The Functional Study Of MUCI70 Gene In Arabidopsis Reproduction

In amgiosperms,cells of the ovule integuments specialize in to multiple cell layers that form tissues of the seed coat after fertilization.In some species,including members from families of the Brassicaceae,Solanaceae,Linaceae and Plantaginaceae,a large amount of pectinaceous mucilage is accumulated during this process.Upon imbibition,the pectinaceous mucilage liberates and envelops the seed to form a gel-like capsule.The physiological role of mucilage remains an enigma.It is thought to help seed dispersal,control germination and resist biotic and abiotic stresses.Arabidopsis mucilage is composed of two layers,a water-soluble non-adherent outer layer and an adherent inner layer.Although the Arabidopsis seed coat mucilage is not essential for seed viability or germination,it represents an excellent genetic model for studying carbohydrate secretion,secondary cell wall formation and regulation.Our previous study proved that TOD1,which encodes an alkaline ceramidase,regulates pollen tube growth by regulating turgor pressure.The mutation of TOD1 gene leads to the reduced seed setting rate due to the decreasing of the pollen transfer efficiency.Using bioinformatics analysis,the homologous gene of TOD1,MUCI70 was identified in the Arabidopsis thaliana genome.In current study,the function of MUCI70gene in plant reproductive development was further studied.Real time quantitative PCR and GUS staining showed that MUCI70 gene is expressed in roots,stems,leaves and flowers.Subcellular localization analysis results showed that MUCI70 is co-localized with Golgi marker,indicating that MUCI70 is a Golgi-localized protein.The homozygous T-DNA insertion mutants of MUCI70（SALK＿129524C）is almost the same as the wild-type in pollen morphology,pollen tube germination and seed setting rate.Ruthenium red staining results showed that the mutation of the MUCI70 gene resulted in the decrease of seed coat mucilage.Seven heterozygous mutants were obtained in T1 generation by CRISPR/Cas9 technology.All of which insert a base T at the same position of the target site,named MUCI70-KO^+/-,which results in the frameshift and premature transcription termination of corresponding MUCI70.The pollens of MUCI70-KO^+/-mutants show obvious atrophy and abortion,and the seed setting rate is about 50%.Five homozygous mutants MUCI70-KO#1～MUCI70-KO#5 were screened out from the progenies of MUCI70-KO^+/-by three different methods.All of which show frameshift and premature transcription termination of corresponding MUCI70,however,they are similar to the wild-type in pollen morphology,vigor,seed setting rate and seed mucilage.It was found that there were differential genes related to m RNA surveillance mechanism between the MUCI70-KO#1 and Col,by analyzing of the transcriptome sequencing results of MUCI70-KO#1 and MUCI70-KO#2.And the down-regulated gene AT5G47010 related to m RNA surveillance mechanism in MUCI70-KO#1 is also down regulated in MUCI70-KO#2,the up-regulated gene related to m RNA surveillance mechanism in MUCI70-KO#1 is also up regulated in MUCI70-KO#2.The co-differential genes of MUCI70-KO#1 and MUCI70-KO#2 encode key protein factors involved in the nonsense m RNA mediated degradation pathway,which provides evidence for the genetic compensation effect activated by nonsense m RNA mediated degradation pathway in MUCI70-KO#1 and MUCI70-KO#2.It was found that there were 44 differential genes related to seed coat mucilage between the SALK＿129524C and Col,by analyzing of the transcriptome sequencing results of SALK＿129524C,and 18 of which were up-regulated and 26 down-regulated in SALK＿129524C.This coincides with existence of mucilage phenotype in SALK＿129524C.There are 45differential genes related to lipids metabolism between the SALK＿129524C and Col,17 of which were up-regulated and 28down-regulated in SALK＿129524C.The lipids involved in metabolism are mainly phospholipids,which are the main lipid molecules of biomembrane.It is speculated that the genes related to lipids metabolism in the SALK＿129524C mutant are involved in the synthesis and secretion of mucilage by participating in endomembrane transport of polysaccharides which make up mucilage.The above results indicate that MUCI70 gene is essential for the accumulation of seed mucilage.However,further studies are needed to elucidate the function of MUCI70 gene in plant reproductive development.