Regulatory Mechanisms Of Double-flower Development In Catalpa Bungei

Catalpa bungei,a chinese originated ornamental and precious timber trees,belongs to Catalpa genus in the Bignoniaceae family,and is cultivated broadly.The double-flower C.bungei,a recently discovered natural variation,only found in the Northwest of Hubei Province.In this study,we used morphology analysis in the single and double flowers of C.bungei,and found the key stage of the petaloid organ in the double flower.We used a comprehensive transcriptomics study(i.e.staminodiums of the single flower,the petaloid organs of the double flower and the normal petals)to identify the crucial transcription factors.Based on the analysis of MADS genes family,we isolated and identified the genes of CabuPI,CabuAP3 and CabuAG,and investigate their expression patterns and functions.Taking the combined advantages of highly sensitive immuno-affinity enrichment and high-resolution liquid chromatography-tandem mass spectrometry,we conducted a global acetylome analysis of C.bungei double flowers,and revealed the function of lysine acetylation in double flower development of C.bungei.The double flowers of C.bungei,have four whorls of floral organs,including sepals in whorl 1,petals in whorl 2,stamens and homeotic conversional petals from staminodiums in whorl 3,and one carpel in whorl 4.The double flower development of C.bungei was classified as 11 development stages,i.e.D1-D11.However,the petaloid organ of the double flower appeared at D8(9 mm),and elongated at D9 and D10.The flowers reached anthesis at D11.We found that there were 13 883 differential expressed genes(DEGs)in the petaloid organs of the double flower and staminodiums of the single flower,including 6 379 significantly upregulated and 7 504 significantly downregulated genes.We identified 2 329 differential expressed genes(DEGs)in the petaloid organs of the double flower and normal petals,including 1 486 significantly upregulated and 843 significantly downregulated genes.Based on the expression abundance of gene expression by Fragments Per Kilobase per Million(FPKM),we identified that MADS genes family differentially expressed in these floral organs.Among them,the expression level of A function MADS-box gene APETALA1(AP1)in the petaloid organs and petals of the double flower was obviously higher than that in staminodiums of the single flower.However,the expression level of C function MADS-box gene AGAMOUS(AG)in the petaloid organs and normal petals of the double flower was obviously lower than that in staminodiums of the single flower.Furthermore,the expression levels of B function MADS-box gene GLO/PI and E function MADS-box gene SEP2 were significantly different during staminodiums of the single flower,the petaloid organs of the double flower,and normal petals.To investigate the molecular mechanisms underlying organ development of woody angiosperm in Catalpa,we isolated and identified a PI homologue,referred to as CabuPI(C.bungei PISTILLATA),from two genetically cognate C.bungei(Bignoniaceae)bearing single and double flowers.Sequence and phylogenetic analyses revealed that the gene is closest related to the eudicot PI homologues.Moreover,a highly conserved PI-motif is found in the C-terminal regions of CabuPI.Semi-quantitative and quantitative real time PCR analyses showed that the expression of CabuPI was restricted to petals and stamens.However,CabuPI expression in the petals and stamens persisted throughout all floral development stages,but the expression levels were different.During the stages of a rapid increase in floral bud differentiation,the expression of CabuPI in double floral buds was obviously higher than that in single floral buds.Later in development of petals,CabuPI expression in single flower was obviously higher than that in double flower.However,CabuPI expression in the petaloid organs of the double flower was higher than that in peal at D8-D11 stages.Later in development of stamens,the level of CabuPI expression in double floral stamens were obviously higher than that in single floral stamens.Compared with the wild Arabidopsis,ectopic expression of the CabuPI in transgenic wild-type or heterozygote pi-1 mutant Arabidopsis produced partially elongated green/white petal-like structures(petaloid sepals)in the first whorl.These first whorl floral parts(i.e.,sepals)of 35S::CabuPI were completely separated and opened immediately after flower.Meanwhile,the cells in the converted white portion of the petaloid sepals were observed to be morphologically similar to the wild-typesecond whorl floral parts(i.e.,petals)epidermal cells opening.Futhermore,in 35S::CabuPI transgenic homozygous pi-1 mutant Arabidopsis,the second and the third whorl floral orgarns produced normal petals and a different number of stamens,respectively.We isolated and identified a AP3 homologue,referred to as CabuAP3 from two genetically cognate C.bungei bearing single and double flowers.Sequence and phylogenetic analyses revealed that the gene is closest related to the eudicot AP3 homologues.Semi-quantitative and quantitative real time PCR analyses showed that the expression of CabuAP3 was restricted to petals and stamens.During the stages of a rapid increase in floral bud differentiation,the expression of CabuAP3 in double floral buds was obviously higher than that in single floral buds.Later in development of petals,CabuAP3 expression in single flower was obviously higher than that in double flower.However,CabuAP3 expression in the petaloid organs of the double flower was higher than that in petals at D8-D11 stages.At the stages of D10-D11,the level of CabuAP3 expression in double floral stamens were obviously higher than that in single floral stamens.Compared with the homozygous ap3-3 mutant Arabidopsis,transgenic homozygous ap3-3 mutant containing 35S::CabuAP3 produced filament-like organs and stamenoid carpels in the third whorl.These data suggested that CabuAP3 can be partially substituted for the AP3 gene in the ap3-3 mutant A.thaliana.In this study,we isolated and identified a AG homologue,referred to as CabuAG from two genetically cognate C.bungei bearing single and double flowers.Sequence and phylogenetic analyses revealed that the gene is closest related to the eudicot AG homologues.Semi-quantitative and quantitative real time PCR analyses showed that the expression of CabuAG was restricted to stamens and carpels.CabuAG expression in the petaloid organ of the double flower was lower than that in stamens of the single flower at D8-D11 stages.At the stages of D6-D11,the level of CabuAG expression in double floral carpels were obviously higher than that in single floral carpels.Compared with the ag-1 mutant Arabidopsis,35S::CabuAG transgenic homozygous ag-1 mutant displayed flower phenotypes with a terminator of unlimited number of perianth and produced carpeloid sepals bearing ovules.These data revealed that CabuAG can be partially substituted for the AG gene in the ag-1mutant A.thaliana.To determine the lysine acetylome in the double flower,we conducted a global acetylome analysis of the double flower in C.bungei.In total,we identified 667 lysine acetylation sites in467 proteins,involving in various biological processes.Analysis of the Gene Ontology enrichment of molecular functions demonstrated that acetylated proteins were mainly associated with structural molecule activity,structural constituent of ribosome,and oxidoreductase activity.Notably,61 and 13 acetylated proteins are involved in glycometabolism and oxidative phosphorylation pathway through functional enrichment analysis,suggesting that lysine acetylation plays a critical role in the regulation of energy metabolism.Accordingly,majority of these interacting proteins were also highly enriched in the process of glucose metabolism and tricarboxylic acid cycle.These data represent the first lysine acetylome in C.bungei flower and serves as an essential basis for exploring the functions of lysine acetylation in the flower development of C.bungei.