Combined Transcriptome Analysis Reveals the Ovule Abortion Regulatory Mechanisms in the Female Sterile Line of Pinus tabuliformis Carr

Abstract

Ovule abortion is a common phenomenon in plants that has an impact on seed production. Previous studies of ovule and female gametophyte (FG) development have mainly focused on angiosperms, especially in Arabidopsis thaliana. However, because it is difficult to acquire information about ovule development in gymnosperms, this remains unclear. Here, we investigated the transcriptomic data of natural ovule abortion mutants (female sterile line, STE) and the wild type (female fertile line, FER) of Pinus tabuliformis Carr. to evaluate the mechanism of ovule abortion during the process of free nuclear mitosis (FNM). Using single-molecule real-time (SMRT) sequencing and next-generation sequencing (NGS), 18 cDNA libraries via Illumina and two normalized libraries via PacBio, with a total of almost 400,000 reads, were obtained. Our analysis showed that the numbers of isoforms and alternative splicing (AS) patterns were significantly variable between FER and STE. The functional annotation results demonstrate that genes involved in the auxin response, energy metabolism, signal transduction, cell division, and stress response were differentially expressed in different lines. In particular, AUX/IAA, ARF2, SUS, and CYCB had significantly lower expression in STE, showing that auxin might be insufficient in STE, thus hindering nuclear division and influencing metabolism. Apoptosis in STE might also have affected the expression levels of these genes. To confirm the transcriptomic analysis results, nine pairs were confirmed by quantitative real-time PCR. Taken together, these results provide new insights into ovule abortion in gymnosperms and further reveal the regulatory mechanisms of ovule development.

Keywords: Pinus tabuliformis Carr.; alternative splicing; auxin response; energy metabolism; ovule abortion; single-molecule real-time sequencing.

Figure 1

(A) Morphologic and microscopic observations of female fertile line (FER) and female sterile line (STE) ovules in Pinus tabuliformis. Cones and scales of different development stages. (B–D) The transverse diameters of cones, scales, and ovules in FER and STE. (E) The micrographs of ovules in different development. FG: female gametophyte; FN: free nucleus; I: integument; N: nucellus; V: vacuole.

Figure 2

Flowchart of the experimental design and analysis for single-molecule real-time (SMRT) sequencing and RNA sequencing. Schematic graph of free nuclear mitosis (FNM) during ovule development in female fertile lines (FER) and sterile lines (STE) of P. tabuliformis collected for SMRT sequencing and Illumina sequencing. Combined PacBio and RNA-seq were used for a series of analyses, including alternative splicing (AS), differentially expressed gene (DEG) analysis, and simple sequence repeats (SSRs). This revealed the transcriptome differences between FER and STE.

Figure 3

(A) Summary of SMRT sequencing proportions of four kinds of the circular consensus sequence (CCS), classified into full-length chimeras reads, full-length nonchimeric reads (FLNC), non-full-length reads, and short reads. (B) DEGs in each FNM period between FER and STE; (C) DEGs with opposite expression patterns during the FNM process in STE and FER, DEGs upregulated in FER and downregulated in STE; (D) DEGs upregulated in STE and downregulated in FER; (E) Gene Ontology (GO) classification of DEGs both upregulated in FER and downregulated in STE; (F) GO classification of DEGs both upregulated in STE and downregulated in FER.

Figure 4

Heat map diagram of expression levels of DEGs, showing obvious differences in different lines. The gene expression level data were normalized to Z-score.

Figure 5

Heat map of DEGs expression levels associated with the auxin response. The gene expression level data were normalized to Z-score.

Figure 6

Comparison of different alternative splicing (AS) among FER and STE. (A) Schematic diagram of Cogent assembly; (B) visualization of seven AS modes; (C) distribution of isoform numbers in FER and STE; (D) different types of AS events in FER and STE; (E) the model graph shows AGPL1/bHLH66/TUBA generating different transcript isoforms of RI detected in two tissues. A3: alternative 3′ splice-site; A5: alternative 5′ splice-site; AF: alternative first exon; AL: alternative last exon; MX: mutually exclusive exons; RI: retained intron; SE: skipping exon.

Figure 7

Comparison of different simple sequence repeat (SSR) among FER and STE. The distribution of SSRs based on the number of repeat units in FER (A) and STE (B); frequency distribution of SSRs based on motif types in FER (C) and STE (D).

Figure 8

Candidate DEGs’ expression levels, as revealed by qRT-PCR. Sucrose synthase (SUS); peroxidase 12 (PER12); ATP-dependent 6-phosphofructokinase 2 (PFK2); clavata 1-like protein (CLV1); leucine-rich repeat receptor-like serine/threonine/tyrosine-protein kinase SOBIR1 (SOBIR1); polyubiquitin-like 10 (UBQ10); tubulin alpha chain (TUBA); ADP-glucose pyrophosphorylase large subunit 1 (AGPL1); tetraspanin-18 (TET18).

Figure 9

Speculative network model revealing the ovule abortion in a sterile female line of Pinus tabuliformis Carr. SUS: Sucrose synthase; AGPL1: ADP-glucose pyrophosphorylase large subunit 1; PFK2: ATP-dependent 6-phosphofructokinase 2; TET18: Tetraspanin-18; RTNLB8: Reticulon-like protein B8; CLV1: Clavata 1-like protein; CYCB: Cyclin B; TUBA: Tubulin alpha chain; DHFR: Dihydrofolate reductase; SOBIR1: Leucine-rich repeat receptor-like serine/threonine/tyrosine-protein kinase SOBIR1; CPI: Cysteine proteinase inhibitor; PER: Peroxidase; CALS3: Callose synthase 3; IAA: Auxin-responsive protein IAA; ARF2: Auxin response factor 2; TIR1: Transport inhibitor response 1.