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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.

SUBMITTER: Yao Y

PROVIDER: S-EPMC6122031 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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

Yao Yang Y Han Rui R Gong Zaixin Z Zheng Caixia C Zhao Yuanyuan Y

International journal of molecular sciences 20180801 8

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 ...[more]

PMID: 30071597

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Project description:During plant sexual reproduction, F-actin takes part in the elongation of the pollen tube and the movement of sperm cells along with it. Moreover, F-actin is involved in the transport of sperm cells throughout the embryo sac when double fertilization occurs. Different techniques for analysis of F-actin in plant cells have been developed: from classical actin-immunolocalization in fixed tissues to genetically tagged actin with fluorescent proteins for live imaging of cells. Despite the implementation of live cell imaging tools, fixed plant tissue methods for cytoskeletal studies remain an essential tool for genetically intractable systems. Also, most of the work on live imaging of the cytoskeleton has been conducted on cells located on the plant's surface, such as epidermal cells, trichomes, and root hairs. In cells situated in the plant's interior, especially those from plant species with thicker organ systems, it is necessary to utilize conventional sectioning and permeabilization methods to allow the label access to the cytoskeleton. Studies about the role of F-actin cytoskeleton during double fertilization in plants with crassinucellate ovules (e.g., Agave, Yucca, Polianthes, Prochnyantes, and Manfreda) remain scarce due to the difficulties to access the female gametophyte. Here, we have developed a straightforward method for analysis of F-actin in the female gametophyte of different Agavoideae sub-family species. The procedure includes the fixation of whole ovules with formaldehyde, followed by membrane permeabilization with cold acetone, a prolonged staining step with rhodamine-phalloidin, and Hoechst 33342 as a counterstain and two final steps of dehydration of samples in increasing-concentration series of cold isopropanol and clarification of tissues with methyl salicylate. This technique allows the analysis of a large number of samples in a short period, cell positioning relative to neighbor cells is maintained, and, with the help of a confocal microscope, reconstruction of a single 3D image of F-actin structures into the embryo sac can be obtained.

Project description:Aposporous apomictic plants form clonal maternal seeds by inducing the emergence of non-reduced (2n) embryo sacs in the ovule nucellus and the development of embryos by parthenogenesis. In previous work, we reported a plant-specific TRIMETHYLGUANOSINE SYNTHASE 1 (TGS1) gene (PN_TGS1-like) showing expression levels positively correlated with sexuality rates in facultative apomictic Paspalum notatum. PN_ TGS1-like displayed contrasting in situ hybridization patterns in apomictic and sexual plant ovules from premeiosis to anthesis. Here we transformed sexual P. notatum with a TGS1-like antisense construction under a constitutive promoter, in order to produce lines with reduced transcript representation. Antisense plants developed prominent trichomes on the adaxial leaf surface, a trait absent from control genotypes. Reproductive development analysis revealed occasional formation of twin ovules. While control individuals typically displayed a single meiotic embryo sac per ovule, antisense lines showed 12.93-15.79% of ovules bearing extra nuclei, which can be assigned to aposporous-like embryo sacs (AES-like) or, alternatively, to gametophytes with a misguided cell fate development. Moreover, around 8.42-9.52% of ovules showed what looked like a combination of meiotic and aposporous-like sacs. Besides, 32.5% of ovules at early developmental stages displayed nucellar cells with prominent nuclei resembling apospory initials (AIs), which surrounded the megaspore mother cell (MMC) or the MMC-derived meiotic products. Two or more concurrent meiosis events were never detected, which suggest a non-reduced nature for the extra nuclei observed in the mature ovules, unless they were generated by proliferation and misguided differentiation of the legitimate meiotic products. The antisense lines produced a similar amount of viable even-sized pollen with respect to control genotypes, and formed an equivalent full seed set (∼9% of total seeds) after self-pollination. Flow cytometry analyses of caryopses derived from antisense lines revealed that all full seeds had originated from meiotic embryo sacs (i.e. by sexuality). A reduction of 25.55% in the germination percentage was detected when comparing antisense lines with controls. Our results indicate that PN_ TGS1-like influences ovule, gametophyte and possibly embryo development.

Dataset Information

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

Publications

RNA-seq Analysis Reveals Gene Expression Profiling of Female Fertile and Sterile Ovules of <i>Pinus</i><i>Tabulaeformis</i> Carr. during Free Nuclear Mitosis of the Female Gametophyte.

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