Functional Studies Of The Arabidopsis Calcium-binding Protein AtCCaP1

The intracellular free calcium concentration([Ca2+]cyt)precisely regulates the plant response to circadian rhythm,the expression of circadian clock genes and the activity of Ca2+ dependent proteins.A large number of studies have found that the circadian endow rhythm expression of[Ca2+]cyt,and affects the physiological activities such as stomatal Covement,flowering and pollen tube growth.Arabidopsis thaliana cytosolic Ca2+binding protein 1(AtCCaP1)is a novel gene encoding a protein with a large number of glutamic acid residues.The bidentate carboxyl group of glutamic acid can effectively react with calcium ion.In this study,the expression pattern of AtCCaP1 was analyzed by GUS fusion gene,in situ hybridization and real-time quantitative PCR.It was found that the AtCCaP1 expression has rhythmicity and expressed in guard cells,trichomes and flowers which was tissue-specific.The physicochemical properties,Ca2+ binding ability and subcellular localization of AtCCaP1 were analyzed.The AtCCaP1 was modified by CRISPR/Cas9 and Gateway technology,to explore the effects of AtCCaP1 on[Ca2+]cyt and stomatal movement,and the potential relationship between circadian rhythm[Ca2+]cyt and physiological activities of growth and development.The main researches and conclusions are as follows:1.The length of AtCCaP1 was 750 bp and consists of two exons and one intron.The length of CDS(coding sequence)was 459 bp.The complete CDS was obtained by PCR,the CDS encoded 152 amino acid residues.The proportion of glutamic acid accounted for 29.6%.Bioinformatics analysis showed that the molecular weight of AtCCaP1 was 16.628 kDa and the isoelectric point was 4.03.The estimated half-life was 30 h and the instability index was 90.05,which was classified as unstable acidic hydrophilic protein.2.The 5' flanking 1990 bp sequence of AtCCaP1 was analyzed.It was found that the region contains a large number of cis-elements including abiotic and biotic stress and hormone response and transcription factor binding sites.It mainly includes light response,guard cell specificity,gibberellin response and auxin response elements which related to light response and growth and development.Using Arabidopsis genome as a template,a sequence containing transcription start site(TSS)of AtCCaP1 was cloned.The GUS fusion of AtCCaP1 promoter showed that AtCCaP1 was mainly expressed in mesophyll cells,epidermal hairs,guard cells,roots,flowers,anthers,pollens and stigmas of cotyledons.and stronger GUS signals could be detected under damaged conditions.GUS signal became stronger with the truncation of AtCCaP1 promoter length.In situ hybridization showed that AtCCaP1 mRNA could be detected in mesophyll cells and guard cells of true leaves.The expression of AtCCaP1 in ccalllhyltocl was no longer rhythmic.These results suggested that the expression of AtCCaPl was tissue-specific and was inhibited by regulation.Real-time quantitative PCR showed that AtCCaP1 was continuously induced by dark,gibberellin,and wounding conditions.The expression of AtCCaP1 was lower in day and higher in night.The expression of AtCCaP1 was no longer rhythmic in the circadian oscillator mutant cca1/llhy/tocl.3.To construct the prokaryotic expression vector of His and GST fusion tag,and purify AtCCaP1.Stains-all and Quin-2 Ca2+ binding probe staining results showed that AtCCaP1 could bind to Ca2+.The results of EMSA showed that the mobility of AtCCaP1 changed in the presence of Ca2+.GFP fusion protein expression vector was constructed and transfected into Arabidopsis t ha liana mesophyll cell protoplast.Subcellular localization showed that AtCCaP1 and GFP fusion green fluorescent protein was located in the cytoplasm of protoplast.These results suggested that AtCCaP1 might affect[Ca2+]cyt as Ca2+ binding proteins in cytoplasm.4.The AtCCaP1 knockout and overexpression vectors were obtained by CRISPR/Cas9 and Gateway technology,respectively.It was found that the stomata of knockout lines could not be closed normally under dark conditions,and the stomatal aperture of knockout lines was higher than that of wild type and overexpression lines.The phenotype of overexpression lines was opposite.Overexpression of.4tCCaP1 could restore the stomatal mutant phenotype.5.The wild-type and mutant homozygous lines stably expressing Ca2+ specific probe R-GECO1 were obtained by genetic transformation.The[Ca2+]cyt of the mutant guard cells was higher than that of the wild type during 1 h of darkness.The results of atomic K+ absorption of mutant guard cell protoplast was higher than that of wild type under 3 h dark condition.The Non-invasive Micro-test Technique(NMT)showed that K+in mutant guard cell changed from efflux to influx under 2 h dark condition.The endogenous NO concentration in guard cells of mutant was higher than that of wild type in 2 h dark treatment.In conclusion,this study verified the calcium binding ability,tissue specificity and rhythmicity of atccapl in cytoplasm.It was further found that AtCCaP1 directly regulated[Ca2+]cyt in guard cells and stomatal movement under dark conditions.NMT and atomic absorption techniques also showed the types of key ion in the mutant guard cells.It provides a basis for further understanding the mechanism of circadian Ca2+oscillations and dark induced stomatal closure.