Functional analysis and characterization of the sMdCAX11 gene in apple

Abstract: 【Objective】Ca2+ /H+ antiporter (CAX) is a type of low-affinity and high-capacity transporter that mainly relies on the transmembrane proton gradient to complete the transport of Ca2+ . This protein may be related to the occurrence of plant calcium deficiency. It is known that Ca2+ /H+ reverse transporter proteins (CAXs) in model plants like Arabidopsis thaliana and tomato play important roles in regulating intracellular Ca2+ distribution and allocation, and maintaining intracellular calcium homeostasis, and overexpression of the CAXs genes in different plants could cause calcium deficiency symptoms in the plants. In the preliminary stage of this study, Honeycrisp apple bitter pit disorder fruits with different degrees of incidence were used as test materials, and the differences in mineral element contents and expression patterns of calcium transport-related genes in disordered fruits were analyzed. The key regulatory genes MdCAX5 and sMdCAX11 (MdCAX11 with the N-terminal autoinhibitory region removed) involved in the development of bitter pit disorder were then identified. However, the function of MdCAX11 protein in apple was still unclear. Meanwhile, it was still unclear whether Ca2+ /H+ reverse transporter proteins were involved in the development of bitter pit disorder in fruit.【Methods】The gene functions of sMdCAX11 were analyzed using experimental methods like genetic transformation. In this study, we first utilized the transient transformation of Honeycrisp apple fruits to verify the calcium transport function of the sMdCAX11 protein. Fruits transiently transformed with the sMdCAX11 gene were sectioned to observe the changes in the flesh tissue near the injection hole, and the mineral element content of the flesh tissue was also determined. Next, we stably transformed sMdCAX11 into the Arabidopsis Col-0 and successfully obtained positive T4 generation transgenic plants. The PCR tests at DNA lev-el and RNA level verified that all obtained were positive plants, and the leaves were analyzed for mineral element detection. In this study, various types of elements contained in the 1500 bp promoter region upstream of the start codon of the apple MdCAX11 gene were also predicted and analyzed. Meanwhile, in order to investigate the effect of the ProCAX11 promoter on the response to Ca2 + , tobacco leaves sprayed with different concentrations (0, 10, 20 and 40 mmol · L^-1 ) of CaCl2 were infiltrated by using Agrobacterium proCAX11::GUS. The effect of calcium ion on the transcriptional activity of MdCAX11 gene promoter was verified by GUS staining and GUS protein activity analysis.【Results】The total calcium content in apple flesh tissues overexpressing the sMdCAX11 gene significantly decreased and continued to decrease with the extension of storage time. By analyzing the total Mg and K contents in the flesh tissues, these two elements showed a rapid increase after a transient decrease at the 3rd day after infestation, reaching the highest value at the 5th day. Further analysis of the elemental ratios of the flesh tissues revealed that the total mineral elements (K+Mg)/Ca significantly increased in the flesh of transiently transformed sMdCAX11 and continued to rise with the extension of storage time. At the 5th day of infestation, (K+Mg)/Ca ratio of water-soluble mineral elements was significantly higher in the flesh tissues of transiently transformed sMdCAX11 genes, while there was no significant change in the elemental ratios of the control. By analyzing the leaf mineral element contents of the four sMdCAX11 transgenic Arabidopsis lines, consistent with the apple flesh material transiently transformed with sMdCAX11, the (K+Mg)/Ca ratios of the total elements, and the ratios of the water- soluble elements appeared to be marked increase and significantly different in the positive plants. A large number of cisacting elements responsive to external environmental conditions were present in the promoter region of the MdCAX11, such as ARE, an element involved in anaerobic induction, as well as the light-responsive elements ATCT-motif, Box 4, G-box, GT1-motif, TCCC-motif and chs-CMA2a. In addition, the MdCAX11 promoter region was characterized by the presence of several transcription factor binding sites, such as the WRKY transcription factor binding sites WBOXNTERF3, WBOXATNPR1 and WRKY710S, as well as the binding sites of transcription factors like MYB. By analyzing the GUS protein activity of tobacco leaves, it was found that ProCAX11 initiation significantly increased in a high calcium environment. The GUS protein activity significantly increased when they were sprayed with different concentrations of CaCl2, and the difference was significant compared with the control. Simultaniously, the GUS protein activity reached the highest value when they were sprayed with 20 mmol·L^-1CaCl2. The transcriptional activity of MdCAX11 promoter was significantly affected by Ca2+ .【Conclusion】The calcium transport capacity of the MdCAX11 gene was influenced by the N-terminal autoinhibitory region, and the sMdCAX11 gene was equipped to transport calcium ions. Overexpression of the sMdCAX11 gene significantly reduced the calcium content in plants and disrupted the balance of mineral element ratios. sMdCAX11 transgenic Arabidopsis thaliana leaves contained significantly lower total Ca content as well as water-soluble Ca content compared with the wild type, and the (K+Mg)/Ca ratio of the total and water-soluble mineral elements was significantly higher than that of the control. In conclusion, these findings provided further evidence that overexpression of the sMdCAX11 gene can cause uneven distribution of elements in plant tissues and imbalance in element proportions.