Identification of MAPK family genes and analysis of their expression patterns in response to cold stress in pomegranate

Abstract: 【Objective】Cold stress is one of the most important factors limiting the progress of pomegranate production. The objectives of this study were to compare the cold hardiness among 31 pomegranate cultivars with wide distribution of seed hardness, and analyze the expression pattern of Mitogenactivated protein kinase (MAPK) family genes in response to cold stress. The results can pave the way for shedding light to the function of Mitogen-activated protein kinase cascades in pomegranate cod tolerance.【Methods】Plant materials were cultivated in the National Horticulture Germplasm ResourcesCenter of China (NHGRC), with conventional cultivation management. Juvenile branches of 31 pomegranate cultivars were sampled in mid- January, the relative electrolyte conductivity (REC) was measured, and the semi-lethal temperature (LT50) was calculated for the evolution of cold hardiness. Seed hardness was detected by the TA-XT texture apparatus. MAPK family genes of Arabidopsis were used as queries to search in the whole pomegranate genome database, and reference genome sequence of ‘Tunisia’was obtained from NCBI database. Potential members of MAPK family were identified. Phylogeny relationship, gene structure and protein physicochemical properties were analyzed. Moreover, we performed real-time quantitative PCR (qRT- PCR) to analyze the expression pattern of 17 MAPK family genes in response to cold stress.【Results】The seed hardness and cold tolerance were evaluated. A total of 17 MAPK family genes were identified, which widely distributed on different chromosomes. All the members of the MAPK family could be mainly divided into three sub-classes, among which the members of subclass A and B mainly included PKc_MAPKK_plant_like and PTZ00024 Domain, subclass C mainly contained the PLN00034 domain, and all members contained a S_TKc domain. The results of analysis of physicochemical properties of proteins showed that the number of amino acid residues of each member distributed from 314 to 860 aa, the number of exons was from 1 to 18, the molecular weight of the protein was from 34 910.05 to 97 965.26 u, and the isoelectric point was from 4.94 to 9.35. Specific primer was designed for each PgMAPKs and PgMAPKKs, and their expression patterns were detected. The results showed that 12 out of 17 members were activated after low-temperature treatment. Yichengfenhongmudan that showed strong cold tolerance, and Malisi with weak cold tolerance, were both selected to compare the expression pattern of MAPKs. PgMKK2, PgMPK6, PgMPK9, PgMPK16 and PgMPK13 were all rapidly activated after low-temperature treatment in Yichengfenhongmudan. PgMKK8, PgMPK1- 1 and PgMKK4 were rapidly activated in Malisi. PgMKK2, PgMPK6, PgMPK9, PgMPK16 and PgMPK13 showed significantly higher expression level in Yichengfenhongmudan than those in Malisi after low-temperature treatment. The expression of PgMPK3, PgMPK12-1, PgMPK20, PgMPK18 and PgMKK6 was not affected by low- temperature treatment. PgMKK8, PgMPK1-1 and PgMKK4 were up- regulated in Malisi than Yichengfenhongmudan. The expression of PgMPK12-2 was not activated after low-temperature treatment in Malisi, but showed gradual increase in Yichengfenhongmudan. The expression level of PgMKK3 increased at first and then decreased during low-temperature treatment in Malisi, whereas it was not detected in Yichengfenhongmudan.【Conclusion】Pomegranate MAPK family genes responded to cold stress signals. PgMKK2, PgMPK6, PgMPK12-2 and PgMPK9 might positively regulate cold tolerance.