Identification and expression analysis of PP2C gene family in Poncirus trifoliata

Abstract: 【Objective】The plant hormone abscisic acid (ABA) plays critical roles in plant growth and developmental responses to various biological and abiotic stresses. Type 2C protein phosphatases (PP2Cs) are negative regulators at early step in the ABA-signaling pathway. Citrus is one of the most important fruit trees in tropical and subtropical areas. To date, the roles of the citrus PP2C gene family has not been systematically studied. We systematically identified the PtrPP2C gene family members, a sequence characteristic, expression patterns in different tissues of Poncirus trifoliata and their responses to abiotic stresses.【Methods】The genome sequences of citrus were retrieved from P. trifoliata genomic database. The gene sequences of PtrPP2C genes were obtained using NCBI-Conserved domain data-base (CDD), Pfam- Batch search (Pfam) and InterPro- Search sequence (InterPro). To investigate the characteristics of putative PtrPP2C proteins, ExPASy-ProtParam tool was used to predict the physical and chemical properties including the number of amino acids, theoretical isoelectric point and molecu-lar weight. The distribution of PtrPP2C family members on chromosome was drawn by Map-MG2C.The phylogenetic analysis was performed by MEGA7.0 to construct neighbor-joining (NJ) phylogenetic analysis with 1000 replicates. The genetic structure and conserved motifs of the PtrPP2C protein se-quences were predicted by Gene Structure Display Server (GSDS) and the MultipleEm for Motif Elici-tation (MEME), respectively. The genetic replication events of PtrPP2C genes were analyzed through TBtools software. The PlantCARE was used to predict cis- acting elements, which were visualized through Excel. The qRT-PCR was used to detect the expression patterns of PtrPP2C gene family mem-bers under drought,（4±1）℃ low temperature as well as（38.0±0.5）℃ high temperature.【Results】A total of 53 PtrPP2C genes were identified in P. trifoliata genomic data, named as PtrPP2C1-PtrPP2C53. in the 53 candidate PtrPP2C genes. They were widely distributed on all the P. trifoliata chromosomes. The lengths of the putative PtrPP2C proteins ranged from 235 to 1184 amino acids, whereas their theoretical pI ranged from 4.82 to 9.47, their protein molecular weights from 25.90 to 127.95 ku, and their instability coefficient from 32.02 to 66.67. The subcellular localization prediction showed that the PtrPP2C proteins were localized at multiple cellular sites, including chloroplast, cyto-plasm, nucleus, mitochondrion, peroxisome, extracellular and plasma membrane. A total of 133 genes from Arabidopsis thaliana and P. trifoliata were used to construct the phylogenetic tree, which were di-vided into 12 subfamilies (Subfamily A-L), of which the PtrPP2C proteins existed in the 10 subfami-lies (except for Subfamily G and K). Consistently, most of the genes in the same subfamily possessed similar gene structure and conserved motifs distribution. The collinear analysis identified 10 pairs of fragment duplication events in the PtrPP2C family members, while no gene tandem phenomenon was found. The promoter sequence analysis revealed that the PtrPP2C promoters contained a large number of growth and development, plant hormone and stress-responsiveness related cis-acting elements. The gene expression profiles of PtrPP2C genes had obvious tissue-specific expression level. For example, PtrPP2C9 was highly expressed in the stems, and PtrPP2C9, PtrPP2C30 and PtrPP2C34 were lowly expressed in the fruits; PtrPP2C10 was highly expressed in the fruits and leaves, while lowly expressed in the flower buds, roots and stems; PtrPP2C38 was highly expressed in the roots, while PtrPP2C42,PtrPP2C43 and PtrPP2C44 were lowly expressed, all of which suggested that different PtrPP2C genes might play different roles in growth and development of citrus. A total of 10 PtrPP2C genes (i.e.,PtrPP2C1, PtrPP2C3, PtrPP2C16, PtrPP2C17, PtrPP2C24, PtrPP2C27, PtrPP2C29, PtrPP2C33,PtrPP2C43 and PtrPP2C51) were chosen to analyze their relative expression levels under different abi-otic stresses, including（4±1）℃ low temperature,（38.0±0.5）℃ high temperature and 15% PEG6000. All 10 PtrPP2C genes performed up-regulated under low temperature stress, while 8 out of 10 genes (i.e., PtrPP2C3, PtrPP2C16, PtrPP2C24, PtrPP2C27, PtrPP2C29, PtrPP2C33, PtrPP2C43 and PtrPP2C51) were up-regulated under high temperature stress, and 9 out of 10 genes (i.e., PtrPP2C1, PtrPP2C3, PtrPP2C16, PtrPP2C24, PtrPP2C29, PtrPP2C33, PtrPP2C43 and PtrPP2C51) were up-regulated under drought stress. Above all, 7 out of 10 genes (i.e., PtrPP2C3, PtrPP2C16, PtrPP2C24,PtrPP2C29, PtrPP2C33, PtrPP2C43 and PtrPP2C51) were induced in low temperature and high tem-perature as well as drought stress. The expression levels of PtrPP2C29 were induced under all the three stresses. These results indicated that the different PtrPP2Cs might played different roles in the process of low temperature stress and high temperature stress as well as drought stress in citrus development. In addition, there were different expression patterns of the same gene under the three stresses. The expres-sion of the PtrPP2C1 increased significantly under low temperature stress and drought stress, while de-creased under high temperature stress, and the expression of the PtrPP2C27 increased significantly un-der low temperature stress and high temperature stress, while decreased under drought stress, indicating that the PtrPP2C genes might participate in different regulatory pathways under the different stresses.【Conclusion】A total of 53 PtrPP2C family members were identified in the whole P. trifoliata genome,and they might be correlated with citrus development. The expression of the PtrPP2C genes in the dif-ferent tissues and their expression patterns in the response to the various abiotic stress implied that they might have complex roles in the resistances of citrus. This study provides insights into the PtrPP2C gene family’s evolution, expression pattern and their response to multiple stresses.