Rafflesia possesses unique biological features and known primarily for producing the world’s largest and existing as a single flower. However, to date, little is known about key regulators participating in Rafflesia flower development. In order to further understand the molecular mechanism that regulates Rafflesia cantleyi flower development, RNA-seq data from three developmental stages of floral bud, representing the floral organ primordia initiation, floral organ differentiation, and floral bud outgrowth, were analysed. A total of 89,890 transcripts were assembled of which up to 35% could be annotated based on homology search. Advanced transcriptome analysis using K-mean clustering on the differentially expressed genes (DEGs) was able to identify 12 expression clusters that reflect major trends and key transitional states, which correlate to specific developmental stages. Through this, comparative gene expression analysis of different floral bud stages identified various transcription factors related to flower development. The members of WRKY, NAC, bHLH, and MYB families are the most represented among the DEGs, suggesting their important function in flower development. Furthermore, pathway enrichment analysis also revealed DEGs that are involved in various phytohormone signal transduction events such as auxin and auxin transport, cytokinin and gibberellin biosynthesis. Results of this study imply that transcription factors and phytohormone signalling pathways play major role in Rafflesia floral bud development. This study provides an invaluable resource for molecular studies of the flower development process in Rafflesia and other plant species.

Rafflesia is a unique plant species existing as a single flower and produces the largest flower in the world. While Rafflesia buds take up to 21 months to develop, its flowers bloom and wither within about a week. In this study, transcriptome analysis was carried out to shed light on the molecular mechanism of senescence in Rafflesia. A total of 53.3 million high quality reads were obtained from two Rafflesia cantleyi flower developmental stages and assembled to generate 64,152 unigenes. Analysis of this dataset showed that 5,166 unigenes were differentially expressed, in which 1,073 unigenes were identified as genes involved in flower senescence. Results revealed that as the flowers progress to senescence, more genes related to flower senescence were significantly over-represented compared to those related to plant growth and development. Senescence of the R. cantleyi flower activates senescence-associated genes in the transcription activity (members of the transcription factor families MYB, bHLH, NAC, and WRKY), nutrient remobilization (autophagy-related protein and transporter genes), and redox regulation (CATALASE). Most of the senescence-related genes were found to be differentially regulated, perhaps for the fine-tuning of various responses in the senescing R. cantleyi flower. Additionally, pathway analysis showed the activation of genes such as ETHYLENE RECEPTOR, ETHYLENE-INSENSITIVE 2, ETHYLENE-INSENSITIVE 3, and ETHYLENE-RESPONSIVE TRANSCRIPTION FACTOR, indicating the possible involvement of the ethylene hormone response pathway in the regulation of R. cantleyi senescence. Our results provide a model of the molecular mechanism underlying R. cantleyi flower senescence, and contribute essential information towards further understanding the biology of the Rafflesiaceae family.

Tetrastigma rafflesiae (Miq.) Planch. is a climbing plant species that is known for its unique relationship with holoparasitic plants of Rafflesiaceae. Knowledge on the mitochondrial genes of this species may contribute towards the development of molecular approaches for species identification. This study aims to identify and characterise genes from the T. rafflesiae mitochondrial genome (mitogenome) and assess their potential to discriminate different Tetrastigma species. Mitochondrial-specific sequences were first selected by mapping T. rafflesiae whole-genome sequences to mitogenomes from several reference plant species. De novo assembly of these selected sequences produced a T. rafflesiae mitogenome with a size of 336 kb. Gene annotation revealed that the T. rafflesiae mitogenome contains at least 40 protein coding genes, 20 tRNAs and two rRNAs. Phylogenetic analysis using several mitochondrial genes, namely ccmB, cob, matR, nad6 and rps3 was able to differentiate T. rafflesiae from three other Tetrastigma species, indicating the potential of these genes as species-specific sequence markers. These findings supplement additional genetic information on T. rafflesiae and may aid in the effort of species classification and conservation.

Rafflesia terkenal sebagai tumbuhan yang menghasilkan bunga tunggal yang terbesar di dunia. Namun, ia semakin jarang ditemui dan ialah spesies dalam bahaya. Sistem pengelasan spesies Rafflesia ialah komponen penting dalam usaha pemuliharaan lazimnya bergantung kepada pencirian morfologi bunga. Walau bagaimanapun, pendekatan molekul, termasuk yang berasaskan kepada jujukan genom mitokondria (mtDNA), berupaya menyediakan kaedah pengelasan yang lebih berkesan. Untuk meneroka kemungkinan ini, jujukan mtDNA empat spesies Rafflesia di Semenanjung Malaysia, iaitu R. cantleyi, R. azlanii, R. kerrii dan R. sharifah-hapsahiae telah dihimpun dan dicirikan dalam kajian ini. Bacaan jujukan mtDNA untuk setiap spesies kajian pada mulanya telah ditentukan masing-masing daripada set data genom keseluruhan menggunakan pendekatan pemetaan berbantukan rujukan. Proses penghimpunan secara de novo dan perancahan kemudiannya telah dijalankan ke atas bacaan jujukan yang telah dikenal pasti untuk menghasilkan jujukan mtDNA bagi R. cantleyi (441,992 pb), R. azlanii (472,723 pb), R. kerrii (500,932 pb) dan R. sharifah-hapsahiae (453,747 pb). Seterusnya, anotasi mtDNA bagi setiap spesies telah mengenal pasti sekurang-kurangnya 31 gen pengekodan protein, enam gen tRNA dan tiga rRNA. Perbandingan gen mitokondria mendapati bahawa beberapa gen seperti cob, rpl10, mttB dan ccmB mempamerkan orientasi yang berbeza dalam spesies Rafflesia yang tertentu manakala analisis penjajaran jujukan berganda menunjukkan jujukan gen nad1 adalah berbeza antara keempat-empat spesies Rafflesia yang dikaji. Analisis filogenetik dengan menggunakan jujukan bagi tujuh gen pengekodan protein yang terpelihara berupaya membezakan spesies Rafflesia yang dikaji. Kesimpulannya, hasil pencirian jujukan mtDNA menunjukkan bahawa jujukan gen mitokondria yang khusus berupaya membezakan spesies Rafflesia yang dikaji dan berpotensi untuk digunakan bagi tujuan pengenalpastian serta pengelasan spesies Rafflesia dalam usaha pemuliharaan organisma yang unik ini.