Identification of Proteoforms Related to Nelumbo nucifera Flower Petaloid Through Proteogenomic Strategy

Abstract

Nelumbo nucifera is an aquatic plant with a high ornamental value due to its flower. Despite the release of several versions of the lotus genome, its annotation remains inefficient, which makes it difficult to obtain a more comprehensive knowledge when -omic studies are applied to understand the different biological processes. Focusing on the petaloid of the lotus flower, we conducted a comparative proteomic analysis among five major floral organs. The proteogenomic strategy was applied to analyze the mass spectrometry data in order to dig out novel proteoforms that are involved in the petaloids of the lotus flower. The results revealed that a total of 4863 proteins corresponding to novel genes were identified, with 227 containing single amino acid variants (SAAVs), and 72 originating from alternative splicing (AS) genes. In addition, a range of post-translational modifications (PTMs) events were also identified in lotus. Through functional annotation and homology analysis with 24 closely related plant species, we identified five candidate proteins associated with floral organ development, which were not identified by ordinary proteomic analysis. This study not only provides new insights into understanding the mechanism of petaloids in lotus but is also helpful in identifying new proteoforms to improve the annotation of the lotus genome.

Keywords: Nelumbo nucifera; new proteoforms; petaloidy; proteogenomics.

Figure 1

The floral organs of the sacred lotus ‘Sleeping Beauty’. P, petal; Sp, stamen petaoidy; St, stamen; C, carpel; and Cp, carpel petaloidy. The bar indicates 1 cm.

Figure 2

Summary of differentially expressed proteins (DEPs). (A) The unique and overlapped DEPs in the comparisons of P vs. Sp, St vs. P, and St vs. Sp; C vs. P, C vs. Cp, and P vs. Cp. (B) The number of up-regulated and down-regulated DEPs in each comparison.

Figure 3

Relationship between transcriptomic and proteomic data. (A) Scatterplots depicting the correlation of the relationship between proteomic and transcriptomic datasets. (B) Scatterplots and correlation coefficients illustrate the relationship between DEPs and DEGs. Scatterplots and correlation coefficients show the relationship between protein and transcript expression ratios with either similar (C) or opposite (D) changing trends.

Figure 4

Diagram of the results in GAPE tool identification. (A) Overview of the identifications from the present study. (B) Total number and overlap of identified gene loci in novel genes, tissue-specific novel genes, conserved novel genes, and conserved novel tissue-specific genes.

Figure 5

Discovery of novel genes, novel alternative splicing events, and SAAVs. (A) Novel peptides located in intergenic regions. Two peptides were pinpointed within a region on scaffold NW_010729074_1 of the N. nucifera genome, devoid annotation of genes. (B) Detection of novel exons on scaffold NW_010729085_1. A splicing peptide was identified in an intronic region in a new locus. Examination of the transcripts also indicates the presence of a spliced variant for this locus. (C) Detection of SNPs on Scaffold NW_010729076_1. An existing gene was unraveled through the identification of one single SNP-containing peptide as well as RNA-seq data. (D–F) Validation of three novel peptides (comprising a novel gene peptide, a novel splice junction peptide, and a novel SNP-containing peptide) by comparing the MS spectra of the identified peptides from proteogenomic analysis. The MS spectra of the novel peptides (HKNTKKNAK [D], MSSLNAEQNDNICCYSPMDK [E], and TFTLIIFQPFK [F]) are shown.

Figure 6

Overview of proteogenomic analysis. (A) Bar chart showing the length of all identified proteins in proteogenomic analyses. (B) Bar chart showing protein sequence coverage. (C) Bar chart showing the GC content of novel proteins via proteogenomic approach. (D) Bar chart showing the distribution of translation start codon of the coding genes of identified novel protein via proteogenomic approach.

Figure 7

Summary of post-translational modification of identified proteins. (A) The number of conserved novel genes in lotus compared to other plants. (B) The identified novel genes annotated to involve in GO biological process, molecular function, and cellular localization terms. (C) The identified novel genes were classified by COG function.

Figure 8

Conserved domain analysis of novel proteins associated with flower organ development. (A) Analysis of conserved domains of three novel tissue-specific genes in Cp. (B) Analysis of conserved domains of two novel tissue-specific genes in St.