| R.rugosa(Rosa rugosa Thunb.)is an erect deciduous bush of the genus Rosaceae,with fragrant flowers and high oil content in petals.The essential oil extracted from R.rugosa is known as ‘liquid gold’ and is one of the raw materials for the preparation of high-end perfumes and cosmetics.In addition,R.rugosa can also be eaten and are used in the production of rose cakes,rose tea,rose sauce,etc.,and are very popular among people.Therefore,R.rugosa is an important special economic plant in China.But most R.rugosa are once flowering varieties,and flowering time is from April to May,which seriously affects its economic value.In Arabidopsis thaliana,TFL1 acts as a flowering inhibitor,maintaining its normal flowering time and indeterminate inflorescence growth in the apex,and tfl1 mutants flower earlier and their inflorescences are converted into determinate ones with the rapid production of a terminal flower.Rose KSN,a homolog of TFL1 of Arabidopsis thaliana,which is closely related to flowering methods including once flowering(OF)and continuous flowering(CF)as a flowering repressor.The CF rose Rosa chinensis variety Old blush contains a 9 kb Copia retrotransposon insertion in the second intron of KSN,that blocks its transcription.In the occasionally re-blooming(OR)rose cultivar ‘Pompon de Paris Climbing’,the insertion of a1 kb long terminal repeat(LTR)in the second intron leads to an unstable KSN and secondary flowering in autumn.Purple branch is a rare continuously flowering variety of R.rugosa that flowers from April to October,it is generally considered to have derived from an interspecific cross between R.rugosa Plena and Rosa davurica.Intriguingly,its presumptive parents are OF rose species.thus,its CF behavior remains unexplained.Elucidating the molecular mechanism of the CF trait in Purple branch will provide a theoretical basis for breeding new varieties with different flowering phenotypes.The main contents and conclusions of this study are as follows:1.Cloning and sequence alignment from CF Purple branch,OF Plena,and R.davurica showed that the diploid Purple branch is heterozygous for KSN.Among them,wild-type allele(KSN-Wt-Pb)was almost the same as that from Plena,except for a few SNPs and a 44 bp insertion in the second intron.Genetic evidence indicated that KSN-Wt-Pb could complement the tfl1-14 mutants with normal function.The other allele(KSN-Copia)was similar to that from CF R.chinensis Old blush and contained a 9 kb Copia retrotransposon insertion in the second intron,resulting in blocked transcription.q RT-PCR analysis showed that the expression of KSN in CF Purple branch was significantly lower than that of the OF R.rugosa Plena and R.davurica in March,April and September.These results indicate that the heterozygous KSN allele and low expression level in Purple branch are closely related to its continuous flowering habit.2.To investigate the effect of the insertion of Copia in Purple branch on the chromatin structure of KSN-Wt-Pb allele,we performed studies on DNA methylation and histone methylation modifications.The results showed that the degree of methylation in the promoter region of the functional KSN-Wt-Pb allele was significantly higher than that of the OF R.rugosa Plena.Ch IP-q PCR results showed that the degree of histone methylation modification of H3K9me3 and H3K27me2 at KSN loci in Purple branch was significantly increased compared with the OF R.rugosa Plena,while the methylation modification of H3K4me3 was slightly reduced.Silencing of the DNA methyltransferase genes MET1 and CMT3 and the histone methyltransferase gene SUVR5 in Purple branch led to enhanced KSN expression,and DNA methylation and histone modification levels were significantly increased.Therefore,the CF habit of Purple branch may be due to reduced expression of KSN caused by the halved dose and may be associated with epigenetic modifications together with retrotransposon insertions along the chromosome.3.Since the differences in KSN of different roses varieties all occur in introns,the introns may play a very important role.In order to further study the influence of the intron of KSN on itself,we carried out a study on the three introns of KSN.Firstly,it was verified that the three introns of KSN have promoter activity by the expression of the intron-driven reporter gene LUC.Secondly,through the 3’-end deletion assays,we determined that the first intron had a positive regulatory function,and the enhancement effect is closely related to the position and orientation of the intron;Finally,through the 3’-end deletion and mutation assays,it was identified that two reverse complementary cis-elements ARR1 binding elements(CGATT/AATCG)within the first intron responsible for the introns’ function.Mutation or deletion of ARR1 box caused the disappearance of enhanced functions.4.To investigate how the ARR1 box is involved in the enhancement of the first intron,we studied the transcription factor ARR1,which binds the ARR1 elements.ARR1 can bind to two ARR1 elements by EMSA experiments,and further Ch IP experiments showed that ARR1 preferentially binds to the first intron compared to ARR1 box on KSN promoter.However,the direct regulation of ARR1 on KSN was not identified by luciferase experiments.Further the ARR1-interacting protein JMJ12 was screened by the yeast two-hybrid screening library,the interaction between ARR1 and JMJ12 was verified by yeast two-hybrid,Bi FC and split luciferase complementation experiments.Luciferase experiments suggested that the first intron may recruit JMJ12-ARR1 complex to facilitate the histone modification at KSN gene locus and promote the gene expression.In summary,this study found that the CF habit of Purple branch may be due to reduced expression of KSN caused by the halved dose and may be associated with epigenetic modifications.Further research on the intron of KSN gene revealed that the first intron of KSN could recruit the ARR1-JMJ12 complex to epigenetically modify its chromatin structure,thereby affecting the expression of KSN.The results of this study reveal a new mechanism of continuous flowering,which is of great significance to targeted breeding. |
