Ovule Transcriptome Analysis Discloses Deregulation of Genes and Pathways in Sexual and Apomictic Limonium Species (Plumbaginaceae)

Abstract

The genus Limonium Mill. (sea lavenders) includes species with sexual and apomixis reproductive strategies, although the genes involved in these processes are unknown. To explore the mechanisms beyond these reproduction modes, transcriptome profiling of sexual, male sterile, and facultative apomictic species was carried out using ovules from different developmental stages. In total, 15,166 unigenes were found to be differentially expressed with apomictic vs. sexual reproduction, of which 4275 were uniquely annotated using an Arabidopsis thaliana database, with different regulations according to each stage and/or species compared. Gene ontology (GO) enrichment analysis indicated that genes related to tubulin, actin, the ubiquitin degradation process, reactive oxygen species scavenging, hormone signaling such as the ethylene signaling pathway and gibberellic acid-dependent signal, and transcription factors were found among differentially expressed genes (DEGs) between apomictic and sexual plants. We found that 24% of uniquely annotated DEGs were likely to be implicated in flower development, male sterility, pollen formation, pollen-stigma interactions, and pollen tube formation. The present study identifies candidate genes that are highly associated with distinct reproductive modes and sheds light on the molecular mechanisms of apomixis expression in Limonium sp.

Keywords: Illumina sequencing; MADS-box; apomixis; floral development; functional annotation; male sterility; sea lavenders; sexual reproduction; transcription factors.

Figure 1

Weighted Venn diagrams of specific and overlapping differentially expressed genes (DEGs) found in the ovules of apomictic L. multiflorum (M), facultative apomictic L. dodartii (d), and sexual L. auriculifolium (a) and L. ovalifolium (o). DEGs were filtered by |log2 fold-change (log2FC)| > 2. Number of overlapping and specific DEGs in: A. L. multiflorum in S1 relative to L. auriculifolium in S1 (M1a1; green) and to L. ovalifolium in stage S1 (M1o1; purple); B. L. multiflorum in S1 relative to L. auriculifolium in S2 (M2a2; green) and to L. ovalifolium in S2 (M1o2; purple); C. L. multiflorum in S2 relative to L. auriculifolium in S3/S4 (M2a4; green), to L. ovalifolium (M2o4; purple) and to L. dodartii in S4 (M2d4; red).

Figure 2

Distribution of differentially expressed genes related to floral development in ovules from apomictic L. multiflorum (M), sexual L. auriculifolium (a) and L. ovalifolium (o), and facultative apomictic L. dodartii (d). Differentially expressed MADS-box genes APETALA (A-class), PISTILLATA (B-class), AGAMOUS (C-class), SEPALLATA (E-class), and other MADS-box genes are represented. DEGs were found in apomictic ovules in S1 relative to sexual ovules in S1 (M1a1 and M1o1) and S2 (M1a2 and M1o2), and relative to sexual ovules in S3/S4 (M2a4 and M2o4), and facultative apomictic in S4 (M2d4).

Figure 3

Distribution of differentially expressed transcription factors potentially related to male sterility is classified into the 10 families with the highest number of differentially expressed genes (DEGs) in ovules: Apomictic L. multiflorum (M), sexual L. auriculifolium (a) and L. ovalifolium (o), and facultative apomictic L. dodartii: AP2/ERF, bHLH, bZIP, C2C2, C2H2, HB, MADS, MYB, NAC, and WRKY.

Figure 4

Over-representation analysis (ORA) performed by gProfiler of differentially expressed genes (DEGs) in ovules from apomictic L. multiflorum (M), sexual L. auriculifolium (a) and L. ovalifolium (o), and facultative apomictic L. dodartii. DEGs were filtered by |log2 fold-change (log2FC)| >2. A.s thaliana, the most similar homolog of each differentially expressed gene (DEG) was mapped to the respective functional annotation, and enriched terms were summarized using REVIGO. Significantly (FDR < 0.01), gene ontology (GO) and biological processes (BP) terms are among DEGs from (A) apomictic in S1 relative to sexual ovules in S1 (M1a1 and M1o1) or S2 (M1a2 and M1o2), and from (B) apomictic in S2 relative to sexual ovules in S3/S4 (M2a4 and M2o4), and facultative apomictic in S4 (M2d4). The dot’s size indicates the number of DEGs annotated with each term (counts), and the color shows the differential expression (red: up-regulated; blue: down-regulated). Enriched terms are grouped by their respective ancestors (ontology level 2).

Figure 5

Regulation of up- and down-regulated differentially expressed genes (DEGs) in ovules in stages S1 (1), S2 (2), and S3/S4 (4) from apomictic L. multiflorum (M), sexual L auriculifolium (a) and L. ovalifolium (o), and facultative apomictic L. dodartii (d), annotated with female-related Gene Ontology (GO) terms. DEGs represent the number of significant genes found to be differently expressed in each comparison.

Figure 6

Regulation of up- and down-regulated differentially expressed genes (DEGs) in ovules in stages S1 (1), S2 (2), and S3/S4 (4) from apomictic L. multiflorum (M), sexual L auriculifolium (a) and L. ovalifolium (o), and facultative apomictic L. dodartii (d), annotated with Gene Ontology (GO) terms related to pollen. DEGs represent the number of significant genes found to be differently expressed in each comparison.