Screening of Differentially Expressed Genes and Localization Analysis of Female Gametophyte at the Free Nuclear Mitosis Stage in Pinus tabuliformis Carr.

Female sterility is a common phenomenon in the plant world, and systematic research has not been carried out in gymnosperms. In this study, the ovules of No. 28 sterile line and No. 15 fertile line Pinus tabuliformis were used as materials, and a total of 18 cDNA libraries were sequenced by the HiSeqTM 4000 platform to analyze the differentially expressed genes (DEGs) and simple sequence repeats (SSRs) between the two lines. In addition, this study further analyzed the DEGs involved in the signal transduction of plant hormones, revealing that the signal pathways related to auxin, cytokinin, and gibberellin were blocked in the sterile ovule. Additionally, real-time fluorescent quantitative PCR verified that the expression trend of DEGs related to plant hormones was consistent with the results of high-throughput sequencing. Frozen sections and fluorescence in situ hybridization (FISH) were used to study the temporal and spatial expression patterns of PtRab in the ovules of P. tabuliformis. It was found that PtRab was significantly expressed in female gametophytes and rarely expressed in the surrounding diploid tissues. This study further explained the molecular regulation mechanism of female sterility in P. tabuliformis, preliminarily mining the key factors of ovule abortion in gymnosperms at the transcriptional level.

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

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.

Integrative transcriptomics and proteomics analysis constructs a new molecular model for ovule abortion in the female-sterile line of Pinus tabuliformis Carr.

Ovule development is critical to plant reproduction and free nuclear mitosis of megagametophyte (FNMM) is vital for ovule development. However, most results of ovule development were based on the studies in angiosperms, and its molecular regulation remained largely unknown in gymnosperms, particularly, during FNMM. In this context, we studied the genome-wide difference between sterile line (SL) and fertile line (FL) ovules using transcriptomics and proteomics approaches in Pinus tabuliformis Carr. Comparative analyses revealed that genes involved in DNA replication, DNA damage repair, Cell cycle, Apoptosis and Energy metabolism were highlighted. Further results showed the low expressions of MCM 2-7, RRM1, etc. perhaps led to abnormal DNA replication and damage repair, and the significantly different expressions of PARP2, CCs1, CCs3, etc. implied that the accumulated DNA double-stranded breaks were failed to be repaired and the cell cycle was arrested at G2/M in SL ovules, potentially resulting in the occurrence of apoptosis. Moreover, the deficiency of ETF-QO might hinder FNMM. Consequently, FNMM stopped and ovule aborted in SL ovules. Our results suggested a selective regulatory mechanism led to FNMM half-stop and ovule abortion in P. tabuliformis and these insights could be exploited to investigate the molecular regulations of ovule development in woody gymnosperms.

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

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.

Differential proteomic analysis revealing the ovule abortion in the female-sterile line of Pinus tabulaeformis Carr.

Ovule abortion affects the yield and quality of Pinus tabulaeformis Carr. seeds. Research into ovule abortion has importance for improving the seed setting rate and establishing artificial seed production techniques. Fertile line (FL) ovules (FL-E) and sterile line (SL) ovules (SL-E) in the early stage of free nuclear mitosis of megagametophyte (FNMM), FL ovules (FL-L) and SL ovules (SL-L) in the late stage of FNMM of P. tabulaeformis were collected as materials. 4192 proteins were identified by isobaric tags for relative and absolute quantitation (iTRAQ)-based analysis. Bioinformatics analysis implied that in SL ovules, substances and energy might be deficient, perhaps leading to abnormal DNA replication. Because the incomplete antioxidant system and the abnormal expression levels of enzymes involved in cell signal transduction, DNA DSBs probably occurs. Facing the abnormities of DNA replication and damage, the cell cycle was arrested and the DNA damage failed to be repaired, potentially resulting in the occurrence of PCD. Taken together, an inference can be drawn from our study - substance and energy deficiencies, reactive oxygen stress, and the failure of both cell cycle progression and DNA damage repair, which possibly hinder FNMM, leading to ovule abortion in the female-sterile line of P. tabulaeformis.