Functional Analysis Of The ABA Catabolism Gene NnCYP707A1 In Lotus Under Copper Stress

Heavy metals are widely present in the atmosphere,soil,and water,and heavy metal pollution is a significant challenge in the field of environmental restoration.Copper is a common heavy metal pollutant in wastewater,and the accumulation of excess copper in water not only inhibits the normal growth and development of plants,but also constrains the construction of the ecological environment.Plant restoration is an effective method for dealing with heavy metal pollution,and lotus is becoming a potential plant choice for copper pollution prevention and control due to its high ornamental and application value and strong ability to accumulate heavy metals.Based on the analysis of the transcriptional data of lotus under copper stress in the previous study of the research group,this study screened and cloned the key gene NnCYP707A1 under copper stress in lotus.By using methods such as transformation of brewing yeast,transient transformation of lotus,and heterologous transformation of Arabidopsis,the molecular mechanism of NnCYP707A1 gene involved in copper stress was analyzed,aiming to provide high-quality genetic resources for lotus to participate in plant restoration for copper pollution prevention and control.The results of the study are as follows:(1)The ABA 8’-hydroxylase gene NnCYP707A1 was cloned from the leaves of the lotus cultivar ’Yanyangtian’.The ORF c DNA of the gene is 1065 bp in length and encodes a protein of 354 amino acids.The protein contains the conserved structure domain GXGXHXCPG of the cytochrome P450 family.The NnCYP707A1 protein is a stable hydrophilic protein with a molecular weight of 40,592.17 and an isoelectric point of 8.68.The NnCYP707A1 protein belongs to the CYP707 A subfamily and is closely related to Pt CYP707A1 from Populus trichocarpa.Subcellular localization analysis indicates that the protein is located in both the cytoplasm and endoplasmic reticulum.(2)The overexpressing yeast strain p YES2-CYP707A1 was obtained by transforming the INVSC1 yeast with the NnCYP707A1 gene.Wild-type yeast strains grew faster and healthier than the overexpressing strain on SD-Ura plates containing 0 mmol,0.1 mmol,0.2 mmol,and 0.3 mmol Cu2+,indicating that overexpression of the NnCYP707A1 gene enhanced the sensitivity of the yeast to copper stress.(3)Transient transformation of lotus resulted in overexpression(CYP707A1)and silencing(cyp707a1)plants.Under 100 mg L-1 copper stress,the superoxide dismutase(SOD)activity in the leaves of the control lotus cultivar ’Yanyangtian’ increased,while the peroxidase(POD)activity decreased.The soluble protein and soluble sugar contents also decreased,while the chlorophyll a content increased and the chlorophyll b and total chlorophyll content decreased.The maximum photochemical efficiency of PSII(Fv/Fm)and photosystem activity(Fv/Fo)significantly decreased.The overexpressed lotus(CYP707A1)did not show any improvement in the above physiological parameters,while the silenced lotus(cyp707a1)showed a significant improvement in all physiological parameters.This indicates that silenced lotus plants can alleviate the inhibitory effects of copper stress,and the NnCYP707A1 gene negatively regulates lotus copper resistance.(4)T3 generation overexpressing Arabidopsis thaliana plants were obtained through heterologous transformation.Under normal growth conditions,overexpression of the NnCYP707A1 gene enhanced the seed germination rate in Arabidopsis.However,following copper stress treatment,both wild-type and overexpressing Arabidopsis lines exhibited a decrease in seed germination rate.Under 150 μmol Cu2+ treatment,the germination rate and root length of wild-type Arabidopsis thaliana were significantly inhibited,with accumulation of superoxide anions(O2-)in the leaves and a significant increase in abscisic acid(ABA)content.However,the germination rate and root length of the overexpressing Arabidopsis thaliana were more severely inhibited,with more accumulation of O2-and no significant change in ABA content.These results indicate that the overexpressing Arabidopsis thaliana is more sensitive to copper stress,and the NnCYP707A1 gene negatively regulates Arabidopsis thaliana’s copper resistance by affecting the decomposition and metabolism of ABA,reducing the accumulation of endogenous ABA in plants.