Analysis Of Interaction Proteins And Expression Patterns Of Aluminum Toxicity Response Factor ALS3 And CAD In Citrus

Citrus is an evergreen fruit tree and one of the most important economic fruit trees in China.Citrus is cultivated in humid,semi-humid tropical,subtropical and temperate regions,where the soil is mostly acidic,and is susceptible to Al toxicity.ALS3 and CAD proteins are two key proteins in response to aluminum toxicity,but the mechanisms of ALS3 and CAD in Al tolerance of citrus is not yet understood.Based on our previous work,two citrus species Citrus sinensis(Al-tolerance)and C.grandis(Al-sensitive)were used as materials.The changes of biomass,photosynthetic rate and root lignin content of Citrus sinensis and C.grandis were investigated in this study.The interaction proteins of ALS3 and CAD were screened by yeast two-hybrid test,and the sub-cellular location and expression mode of candidate interaction proteins were further validated by bimolecular fluorescence complementarity(BiFC),western hybridization and RT-qPCR.The main results are listed as follows:(1)Aluminum stress affects the growth of C.sinensis and C.grandis by increasing the oxidative stress and lignification of citrus roots.The results showed that Al treatment decreased Fv/Fm,Fv’/Fm’and ETR values in both C.sinensis and C.grandis,while increased qNP and NPQ values,indicating that the utilization of light energy of C.sinensis and C.grandis under Al toxicity was low.In addition,Al treatment increased the content of hydrogen dioxide and lignin in C.sinensis and C.grandis roots,as compared with(2)control.Under Al treatment,the root biomass of C.grandis significantly decreased,the root system was sparse and accumulated high amount of Al.This may be the reason that Al toxicity increases the lignin content in the cell wall of C.sinensis and C.grandis roots.Increaseed accumulation of Al in roots restricts the elongation of root cells and thus inhibits plant growth.(3)The expression levels of ALS3 and CAD were analyzed by RT-qPCR using root cDNA as templates.The expression level and protein abundance of ALS3 in C.sinensis and C.grandi root under Al treatment was analyzed by Western Blotting.The results showed that Al treatment increased the expression level of ALS3 and CAD in C.sinensis and C.grandis.Furthermore,Al treatment increased the protein level of ALS3 in C.sinensis and C.grandis roots.(4)Fifteen interacting proteins of ALS3 and eleven interacting proteins of CAD were obtained by yeast two-hybrid test.BiFC test was used to exclude the false positive in yeast hybrids.Finally,six ALS3-interacting proteins and seven CAD-interacting proteins were screened and verified.The interacting proteins of ALS3 were glutathione-S-transferase(GST),V-ATPase,plasma membrane aquaporin PIPs(PIP2),serine/threonine protein kinase(UCT13),hypothetical dicyanine blue copper protein(DCBC),hypothetical protein OSJNB0071O2 1.22(UP2),and the interacting proteins of CAD were GST,V-ATPase,cytochrome P450 protein,ALS3,PIP2,hypothetical protein SB01G006870(UP1)and UP2.Furthermore,GST,V-ATPase,PIP2 and UP2 interacted with ALS3 and CAD simultaneously.RT-qPCR analysis showed that the gene expression of these interacting proteins was mainly up-regulated in the root of C.grandis under Al treatment,but not in C.sinensis.In conclusion,this study explored the molecular regulation mechanism of Al toxicity on lignin accumulation and Al ion redistribution in Citrus roots at physiological,biochemical and molecular levels,which provided scientific basis for high yield and quality in citrus cultivation.