Transcriptome analysis reveals that regulation network of the genes related to unique double flowers in tropical viviparous water lily

Abstract

Among tropical viviparous water lilies (Nymphaea L.), double-petaled cultivars are exceptionally rare, and the molecular mechanisms underlying their floral organ development remain largely unexplored. In this study, we identified Nymphaea 'Candy Rain' as the sole known tropical water lily cultivar exhibiting a double-petaled phenotype, characterized by complete petaloid conversion of innermost stamens. To elucidate the genetic basis of this unique floral morphology, we performed comparative transcriptome analysis of three distinct petal types in N. 'Candy Rain'. Our investigation further extended to comprehensive identification and characterization of MADS-box gene family members in the reference genome of the tropical viviparous water lily Nymphaea 'Paul Stetson'. Genome-wide analysis revealed 65 NcMADS-box genes unevenly distributed across 25 chromosomes. Phylogenetic and expression analyses implicated several MADS-box gene clades (particularly AGL6, AP2, and AG) in the development of the double-petaled phenotype. Additionally, our data suggest potential involvement of other transcription factor families including WOX, NAC, TCP, MYB, and bHLH in this specialized floral morphogenesis. Notably, we identified multiple phytohormone-related genes (such as IAA16, YUC6, and TCH4) that may participate in the formation of double-petaled flowers, indicating complex hormonal regulation of this trait in tropical viviparous water lilies.

Keywords: ABCE gene model; Double flower; MADS-box gene family; Stamen petaloid; Tropical viviparous water lily.

Fig. 1

Floral phenotype of N.’Candy Rain’. (A) Complete flower phenotype. (B) Three petal types: P1 (normal petal), P2 (slender petal), and P3 (stamen petaloid). Scale bar = 1 cm.

Fig. 2

Analyses of DEGs at three development stages of normal petals (P1), slender petals (P2), and petaloid stamen (P3). (A) The number of up- and down-regulated DEGs in 3 comparisons. (B) Venn diagram of DEGs in P1 vs P2, P1vs P3 and P2 vs P3. (C) KEGG pathwayenrichment analysis of DEGs for P1 vs P2, P1vs P3 and P2 vs P3.

Fig. 3

Expression heat maps of DEGs involved in the biosynthesis and signaling pathways of phytohormone as auxin, CK cytokinin, GA gibberellic acid, BR brassinolide, and JA jasmonic acid. Gene expression level (log₂FPKM) are represented by color gradation.

Fig. 4

Expression heat maps of key transcription factor genes encode DEGs regulating Unique Double Flower phenotype. The heatmap displays three petal types (P1, P2, P3), each with three biological replicates (e.g., P1-1, P1-2, P1-3 for P1). (A) AP2-like transcription factor. (B) MADS-box transcription factor. (C) WOX transcription factor. (D) NAC transcription factor. (E) TCP transcription factor. (F) MYB transcription factor. (G) bHLH transcription factor. Gene expression level (log₂FPKM) are represented by color gradation.

Fig. 5

Validation of RNA-Seq results by qRT-PCR. Error bars represent standard deviation (SD) of biological replicates. Green bars show the relative expression level(2^−∆∆CT), solid red circles represent fpkm values, red lines represent fpkm trends.

Fig. 6

The phylogenetic tree of MADS-box proteins based on the N. ‘Paul stetson’ and A. thaliana protein sequences reconstructed with the neighbour-joining (NJ) method.

Fig. 7

Chromosomal distribution of the N. ‘Paul stetson’ MADS-box genes. The chromosome number is markered at the top, and blue gene labels denote the Type I, blue gene labels denote the Type II. The chromosome length scale is located on the left.

Fig. 8

NcMADS-box conserved protein motifs and gene structure.

Fig. 9

Analysis of NcMADS-box gene family cis-acting elements (A) and transcription factor binding sites (B) in promoter.

Fig. 10

Synteny analysis of the MADS-box genes. (A) Genomic localization and gene duplication of NcMADS-box genes on N. ‘Paul stetson’ chromosomes. Gray lines indicate all syntenic blocks in the N. ‘Paul stetson’ genome; red lines indicate the interchromosomal relationships of MADS-box genes. (B) Synteny analysis of MADS-box genes between N. ‘Paul stetson’, A. thaliana and A. trichopoda species. The collinear blocks between N. ‘Paul stetson’ and other two plant genomes are shown as the gray lines in the background, while the syntenic MADS-box gene pairs are highlighted with red lines.

Fig. 11

Inference model of gene regulatory network for unique double flower phenotype of the tropical viviparous water lily.