Identification and cloning of NRAMP genes from two citrus rootstocks and analysis of their expression patterns under nutrient stresses

Abstract: 【Objective】Citrus is one of the most important fruit crops in the world, and in some regions like some mountainous areas of China, citrus production is regarded as a mainstay industry.However the frequent occurrence of nutrient stresses such as K, Mg, B and Fe deficiency, as well as Mn, Cu and Cd toxicity have severely limited citrus production.The NRAMP (natural resistance-associated macrophage protein) family of genes plays important roles in mineral nutrient homeostasis;however, related information regarding their roles in citrus plants is limited.It is possible that NRAMP genes of citrus play roles in the response to these nutrient stresses as in model plants and may therefore have the potential to improve citrus tolerance to nutrient stresses.Therefore, the objective of this experiment is to study genome-wide identification and cloning of NRAMP family genes in citrus and to analyze their expression patterns under different metal nutrient stresses.【Methods】Two commonly used rootstocks in China (trifoliate orange[Poncirus trifoliata (L.) Raf.]and‘Ziyang Xiangcheng’[Citrus junos‘Ziyang Xiangcheng’]) , were selected as materials in this study.The seed coats of trifoliate orange and Ziyang Xiangcheng were removed and seeds germinated at a relative humidity of 70%and a temperature at28℃under darkness for 7 d.Subsequently, the germinated seedlings were transferred to a modified Hoagland solution composed of 2 mmol·L-1Ca (NO3) 2, 3 mmol·L-1KNO3, 0.5 mmol·L-1NH4H2PO4, 1mmol·L-1MgSO4, 20µmol·L-1H3BO3, 10µmol·L-1MnSO4, 5µmol·L-1ZnSO4, 1µmol·L-1CuSO4, 1µmol·L-1H2MoO4and 50µmol·L-1Fe-EDTA at 25℃under a 16-h photoperiod (50µmol·m-2·s-1) for30 d of normal growth and development.For nutrient excess treatments, the seedlings were transferred to new hydroponic solutions containing 0.5 mmol·L-1ZnSO4 (++Zn) , 1.0 mmol·L-1MnSO4 (++Mn) , 0.1mmol·L-1CuSO4 (++Cu) and 0.38 mmol·L-1CdSO4 (++Cd) .For nutrient deficiency treatments, the seedlings were transferred to new hydroponic solutions containing 10µmol·L-1MgSO4 (-Mg) , 0.2µmol·L-1H3BO3 (-B) , 0.5µmol·L-1Fe-EDTA (-Fe) and 30µmol·L-1KNO3 (-K) , while those grown in normal medium were used as control (CK) .After 2 d and 5 d of nutrient excess treatments or 4 d and 10 d of nutrient deficiency treatments, the roots and leaves were collected and immediately frozen in liquid nitrogen then stored at-80℃for analyzing the expression patterns of NRAMP genes.In addition, physiologically mature leaves were collected from the trifoliate orange and‘Ziyang Xiangcheng’seedlings, the total chlorophyll content and malondialdehyde amount were evaluated after 15 days of the nutrient stress treatments.Meanwhile, NRAMP genes were identified from sweet orange (Citrus sinensis) by performing BLASTP search with sweet orange genome database using the known Arabidopsis thaliana NRAMP proteins as queries.Subsequently, these genes were isolated from two commonly used rootstocks trifoliate orange and‘Ziyang Xiangcheng’, respectively, and were named and sequence analyzed according to the similarity to the AtNRAMPs.Moreover, their expression patterns were analyzed in roots and leaves of the two rootstocks under excess of Zn, Cu, Cd and Mn and deficiency of Mg, B, Fe and K treatments.【Results】After BLASTP search and removing redundant sequences and alternative splices of the same gene, four potential NRAMPs were identified from sweet orange.Subsequently, the NRAMP genes were PCR-amplified, cloned and sequenced from trifoliate orange and Ziyang Xiangcheng using sweet orange sequences as references.Following sequencing, the genes were denoted PtNRAMP2, PtNRAMP3, PtNRAMP6.1, PtNRAMP6.2, CjNRAMP2, CjNRAMP3, CjNRAMP6.1, and CjNRAMP6.2 based on their sequence similarity to A.thaliana NRAMPs.The PtNRAMPs/CjNRAMPs were predicted to encode proteins of 512 to 559 amino acids in length that included 10 to 12 putative transmembrane domains.Multiple sequence alignment revealed a high similarity among PtNRAMP CjNRAMP and AtNRAMP proteins.PtNRAMP2/CjNRAMP2 and PtNRAMP3/CjNRAMP3 showed the highest similarly with AtNRAMP2 (78.9/78.7%) and AtNRAMP3 (79.3/79.5%) , respectively.Both PtNRAMP6.1/CjNRAMP6.1 and PtNRAMP6.2/CjNRAMP6.2 were most similar to AtNRAMP6When compared between PtNRAMPs and CjNRAMPs, PtNRAMP2 and CjNRAMP2 were different in seven amino acids, PtNRAMP3 and CjNRAMP3 different in two amino acids, PtNRAMP6.2 and CjN-RAMP6.2 different in six amino acids, while there were no difference between PtNRAMP6.1 and CjN-RAMP6.1.Phylogenetic analysis revealed that the PtNRAMPs and CjNRAMPs were divided into two groups, similar to the AtNRAMPs.The NRAMP proteins in the same cluster often shared a similar gene structure and conserved motifs.The phenotypic and physiological determination of chlorophyll and malondialdehyde suggested that excess Zn, Mn, Cu and Cd, and deficiency of Mg, B, Fe and K treatments significantly limited the growth of the two rootstocks relative to CK.Expression analysis of the PtNRAMP and CjNRAMP genes showed that NRAMP2 and NRAMP3 were up-regulated in response to most nutrient excesses, while NRAMP6.2 was down-regulated in the two rootstocks.Moreover NRAMP3 and NRAMP6.2 were primarily up-regulated under Fe and K deficiency.【Conclusion】Four PtNRAMP and four CjNRAMP genes were isolated from trifoliate orange and‘Ziyang Xiangcheng’, respectively.These genes were highly similar to Arabidopsis thaliana orthologs at the protein sequence level and in terms of putative functions.Based on a comprehensive comparison of gene expression patterns and the findings of previous reports, we speculated that the PtNRAMPs and CjNRAMPs played potential roles in different nutrient stresses.The present study provides essential information regarding citrus NRAMP genes, but more in-depth investigations on their exact functions are warranted.