Functional characterization of MIKC type MADS box transcription factors controlling flower development in Petunia (Petunia hybrida)”

Abstract

Petunia hybrida, with its diversity of colour and morphology is the world’s most popular bedding plant belonging to the solanaceae family which includes other agronomically important species such as S. tuberosum, L. esculentum, C. annuum and N. tabacum. The MADS box family of TFs characterized by a conserved ~58 Amino acid domain, known to plays significant role during the development of floral organs in plants. In present study, we focused on molecular and functional characterization of PhFBP9, PhFBP26 and PhFBP29 MIKC type MADS box genes in P. hybrida, to enrich our knowledge about their role in regulation of flower development and reproductive transitions. For the molecular characterization, we amplified the CDS (738 bp for each) region of PhFBP9, PhFBP26 and PhFBP29 genes from the cDNA of flower bud of P. hybrida, each encoding 245 Aa and revealed the association with other MIKC Type MADS Box genes in BLAST analysis. The protein multiple sequence alignment and conserved motif analysis of PhFBP9, PhFBP26 and PhFBP29 revealed the presence of highly conserved MADS-box and a K-box domain respectively. The expression pattern of PhFBP9, PhFBP26 and PhFBP29 genes from vegetative (leaf) and reproductive part of P. hybrida revealed that PhFBP9 expressed in reproductive part exclusively with quite variable transcripts range which may suggest their role during floral transition. The PhFBP26 gene was recorded to express in both vegetative and reproductive parts with nearly similar transcript abundance, suggesting their requirement in all over developmental processes while, the PhFBP29 transcripts were detected in leaf and all floral samples with a decreased level in petals of petunia flowers which may suggest, its role in growth and aging processes. The 3-D proteins structure modeling of petunia hybrida (PhFBP9, PhFBP26 and PhFBP29) MADS box proteins were computed by PHYRE2 showed α-helix region 43%, 46% and 42% while, β-sheets 5%, 5% and 7% respectively. The in-silico functional analysis of modelled structure of PhFBP9, PhFBP26 and PhFBP29 proteins revealed the high conservance, good alignment with other reported template, low to average mutational sensitivity and significant protein pocket in active site. Further, in-silico analysis for miRNA targets within PaMIKC genes revealed that a total of 17 PaMIKC genes were targeted by a total of 13 miRNAs out of 71 mature sequences of miRNAs. Moreover, a total of 11, 14 and 15 regulatory TFs were predicted for FBP9, FBP26 and FBP29 based on promoter scanning of respective genes. For VIGS based functional analysis, we developed and validated transgene constructs (TRV2:PhFBP9: CHS, TRV2:PhFBP26: PDS, TRV2:PhFBP29: PDS, TRV2: CHS & TRV2: PDS) in pTRV2 vector backbone using PhFBP9, PhFBP26 and PhFBP29 genes as transgene along with CHS & PDS as scorable marker for efficient symptomatic (phenotypic) validation. During experimental observations we reported the silencing of CHS & PDS genes in terms of their corresponding visible phenotypic symptoms in agro-inoculated plants with/without concern TFs. No remarkable phenotypes related to VIGS of TFs under study were observed in agro-inoculated plants which, may suggest redundant nature of analysed genes.