Identification And Functional Analysis Of Citrus CAMTA Genes

Citrus is one of the most important economical fruit crops,which is widely grown all over the world.In china,it has a long history for citrus cultivation,which also has all kinds of varieties.Meanwhile,China is a large citrus producing and consuming country in the world.In the southern part of China,the citrus industry has become a pillar industry for farmers to increase their incomes and possesses a pivotal position in the regional economy promotion.In recent years,significant process has been made in citrus industry.However,the developments of quality and yield are impeded by various biotic and abiotic stresses.Therefore,it is important to obtain cultivars with enhanced stress tolerance.However,traditional breeding has various limitations and difficulties,and it is difficult to obtain stress-tolerant germplasm in a short time.The use of genetic engineering techniques to transfer some important resistance genes into citrus is the most efficient and fast ways.That requires deep exploration and utilization of citrus resistant functional genes.CAMTA(calmodulin-binding transcription activator)transcription factor is a type of the target proteins of CaM in all the multicellular eukaryotes and plays an important role in plant stress response.However,in citrus,this gene family,to date,has not been systematic reports.Based on the clementine and Sweet Orange genomes(http://www.phytozome.net),in this study,citrus CAMTA members have been systematically identified for the first time.Their gene structure,phylogenetic relationship and the expression patterns in different tissues and stress treatments were investigated in detail.The CitCAMTA8 and CitCAMTA9 overexpression vectors were constructed and transferred into tobaccos,and their stress tolerance were studied.The main results are as follows:(1)Based on citrus genomic sequences and using Arabidopsis CAMTAs as seed sequences,A total of 9 CAMTA members were identified in citrus,which were named CitCAMTA1-9,respectively.The results showed that the physico-chemical characteristics among different CitCAMTAs were similar,with 1 or 2 exceptions.all members are unstable proteins with the exception of CAMTA6 and CAMTA9.CAMTA1,2 and 5 are acidic proteins,the rest are basic proteins.Exept for CitCAMTA4,all proteins are hydrophilic protein.The Conserved domain analyses revealed that all CitCAMTA members contained a typical CaMBD domain.Besides that,most members also have other conserved domains,such as CG-1 domain,TIG domain,ankyrin repeats and IQ motifs.(2)Most CitCAMTA members together with other plant CAMTA members were clustered into 5 groups,which indicated that most CitCAMTA genes highly conserved during evolution.Interestingly,CitCAMTA 7,8 and 9 form a separate group,indicating the isolation of new CAMTA members during evolution.These CitCAMTA members may possess distinct functions in citrus growth and development and stress responses.(3)Tissue-specific expression analysis showed that CitCAMTA3 was expressed at a high level in all types of tissue,particularly in cotyledon and seed.CitCAMTA1 was dominantly expressed in cotyledon and seed.The highest expression level of CitCAMTA8 was observed in peel.CitCAMTA2,6,7 and 9 were mainly expressed in cotyledon or/and seed at a relative low level.Notably,CitCAMTA4 was expressed at an undetectable level in all investigated tissues.Taken together,these data reflect the difference of tissue-specific expression among CitCAMTA members,implying that CitCAMTAs play an important role in different tissues of citrus.(4)Under abiotic stress(NaCl and dehydration)and hormones(SA,ETH,IAA,6-BA,ABA,MeJA and GA3),these CitCAMTA genes-with the exception of CitCAMTA4-could be expressed by one or more treatments,indicating that CitCAMTAs play an important role in citrus stress response and growth.Under NaCl stress,CitCAMTA1,5 and 9 were significantly upregulated.The expression level of CitCAMTA7 was increased first and then decreased.CitCAMTA3 was suppressed.CitCAMTA2 and CitCAMTA8 showed no obvious changes.Under dehydration stress,the expression levels of CitCAMTA5,6 and 8 were slightly decreased,while CitCAMTA3 and 9 were noticeably upregulated and reached maximum at 12 h.Dehydration moderately increased the expression level of CitCAMTA2 and did not obviously alter that of CitCAMTA1.The transcripts of CitCAMTA7 were nearly undetected under dehydration treatment.CitCAMTA1,5 and 7 exhibited a positive response to all hormones,while CitCAMTA3 showed a negative trend.CitCAMTA8 gene was repressed by MeJA,ABA and ETH,and showed no obvious regular change under other hormone treatments.Further analysis revealed that there are many cis-acting elements associated with stress,hormones and light responsiveness in the CitCAMTAs promoter region,such as G-box,MBS and TCA element.(5)Transgenic tobacco plants overexpressing CitCAMTA8 were shown to be more tolerant to drought stresse than the wild type(WT).Under drought stress,the transgenic tobacco had higher proline content compared to the WT.Meanwhile,resistance related gene of NtCAT,NtSOD and NtP5 CR expression level were higher.