Effects of cyclanilide on growth and branching characteristics of 1-yearold apple plants

Abstract: 【Objective】The divalentions of calcium (Ca2+) play a key role as core transducers and regula- tors in response to environmental stimuli and processes related to development of plants. Ca2 + signals are decoded into appropriate physiological responses and transmitted to different load states. In plants, there are three main classes of Ca2+ sensors to decode and transmit the Ca2+ signals, including calmodu- lin (together with calmodulin-like proteins) (CaMs/CMLs), calcium-dependent protein kinases (CD- PKs) and calcineurin B-like proteins (CBLs). CaM, in a Ca2+-dependent manner, regulates several tran- scription factors (TFs) that are implicated in various molecular, physiological, and biochemical func- tions in cells. The CAMTA (calmodulin-binding transcription activator) is a member of the Ca2+-loaded CaM-dependent family of TFs. The CAMTA proteins are characterized by several conserved domains, including a unique DNA-binding domain (CG-1), a transcription factor immunoglobulin-like DNA bind- ing domain (TIG), ankyrin repeats (ANK), IQ motifs(IQXXXRGXXXR) and a Ca2+-dependent calmod- ulin binding domain (CaMBD). The CAMTA in a Ca2+/CaM-driven modus has been involved in carry- ing out important functions by modulating plant stress responses and overall development. Mango (Mangifera indica) is a new functional fruit tree which has been widely cultivated in tropical region. Al- though the market and planting area of mango have an increasing trend, there is still a gap in the under- standing of its growth and development regulation. The CAMTA transcription factor genes are the cen- tral element mediating plant development. Hence, we performed the genome-wide analysis of the CAM- TA transcription factor gene family of mango to provide sequence resource for further functional verifi- cation.【Methods】In order to identify the CAMTA gene family genome wide, the mango genomic data were downloaded from the NCBI. The obtained protein sequences were checked by the NCBI-Con- served domain database (CDD), and the proteins without characteristic domain were removed. The re- dundant sequences containing complete CG-1, ANK repeats and the IQ domain were further removed by alignment, and the remainder were considered as putative CAMTA genes. All the obtained sequences were sorted as unique sequences for further protein domain search in the Pfam database. The CAMTA protein sequences of Arabidopsis thaliana, Populus trichocarpa, Nicotiana tabacum, Malus domestica,Ananas comosus were obtained from the Plant Transcription Factor Database (PlantTFDB v5.0), respec- tively. The ExPASy-ProtParam tool was used to predict the physical and chemical properties of Mi- CAMTAs, including the number of amino acids, gene length, molecular weight and theoretical isoelec- tric point. The secondary structure prediction of MiCAMTAs protein sequence was carried out through the online site SPOMA secondary structure prediction. Phylogenetic analysis was performed by MEGA v7.0 program with the neighbor-joining (NJ) method and the bootstrap test was carried out with 1000 replicates, and use the online website iTOL to beautify the phylogenetic tree. The conserved motifs and domains of the MiCAMTA protein sequences were predicted by the MultipleEm for Motif Elicitation (MEME) and Prosite software, respectively. And the conserved motifs and conserved domains were vi- sualized through the TBtools software. qRT-PCR was used to infect MiCAMTAs against Colletotrichum gloeosporioides (Cg) and Xanthomonas citri pv. mangiferaeindicae (Xcm) and treatment with salicylic acid (SA) and methyl jasmonate (MeJA) and the gene expression profile was visualized through the TB- tools software.【Results】A total of 8 putative CAMTA candidate genes were identified in mango ge- nome. The number of amino acids in the MiCAMTAs protein of mango was 86-1075, the molecular weight was 59 783.87-120 146.13, and the isoelectric point was 4.78-9.93, and all of them were labile and hydrophilic proteins. The secondary structure analysis showed that the helixes were the major part of the CAMTA protein. In order to understand the structural diversity of the MiCAMTA protein, the conserved motifs and conserved domains were analyzed. The results showed that all the MiCAMTA (1, 2, 3, 5, 7) contained motif 1-10, MiCAMTA6 did not contain motif3, and MiCAMTA4 only contained motif (3, 8, 9), but no conserved motif was detected in MiCAMTA8. All MiCAMTAs contained CG-1 domains, except for MiCAMTA4 and MiCAMTA8 containing AnK and IQ conserved domain. They could be divided into two categories according to whether they contained TIG domain, both MiCAM- TA3 and MiCAMTA5 contained TIG conserved domain. In addition, the conserved motifs of proteins with close relationships were basically the same, and the conserved domains of members with close phylogenetic relationships were basically similar. The MiCAMTA gene family was highly conserved dur- ing plant evolution. A NJ phylogenetic tree was constructed using the CAMTA proteins of mango, Ara- bidopsis thaliana, Populus trichocarpa, Nicotiana tabacum, Malus domestica, Ananas comosus, and the CAMTAs were clustered into 13 groups (GroupI-XIII). The results showed that the MiCAMTAs were closely related to apples, poplars and tobacco. qRT-PCR was used to determine the expression level of the MiCAMTAs under different pathogen infections and hormone treatments, and the gene expression profile was visualized by the TBtools software. The qRT-PCR expression analysis showed that 8 Mi- CAMTA genes in 4 treatments (Cg, Xcm, SA, MeJA) had different levels of expression, indicating that the different MiCAMTA gene members had different functions related to the resistance of mangoes to pathogenic bacteria and hormone response. The expression levels of the MiCAMTA (1, 2, 3) under Cg and Xcm infections were opposite, while the MiCAMTA (5, 6, 7, 8) expression levels were up-regulated under Cg and Xcm infections. However, the MiCAMTA4 did not change significantly during the 72- hour surveillance period after infection. It was found that MiCAMTAs were up-regulated or down-regu- lated to varying degrees in the treatment of mango leaves with hormones (SA, MeJA), and the overall expression of the MiCAMTAs under SA treatment was higher than that under MeJA treatment【. Conclu- sion】There were 8 MiCAMTA family members in the whole mango genome, with typical CaM binding domains, including 10 motifs, which could be activated by pathogenic bacteria and disease-resistant sig- nal molecules to varying degrees, laying a foundation for studying its disease-resistant mechanism. Our findings would provide new insights of mango CAMTA gene family, and lay a foundation for further re- search on the role of the MiCAMTA genes in mango development and growth and stress response.