RNA-seq Analysis Reveals Gene Expression Profiling of Female Fertile and Sterile Ovules of PinusTabulaeformis Carr. during Free Nuclear Mitosis of the Female Gametophyte

Abstract

The development of the female gametophyte (FG) is one of the key processes of life cycle alteration between the haploid gametophyte and the diploid sporophytes in plants and it is required for successful seed development after fertilization. It is well demonstrated that free nuclear mitosis (FNM) of FG is crucial for the development of the ovule. However, studies of the molecular mechanism of ovule and FG development focused mainly on angiosperms, such as Arabidopsis thaliana and further investigation of gymnosperms remains to be completed. Here, Illumina sequencing of six transcriptomic libraries obtained from developing and abortive ovules at different stages during free nuclear mitosis of magagametophyte (FNMM) was used to acquire transcriptome data and gene expression profiles of Pinus tabulaeformis. Six cDNA libraries generated a total of 71.0 million high-quality clean reads that aligned with 63,449 unigenes and the comparison between developing and abortive ovules identified 7174 differentially expressed genes (DEGs). From the functional annotation results, DEGs involved in the cell cycle and phytohormone regulation were highlighted to reveal their biological importance in ovule development. Furthermore, validation of DEGs from the phytohormone signal transduction pathway was performed using quantitative real-time PCR analysis, revealing the dynamics of transcriptional networks and potential key components in the regulation of FG development in P. tabulaeformis were identified. These findings provide new insights into the regulatory mechanisms of ovule development in woody gymnosperms.

Keywords: conifer; female gametophyte development; megagametogenesis; ovule abortion; transcriptome sequencing.

Figure 1

Genes expressed in the female fertile line (FL) and sterile line (SL) ovules at three developmental stages.

Figure 2

Significantly enriched GO terms in ‘Biological process’ from DEGs in comparison between fertile and sterile ovules in Pinus tabulaeformis during free nuclear mitosis. The significance of effects was determined by examining enrichment of gene ontology (GO) terms associated with differentially expressed genes (DEGs) versus the respective GO terms in the whole genome distribution using a hypogeometric test and a threshold of the Benjamini and Hochberg false discovery rate (FDR) corrected to p < 0.05. The y-axes indicate the value of average log2 (fold change) (AFC), which suggest the fold change in expression level between the FL and SL for all of the genes that belong to the respective GO term. Bars in a specific GO term represent AFC at different developmental stages. Other ontologies are shown in supporting information Table S3.

Figure 3

GO analysis for DEGs preferentially expressed in FL ovules. DEGs were grouped to the secondary classification of hierarchical GO terms.

Figure 4

Specific temporal changes of cell cycle-associated genes during FNM. For the full names and expression and annotation data of the genes, see Tables S2 and S5. Data for the gene expression level were normalized to Z-score.

Figure 5

Significant DEG expression profiles (A–D) and their GO classification (E) in female fertile ovules. Profile 1 (A) and profile 0 (B) indicates a down-regulated trend and profile 6 (C) and profile 7 (D) indicates an up-regulated trend from FNM1 to FNM3 stage. GO analysis for these unigenes was shown (E). The down-regulated unigenes are union of profile 0 and profile 1. The up-regulated unigenes are union of profile 6 and profile 7.

Figure 6

Heat map diagram of expression levels of DEGs annotated in the plant hormone signal transduction pathways analyzed by KEGG. Data for the gene expression level were normalized to Z-score.

Figure 7

Candidate unigenes expression levels revealed by qRT-PCR and RNA-seq. Data from qRT-PCR are the means of three technical replicates and bars represent SE. RPKM values from RNA-seq were generated from pooled ovule samples.

Figure 8

Coefficient analysis of fold change data between qRT-PCR and RNA-seq. 14 unigenes were selected for qRT-PCR. Data indicating the relative transcript level from qRT-PCR are the means of three replicates. Scatterplots were generated by the log2 expression ratios from RNA-seq (x-axis) and RT-qPCR (y-axis).

Figure 9

Diagram of the putative regulatory mechanisms of the FNM process. External signals are sensed by FG and may trigger the FNM process. The DEGs, which are found in comparison between developing and abortive ovules, are involved in phytohormone pathways and the cell cycle process. The phytohormone pathway contains the following genes involved in auxin biosynthesis (YUC and ASA), auxin signal transduction (AUX1, ARF, AUX/IAA and SAUR), cytokinin degradation (CKX) and signal transduction (CER1/AHK, AHP and A-ARR), ABA biosynthesis (NCED) and signal transduction (PYR/PYL and ABF). The gene expression may also regulate the cell cycle process by up- or down-regulated key genes, such as genes encoding CDKB, ICK1, APC/C and MCM proteins. The line arrow indicates direct interaction between subjects. The dot line arrow suggests the indirect influence or abbreviated multi-step between two subjects. ABF, ABA responsive element binding factor; AHK, Arabidopsis histidine kinase; AHP, Arabidopsis histidine phosphotransfer protein; ARF, auxin response factor; AUX1, AUXIN1; APC/C, anaphase-promoting complex/cyclosome; ASA, anthranilate synthase; CDK, cyclin-dependent kinase; CKX, cytokinin oxidases/dehydrogenases; CRE1, cytokinin response 1; CYC, cyclin; ICK, CDK inhibitory protein; MCM, minichrome maintenance; NCED, 9-cis-Epoxycarotenoid dioxygenase; PYL, PYR1-Like protein; PYR, pyrabactin resistance 1 protein; SAUR, small auxin-up RNA; YUC, YUCCA.