Patterning Of The Chrysanthemum Inflorescence:Roles Of B-Class And CYC2 Subclade Genes

Flowers constitute one of the most important parts of an ornamental plant.Floral organ identity in the model species Arabidopsis and Antirrhinum follow the classic ABCE model where combinatorial activities of these genes in higher order fashion effect organ identity.In chrysanthemum,unlike the model species whose flowers are simple and solitary,the capitulum is composed of hundreds of tightly packed marginal ray and central disc florets.The chrysanthemum cultivars are classified majorly by the number of rows of ray florets.In addition to this,the disc flowers morphologies contribute to classification.Several cultivars including the double ray flowered cultivar,radiated cultivar,anemone cultivar and nonanemone cultivar are widely cultivated.However,the molecular basis for variations of the inflorescence morphology remains scanty.In order to understand the regulatory mechanism that govern flower and inflorescence patterning in chrysanthemum,an exploratory study aimed at investigating whether B class and CYC2 subclade genes are involved in the patterning of inflorescence of the chrysanthemum cultivars was undertaken.In the second part of this study,we cloned and functionally characterized a chrysanthemum B class gene,CDM19.Here,are the main findings and summary of the study.1.The expression of B and CYC2 subclade genes were analyzed in cultivars bearing elongated and showy disc flowers(anemone type)against those with short and often less conspicuous disc flowers(non-anemone type).Results showed that CmCYC2d and CmCYC2f had significantly higher expression in the disc florets of anemone type compared to that of non-anemone type.The expression of the B class genes showed inconsistencies in their expression.2.Additionally,cultivars with very few disc flowers(double ray flowered cultivars),were compared with cultivars possessing large numbers of disc flowers(radiate cultivars)for the expression of both B and CYC2 subclade genes.Here,the expression of B class genes CDM19,CmAP3L1.1 and CDM86 was low in disc flowers of double ray flowered cultivars compared with radiate cultivars.Thus,B class genes are likely involved in regulation of florets number and inflorescence morphology.The relative expression of CYC2 subclade genes was irregular and therefore they could not be implicated as important factors that regulate floret numbers in chrysanthemums.3.Chrysanthcmum B class gene,CDM19 was cloned and functionally characterized.CDM19 sequence has an open reading frame(ORF)containing 696bp predicted to encode a 232 amino acids product.CDM19 does not have transcription activation ability and its transient expression in onion epidermal cells showed that it is expressed throughout the cell.Sequence and phylogenetic analyses revealed that CDM19 belongs to the DEF/AP3 lineage possessing the characteristic MIKC-type ? structure.Expression analysis showed that CDM19 was transcribed in ray petals and disc petals and stamens with weak expression in the carpels.4.Ectopic expression of CDM19 in Arabidopsis wild-type background altered carpel development resulting in multi-carpel siliques.CDM19 could only partially rescue the ap33 mutant.Constitutive expression in 'Jinba' generated flowers with homeotic replacement of third whorl stamens with petaloid organs.The expression of AGAMOUS-like gene,CDM37,was not significantly affected indicating that changes in CDM19 transcripts were responsible for the homeotic changes.Morphological changes of disc florets in the 35S::CDM19 overexpression lines resulted in shifts in flower symmetry.5.Analysis of transcripts abundance of known flower symmetry genes,CYC2 subclade genes revealed high expression of CmCYC2c and CmCYC2d indicating that CDM19 may positively regulate these flower symmetry genes thus causing symmetry shifts in chrysanthemum florets.At the inflorescence level,the marginal ray flowers of the 35S::CDM19 were incurving unlike the outcurving WT ray flowers and the number of disc florets was decreased.The inflorescences of 35S::CDM19 plants more compact compared to the WT.Taken together,the results suggest that CDM19 is involved in a novel function in carpel development in addition to specifying the identities of petals and stamens.Besides,CDM19 can positively regulate the expression of CYC2c and CYC2d genes which are involved in flower symmetry.These results provide foundational data on which further studies can be built on with the purpose of understanding the diversities observed in the chrysanthemum cultivars.