The Response To Water And Cadmium Stress In Arabidopsis Plants Overexpressing Populus Euphratica PeAnn1

Annexins are a large class of annexin families commonly found in prokaryotes and eukaryotes,and can participate in many stress response processes such as oxidative stress,heat,drought and salt stress.At present,the function of annexins in Populus euphratica in response to abiotic stress is still poorly understood.The objective of this study is to investigate the role of Populus euphratica Anneixn1 in plant response to water and cadmium stress.In this study,Pe Ann1-overexpressed Arabidopsis（Pe Ann1-OE1 and Pe Ann1-OE2）,Annexin1 mutant（Atann1）,and wild-type Arabidopsis（WT）,were used as experimental materials.These genotypes were respectively subjected to osmotic stress,soil drought and rewatering,or heavy metal（cadmium）treatment.Then physiological,biochemical and molecular biological measurements were carried out to compare the various responses of these Arabidopsis genotypes under water and heavy metal stress.The main results are obtained in the following:The overexpression of Pe Ann1 gene increased the sensitivity of Arabidopsis plants to water stress.The phenotype results showed that under mannitol stress,the seed germination rate and root length of all tested genotypes were inhibited,and the inhibition increased with the concentration of mannitol（0,150,200,250,300 m M）.However,the seed germination rate and root growth of Pe Ann1-overexpressed Arabidopsis were significantly lower than those of WT and Atann1 mutant.Similar findings were observed when these Arabidopsis genotypes were exposed to drought treatment.After 8 days of soil drought,the chlorophyll content,maximum photochemical efficiency of PSⅡ（Fv/Fm）,actual photosynthetic quantum yield（YⅡ）and relative electron transfer rate（ETR）of Pe Ann1-transgenic Arabidopsis were lower than those of WT and Atann1 mutant（P<0.05）.After rehydration,the recovery of photosynthetic parameters of Pe Ann1-transgenic plants was significantly lower than that of WT and Atann1 mutant.The increased sensitivity to water stress in transgenic plants is related to the decreased capacity in reactive oxygen species（ROS）scavenging,water uptake,and Ca²⁺signal transduction under water deficits.（1）Under 250 m M mannitol treatment,the relative electrolyte leakage and H₂O₂ levels in Pe Ann1-overexpressed plants were significantly higher than those of WT and Atann1 mutant,whereas the activity of antioxidant enzymes,such as superoxide dismutase（SOD）,peroxidase（POD）,catalase（CAT）,and the expression level of their encoding genes were significantly lower than that of WT and Atann1 mutant.Therefore,the antioxidant enzyme system in transgenic plants was unable to scavenge ROS under osmotic stress,leading to an increased permeability of cell membrane.As a result,the sensitivity to water stress increased in Pe Ann1-transgenic Arabidopsis plants.（2）RT-q PCR data showed that the expression of water channel genes,At PIP1;2,At PIP2;1,was significantly upregulated in WT plants under mannitol treatment（250 m M）.While At PIP1;2,At PIP2;1 expression was lower in Pe Ann1-transgenic plants than in WT and Atann1 mutant.Moreover,At PIP2;1 transcript was declined in Pe Ann1-OE1 under osmotic stress.This would affect the water uptake and plant water status under conditions of water deficit.（3）The Ca²⁺levels in root cells of WT and Atann1 mutant was elevated under osmotic stress,whereas there was no corresponding changes in Pe Ann1-transgenic lines.This indicates that transgenic plants was unable to sense and transduce stress signals after the onset of stress,thus limiting the plant ability adapting to water stress environments.The overexpression of Pe Ann1 gene increased the sensitivity of Arabidopsis plants to heavy metal stress,cadmium.The phenotype tests showed that seed germination and root growth of all tested lines were lowered by cadmium treatment,and the inhibition increased with the increasing of Cd Cl₂concentration（0,50,75,100μM）.The cadmium inhibition of growth was more pronounced in Pe Ann1-transgenic lines than in WT and Atann1 mutant.The increased sensitivity to heavy metal in transgenic plants is due to the decreased capacity in antioxidant defense,water uptake,and Ca²⁺signal transduction under heavy metal stress.（1）Under 100μM Cd Cl₂ treatment,the antioxidant enzymes,such as SOD,POD,CAT,and gene expression of these antioxidant enzymes were significantly lower in transgenic plant than in WT and Atann1 mutant.Therefore,the antioxidant defense system in transgenic plant was unable to scavenge ROS under heavy metal stress,leading to an increased H₂O₂ level and electrolyte leakage in root cells.（2）The expression of water channel genes,At PIP1;2,At PIP2;1 was significantly lower in Pe Ann1-transgenic plants than in WT and Atann1 mutant.Moreover,a marked decline of At PIP2;1 transcript was observed in transgenic plants under cadmium stress.This would reduce the water uptake and plant water status under heavy metal stress.（3）The Cd Cl₂ treatment（100μM）caused an elevation of Ca²⁺levels in root cells,and the effect was more pronounced in of WT and Atann1 mutant,as compared to Pe Ann1-transgenic lines.This is mainly due to competition effect between Ca²⁺and Cd²⁺in entering the cells,i.e.,high concentrations of Cd²⁺（100μM）reduced the entry of Ca²⁺through calcium channels in the plasma membrane.As a result,the Ca²⁺signal transduction was restricted in transgenic plants under cadmium stress,thus limiting the plant ability adapting to heavy metal stress environments.In conclusion,the overexpression of Pe Ann1 gene increased the sensitivity of Arabidopsis plants to water and heavy metal stress.This is resulted from the inhibited light reaction center,decreased capacity in antioxidant defense,water uptake,and Ca²⁺signal transduction under stress conditions.Therefore,P.euphratica Pe Ann1 gene serves as a negative regulator of drought and cadmium resistance.The Pe Ann1-mediated Ca²⁺signaling,water channel gene expression and antioxidant defense need further investigations.