Analysis of Interacting Proteins of Aluminum Toxicity Response Factor ALS3 and CAD in Citrus

Aluminum (Al) treatment significantly decreased the dry weight (DW) of stem, shoot and whole plant of both Citrus sinensis and C. grandis, but did not change that of root. Al significantly decreased leaf DW of C. grandis, increased the ratio of root to shoot and the lignin content in roots of both species. The higher content of Al in leaves and stems and lignin in roots of C. grandis than that of C. sinensis might be due to the over-expression of Al sensitive 3 (ALS3) and cinnamyl alcohol deaminase (CAD) in roots of C. grandis, respectively. By using yeast-two-hybridazation (Y2H) and bimolecular fluorescence complementation (BiFC) techniques, we obtained the results that glutathione S-transferase (GST), vacuolar-type proton ATPase (V-ATPase), aquaporin PIP2 (PIP2), ubiquitin carboxyl-terminal hydrolase 13 (UCT13), putative dicyanin blue copper protein (DCBC) and uncharacterized protein 2 (UP2) were interacted with ALS3 and GST, V-ATPase, Al sensitive 3 (ALS3), cytochrome P450 (CP450), PIP2, uncharacterized protein 1 (UP1) and UP2 were interacted with CAD. Annotation analysis revealed that these proteins were involved in detoxification, cellular transport, post-transcriptional modification and oxidation-reduction homeostasis or lignin biosynthesis in plants. Real-time quantitative PCR (RT-qPCR) analysis further revealed that the higher gene expression levels of most of these interacting proteins in C. grandis roots than that in C. sinensis ones were consistent with the higher contents of lignin in C. grandis roots and Al absorbed by C. grandis. In conclusion, our study identified some key interacting components of Al responsive proteins ALS3 and CAD, which could further help us to understand the molecular mechanism of Al tolerance in citrus plants and provide new information to the selection and breeding of tolerant cultivars, which are cultivated in acidic areas.