite,and it increased significantly over the next day.In addition,the experiments examined the expression levels and enzyme activities of genes related to the synthesis and regeneration of ASA and GSH.Under Cu stress,the expression levels of the genes GMP,GME and GaILDH in the Vc synthesis pathway of tea plants showed a trend of increasing first and then decreasing.Both GMP and GaLDH genes showed an upward trend under Cd stress.Under Cu and Cd stress.GGP and GPP genes showed a continuous downward trend.The expression levels of Csy-ECS and CsGSHS in the glutathione synthesis pathway under Cu and Cd stress were significantly higher than those in the control group.The APX gene and MDHAR gene in the AsA-GSH circulation pathway of tea plant were induced to express under Cu stress,which were increased by 1.9 times and 2.2 times respectively.Under Cd stress,the APX gene reached its peak on the third day,the peak value was 1.9 times that of the control group;while the MDAHR gene significantly increased,the highest peak was on the seventh day,and the peak value was 4 times the control group.In the AsA-GSH cycle,the DHAR and GR genes were significantly suppressed under Cu and Cd stress.After the Cu and Cd stress of tea plants,the antioxidant enzyme-related enzyme activities were measured.After 1 day of Cu stress,the CAT and POD enzyme activities in tea trees increased by 39.3%and 10.8%,respectively.Cd stress increased POD by 6.3%on the first day.In the AsA-GSH cycle,APX and MDHAR enzymes play an important role in the stress relief of tea plants in the early stage of copper and cadmium stress.APX activities were increased by 81.2%and 110.2%on the first day.The activities of MDHAR were increased by 79%and 29%on the first day.With the prolonged stress,DHAR and GR enzymes related to GSH regeneration were significantly inhibited in the AsA-GSH cycle,resulting in the destruction of GSH regeneration pathway.