The Role Of ELONGATED HYPOCOTYL5 In The Response Of Plants To Abiotic Stress

Plants are often exposed to abiotic stresses because they are sessile,and have evolved multiple response pathways to adapt to abiotic stresses.The adaptation depends largely on the coupling of light signals and stress response pathways.As a central transcription factor of light signal network,ELONGATED HYPOCOTYL5（HY5）,plays an important role in the integration of light signals in plants to regulate the responses of plants to abiotic stress.To determine HY5 can act as a signal molecule for plants to respond to abiotic stresses,this study was conducted to investigate the role of HY5 in soybean and rice in response to abiotic stresses,e.g.high temperature（37℃）,low temperature（4℃）,salt（100 m M NaCl）and UV-B（10μw/cm²）.Next,an indirect competitive enzyme immunoassay（ic-ELISA）for the detection of HY5 was developed,the parameter optimization,sensitivity,specificity,accuracy and feasibility evaluation of ic-ELISA were completed.Based on the established ic-ELISA method,the role of HY5 in emerging contaminants was investigated.The main results are as follows:（1）HY5 was found to act as a signal molecule for plants in response to abiotic stresses.The visual analysis of existing literature on keyword co-occurrence network,clustering and timeline map showed that HY5 can participate in plant response to stress as a transcription factor,and HY5 can influence plant response to stress through its own accumulation level,which theoretically proves that HY5 can be used as a signal molecule for plant response to abiotic stresses.The relative levels of HY5 changed rapidly and significantly in soybean and rice treated with high temperature,low temperature,salt,UV-B for 1 h,3 h,12 h and 24 h,compared with the traditional signal molecules,such as chlorophyll content,reactive oxygen species content and proline content.In addition,the changes in the relative levels of HY5 showed various regularities for different stresses and similar regularity in the response of different plants to stresses.From the experimental point of view,we demonstrated that HY5 can function as a signal molecule for plants in response to abiotic stresses by changing its own content.（2）An ic-ELISA method was developed for the detection of HY5 content.HY5protein was isolated and purified by immune-precipitation method,and HY5 standard at a concentration of 67 ng·mL^-1 was obtained.In established ic-ELISA method,a coating concentration of 0.333μg·mL^-1 and a HY5 primary antibody concentration of 0.111μg·mL^-1were selected as the working points.The optimal standard dilution p H,methanol and NaCl contents,incubation time of HY5 primary antibody,reaction time of secondary antibody and color development time were 7.5,0.0%methanol and 0.8%NaCl,30 min,30 min and 15 min,respectively.Under the optimal conditions,the half-inhibitory concentration value was 1.598ng·mL^-1,and the minimum detection limit was 0.272 ng·mL^-1.The linear range of detection ranged from 0.523 ng·mL^-1 to 4.878 ng·mL^-1.The results of ic-EILSA showed that the HY5content in overexpressed-HY5 plant（higher mRNA transcript level and protein expression level of HY5）was higher than that in wild-type Arabidopsis（Col-0）,and the HY5 content in hy5 mutant（the loss of HY5 function）was below the detection limit,indicating that ic-ELISA method has good specificity.The results obtained by the newly developed ic-EILSA method and the conventional Western Blot assay for HY5 content were consistent,demonstrating the good accuracy of the ic-ELISA method.The spiked recoveries in the actual samples were in the range of 90%～110%,and the coefficient of variation was less than 10%,indicating that the method is feasible for the actual sample detection.（3）Based on the established ic-ELISA method,the role of HY5 in plant response to the stresses of emerging contaminants was determined.Based on the ic-ELISA method to detect HY5 content,the role of HY5 in plants facing lanthanum[La（Ⅲ）],bisphenol A and polyethylene microplastic stresses was determined using genetics（hy5 mutants）and correlation analysis methods.Specifically,a decrease in HY5 content under low concentrations（30 and 60μM）of La（Ⅲ）treatment led to an increase in hypocotyl length,anthocyanin content,net photosynthetic rate（P_n）and intercellular CO₂ concentration（C_i）;an increase in plant HY5 content under high concentrations（120 and 240μM）of La（Ⅲ）treatment led to a decrease in hypocotyl length,anthocyanin content,P_n and C_i decreased.When plants facing 3,6,12 and 24 mg·L^-1 BPA and 10,20,40 and 80 mg·L^-1 polyethylene microplastics,HY5 content increased,leading to an increase in anthocyanin content and a decrease in hypocotyl length,P_n and C_i.In conclusion,HY5 can be used as a signal molecule for plants in response to abiotic stresses,and the established ic-ELISA method has good sensitivity,specificity,accuracy and feasibility for the functional study of HY5 in emerging contaminants affecting plant growth and development.This study identifies a new signal molecule that can be used to characterize the stress status of plants and provides a new means of detection,offering ideas for rapid detection of plant proteins.