Identification Of The CLE Gene Family In Mulberry And Its Function Under Salt Stress

As sessile organisms,good growth and development of plants cannot be achieved without suitable external environment,and the salt stress caused by excessive salt content in the soil seriously restricts the growth and development of plants.In order to adapt to salt stress,plants have developed complex response mechanisms in the course of evolution to cope with the effects of salt stress.Such response mechanisms include the recognition,transduction and transmission of stress signals as well as the expression of related genes.CLE protein,as a member of the plant peptide hormone family,plays an important role in signal transduction and transmission.Studies have shown that the precursor peptide of CLE gene is cleaved in cells to form a functional peptide fragment of 12-13 amino acid length--CLE-domain.Subsequently,cle-domains are secreted outside the cell and perceived by leucine repeat receptor-like kinases(LRR-RLKs)on the downstream receptor cells.In the recipient cells,the signals further regulate the expression and synthesis of downstream genes through a series of cascade amplification,and finally realize the transmission of inter-cell signals.CLE peptides have been reported to be extensively involved in the growth and differentiation of plant cells in previous studies,and recent studies have shown that CLE peptides are also regulated in response to environmental stresses such as drought,high temperature,and salt stress.To date,the CLE gene family has been identified in Arabidopsis thaliana,radish,soybean,rice,wheat,grape,tomato,poplar and other species.However,studies on CLE peptides currently focus on the regulation of cell differentiation,and there are relatively few studies in the field of abiotic stress such as salt stress,and the mechanisms of which need to be resolved.Mulberry(Morus L.),belonging to the Moraceae family,is an economic tree native to China.It has strong stress resistance to abiotic stresses such as salt,alkali,drought,flood and cold.By BLAST analysis and Hummer search using CLE genes and protein sequences from Arabidopsis thaliana as templates,a total of 28 CLE genes were identified in the Mulberry Genome Database.Subsequently,we studied the CLE gene of mulberry from two aspects of expression analysis and functional verification of CLE polypeptides.The main results are as follows:1.Identification and bioinformatics analysis of CLE family genes in mulberryBased on sichuan mulberry genome database,through the bioinformatics methods such as BLAST and HUMMER,preliminary appraisal to 28 in sichuan mulberry mulberry CLE genes,and then the loci of the 28 CLE structure analysis,function structure domain prediction,key functional areas of appraisal and the analysis of the multiple sequence alignment and evolutionary tree and so on bioinformatics analysis.The results showed that all of the genes contained typical cle-domains,and(86 %)of the genes were relatively simple in structure,containing only one functional motif,without introns,and only composed of a single exon.Multiple sequence alignment showed that the CLE peptides of mulberry were highly conserved in the cle-domain region and less conserved in the non-cle-domain region.The phylogenetic analysis showed that CLE genes was divided into five subfamilies(Ⅰ、Ⅱ、Ⅲ、Ⅳ and Ⅴ).The cle-domains of different subfamilies were analyzed,and it was found that only the first subfamily had histidine(H)as the starting amino acid,while the other first,second,third,and third subfamilies all had arginine(R)as the starting amino acid.2.Cloning and expression analysis of CLE family genes in mulberryAll 28 CLE genes were cloned and identified by PCR in white mulberry.The results showed that the CLE gene of mulberry was highly conserved among different varieties.Phylogenetic tree analysis showed that Ma CLE gene showed a highly conserved clustering pattern among different mulberry cultivars.Further sequence alignment revealed that there were differences in amino acid sequences of three CLE peptides in the cle-domain region,namely,Ma CLE2,Ma CLE3 and Ma CLE45.By using q RT-PCR,we constructed the expression profiles of CLE peptides in roots,stems and leaves of mulberry,and the results showed that there were 5 different expression patterns of CLE gene in mulberry:(1)It is mainly expressed in roots,but is relatively low in stems and leaves.Ma Cl E1,Ma CLE4,Ma CLE5,Ma CLE10,Ma CLE11,Ma CLE13,Ma CLE19,Ma CLE26,Ma CLE27,Ma CLE40,Ma CLE43 and Ma CLE44;(2)Ma CLE14,Ma CLE15,Ma CLE20 and Ma CLE25 were mainly expressed in stems,but were relatively low in roots and leaves;(3)The main expression sites were in roots and stems,and the expression levels were relatively low in leaves,including Ma CLE12,Ma CLE17,Ma CLE18,Ma CLE41 and Ma CLE45;(4)The expression was mainly expressed in leaves,but was relatively low in roots and stems,including Ma CLE2,Ma CLE9 and Ma CLE16;(5)Only specific expression in roots,including Ma CLE3 and Ma CLE6.By comparing with the results of phylogenetic tree,we found that the differential expression pattern of CLE polypeptides in mulberry was related to their positions in phylogenetic tree.CLE peptides in the Ⅰ,Ⅳ and Ⅴ subgroups were mainly expressed in roots,the CLE peptides in the Ⅲ group were mainly expressed in stems,and the CLE peptides in the Ⅱ subgroup were mainly expressed in leaves.Further analysis showed that there were 19 CLE peptides with high expression in roots,accounting for 73 % of the detected genes.The expression profile of Ma CLE peptides induced by salt stress was obtained by sodium chloride treatment.The results showed that only 3 genes were up-regulated by salt stress in the root,accounting for 12.5 % of the genes expressed in the root.In stem,8 genes were induced by salt stress,accounting for 33.3 % of the expressed genes in stem.In leaves,6 genes were induced by salt stress,accounting for 25 % of the gene expression in leaves.The expression levels of other genes in root,stem and leaf were inhibited by sodium chloride treatment.Most of the CLE genes in mulberry showed a trend of down-regulated expression under salt stress.In order to further analyze the function of CLE genes in mulberry,we selected Ma CLE2,Ma CLE4,Ma CLE9,Ma CLE10,Ma CLE13 and Ma CLE17 among the up-regulated genes as candidate genes for functional exploration,while in the down-regulated genes,Macle16,which has a relatively high expression level and a significant response to salt stress,was selected for further functional analysis.3.Study on salt stress function of CLE polypeptides from mulberryIn a preliminary study,we used Arabidopsis seeds as experimental materials to study the effects of mulberry Ma CLE peptides on the germination of Arabidopsis seeds under salt stress.The results showed that the germination of Arabidopsis seeds was almost completely inhibited when the concentration of sodium chloride was 150 m M.Adding Ma CLE13 peptide to the medium could break the inhibition effect of high salt stress on seed germination.When Ma CLE13 peptide concentration reached 1000 n M,seed germination rate of Arabidopsis thaliana significantly increased from 0 to about 20% compared with salt stress and other peptide treatment groups.Through stress treatment of Arabidopsis thaliana and mulberry seedlings,we found that in Arabidopsis thaliana,150 m M Na Cl significantly inhibited the growth of main root and lateral root,and Ma CLE4 reduced the inhibitory effect of salt stress on the growth of main root and lateral root.Ma CLE2 significantly alleviated the growth inhibition of Arabidopsis lateral roots under salt stress,while Ma CLE10,13,16 and 17 significantly enhanced the growth inhibition of lateral roots under salt stress.Further analysis revealed that ROS scavenging enzymes(such as SOD,CAT,and POD)in Arabidopsis thaliana were significantly inhibited by 150 m M Na Cl,while the opposite was true in mulberry.Studies in Arabidopsis thaliana have shown that except Ma CLE4,Ma CLE2,Ma CLE9,Ma CLE10,Ma CLE12,Ma CLE16 and Ma CLE17 can significantly up-regulate SOD activity in Arabidopsis thaliana seedlings under pseudo-salt stress.Ma CLE17 significantly promoted CAT activity in Arabidopsis seedlings under salt stress,while other peptides significantly inhibited CAT activity in Arabidopsis seedlings under salt stress.Ma CLE4,Ma CLE9,Ma CLE13 and Ma CLE16 polypeptides had no significant effect on POD activity of Arabidopsis seedlings under salt stress,while Ma CLE10 and Ma CLE17 were positively regulated and Ma CLE2 was negatively regulated.In terms of MDA and proline accumulation,Ma CLE2,Ma CLE9,Ma CLE13,Ma CLE16 and Ma CLE17 played a positive role in regulating MDA accumulation in Arabidopsis under salt stress,while Ma CLE4 and Ma CLE10 had no significant effect on MDA accumulation.All Ma CLE polypeptides in mulberry significantly increased the accumulation of proline in Arabidopsis under salt stress.The results of treatment of mulberry seedlings showed that Ma CLE9,Ma CLE10 and Ma CLE13 peptides played a positive role in regulating the SOD activity of mulberry seedlings under salt stress,while Ma CLE2,Ma CLE6 and Ma CLE17 significantly inhibited the SOD activity of mulberry seedlings after treatment.Further analysis showed that Ma CLE2,Ma CLE4,Ma CLE13,Ma CLE16 and Ma CLE17 negatively regulated CAT activity under salt stress,and Ma CLE10 promoted CAT activity,while Ma CLE9 had no significant effect on CAT activity.Compared with sodium chloride treatment group,there was no significant change in POD activity of mulberry seedlings treated with Ma CLE4 and Ma CLE13 exogenous polypeptides.Ma CLE2,Ma CLE16 and Ma CLE17 had a negative regulation effect on POD activity of mulberry seedlings under salt.