Functional Study Of The PYR/PYLs- And CAR-like C2-domain Genes In Brassica Rapa And Their Phylogentic Sequence Comparison To Brassica Oleracea And Arabidopsis Thaliana

Abscisic acid(ABA),an important plant hormone,not only regulates the growth and development of plants,but also regulates plant stress responses.With the discovery of PYR/PYL(PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS)type ABA receptors,the molecular mechanisms of ABA perception and ABA signalling have been elucidated.The C2-domain ABA-related(CAR)protein has been found to mediate plasma-membrane recruitment of PYR/PYLs in a Ca²⁺dependent manner in Arabidopsis thaliana.ABA signalling have been studied in Brassica oleracea and Brassica rapa,however,the mechanism of ABA perception and signalling is still relatively scarce.In the current study,genome-wide annotations and functional analysis of PYR/PYL-and CAR-like genes in Brassica oleracea and Brassica rapa were performed.We obtained following results:(1)A total of 24 PYR/PYL-like genes were encoded in the genome of Brassica oleracea and Brassica rapa respectively.Phylogenetic analysis revealed that PYR/PYL-like proteins are also classified into three subclasses.(2)Nearly all the identified 24 Bo PYR/PYL genes were mapped on the C01 to C09chromosomes of Brassica oleracea,except Bol003645(Bo PYR1a)and Bol007552(Bo PYL6a).All of the 24 BrPYR/PYL genes were found to be distributed on the A01 to A10.Moreover,the distribution density in each chromosome was different.(3)Gene structure and protein architecture analysis showed that most of the Bo PYR/PYLs and BrPYR/PYLs displayed similar organization pattern to their Arabidopsis orthologs.(4)The RNA-seq data(from the GEO database)based transcriptional analysis showed that nearly all the Bo PYR/PYLs and BrPYR/PYLs are highly expressed in at least one tissue,except Bo PYL11(Bol006314),Bo PYL13(Bol009397),BrPYL5a(Bra028758)and BrPYL13(Bra013334).(5)The expression of BrPYL1 was induced by ABA in Brassica rapa.(6)Transgenic plants harboring BrPYL1 conferred ABA hypersensitivity to plants.(7)ABA activated the expression of an ABA-responsive reporter(RD29B-LUC)in Arabidopsis protoplasts expressing BrPYL1.(8)We obtained regenerated seedlings of Brassica napus that aims to knock-out Bo PYR/PYLs and BrPYR/PYLs by CRISPR-Cas9-mediated gene editing technology.(9)We also identified 16 CAR-like proteins in Brassica oleracea and Brassica rapa respectively.Phylogenetic relationship and chromosome localization of Bo CARs and BrCARs were examined.We also determined their gene structure,protein architecture and expression level.Furthermore,we also obtained regenerated seedlings of Brassica napus that attempts to knock-out Bo CARs and BrCARs by CRISPR-Cas9-mediated gene editing.In summary,the gene structure and protein architecture of PYR/PYLs and CARs in Brassica oleracea,Brassica rapa and Arabidopsis thaliana were highly similar.Further functional studies revealed that BrPYL1 can act as an ABA receptor to perceive ABA signal.Taken together,our data support a co-linear relationship among Brassica oleracea,Brassica rapa and Arabidopsis thaliana with respect to the molecular basis of early ABA perception and signalling.