an-efficient-clearing-protocol-for-study-seed-development-tomato

An Efficient Clearing Protocol for the Study of Seed Development in Tomato (<em>Solanum lycopersicum</em> L.)

Tomato (Solanum lycopersicum L.) is one of the major cash crops worldwide. The tomato seed is an important model for studying genetics and developmental ...

Chinese Academy of Sciences

During sexual reproduction of flowering plants, the pollen tube grows fast and over a long distance within the pistil to deliver two sperms for double fertilization. Growing plant cells need to communicate constantly with external stimuli as well as monitor changes in surface tension of the cell wall and plasma membrane to coordinate these signals and internal growth machinery; however, the underlying mechanisms remain largely unknown. Here we show that the rice member of plant-specific receptor-like kinase CrRLK1Ls subfamily, Ruptured Pollen tube (RUPO), is specifically expressed in rice pollen. RUPO localizes to the apical plasma membrane and vesicle of pollen tubes and is required for male gamete transmission. K+ levels were greater in pollen of homozygous CRISPR-knockout lines than wild-type plants, and pollen tubes burst shortly after germination. We reveal the interaction of RUPO with high-affinity potassium transporters. Phosphorylation of RUPO established and dephosphorylation abolished the interaction. These results have revealed the receptor-like kinase as a regulator of high-affinity potassium transporters via phosphorylation-dependent interaction, and demonstrated a novel receptor-like kinase signaling pathway that mediates K+ homeostasis required for pollen tube growth and integrity.

Receptor-Like Kinase RUPO Interacts with Potassium Transporters to Regulate Pollen Tube Growth and Integrity in Rice.

Sperm cell (SC) lineage development from the haploid microspore to SCs represents a unique biological process in which the microspore generates a larger vegetative cell (VC) and a smaller generative cell (GC) enclosed in the VC, then the GC further develops to functionally specified SCs in the VC for double fertilization. Understanding the mechanisms of SC lineage development remains a critical goal in plant biology. We isolated individual cells of the three cell types, and characterized the genome-wide atlas of long non-coding (lnc) RNAs and mRNAs of haploid SC lineage cells. Sperm cell lineage development involves global repression of genes for pluripotency, somatic development and metabolism following asymmetric microspore division and coordinated upregulation of GC/SC preferential genes. This process is accompanied by progressive loss of the active marks H3K4me3 and H3K9ac, and accumulation of the repressive methylation mark H3K9. The SC lineage has a higher ratio of lncRNAs to mRNAs and preferentially expresses a larger percentage of lncRNAs than does the non-SC lineage. A co-expression network showed that the largest set of lncRNAs in these nodes, with more than 100 links, are GC-preferential, and a small proportion of lncRNAs co-express with their neighboring genes. Single molecular fluorescence in situ hybridization showed that several candidate genes may be markers distinguishing the three cell types of the SC lineage. Our findings reveal the molecular programming and potential roles of lncRNAs in SC lineage development.

Transcriptomics analyses reveal the molecular roadmap and long non-coding RNA landscape of sperm cell lineage development.

Pollen adhesion and hydration are the earliest events of the pollen-stigma interactions, which allow compatible pollen to fertilize egg cells, but the underlying mechanisms are still poorly understood. Rice pollen are wind dispersed, and its pollen coat contains less abundant lipids than that of insect-pollinated plants. Here, we characterized the role of OsGL1-4, a rice member of the Glossy family, in pollen adhesion and hydration. OsGL1-4 is preferentially expressed in pollen and tapetal cells and is required for the synthesis of very long chain alkanes. osgl1-4 mutant generated apparently normal pollen but displayed excessively fast dehydration at anthesis and defective adhesion and hydration under normal condition, but the defective adhesion and hydration were rescued by high humidity. Gas chromatography-mass spectrometry analysis suggested that the humidity-sensitive male sterility of osgl1-4 was probably due to a significant reduction in C25 and C27 alkanes. These results indicate that very long chain alkanes are components of rice pollen coat and control male fertility via affecting pollen adhesion and hydration in response to environmental humidity. Moreover, we proposed that a critical point of water content in mature pollen is required for the initiation of pollen adhesion.

Deficiency of very long chain alkanes biosynthesis causes humidity-sensitive male sterility via affecting pollen adhesion and hydration in rice.

Potassium homeostasis is essential for pollen development and pollen-pistil interactions during the sexual reproduction of flowering plants. Here, we described the role of a Shaker K channel, OsAKT1.2, in rice pollen germination and growth. OsAKT1.2 is specifically expressed in the tricellular pollen, mature pollen grains and growing pollen tubes. Using CRISPR gene editing, we found that knockout lines did not differ from wildtype in vegetative growth, but showed decreased pollen germination rate both in the germination medium and in vivo. OsAKT1.2-GFP fusion protein was localized in the plasma membrane and enriched at the pollen tube tip. OsAKT1.2 could complement the yeast strain which is deficient in K intake. These findings suggest that OsAKT1.2 is associated with pollen germination and tube elongation in rice.

Pollen germination is impaired by disruption of a Shaker K channel OsAKT1.2 in rice.

During the sexual reproduction of higher plants, DNA methylation and transcription are broadly changed to reshape a microspore into two sperm cells (SCs) and a vegetative cell (VC). However, when and how the DNA methylation of SCs is established remains not fully understood. Here we investigate the DNA methylation (5 mC) dynamics of SC lineage and the VC in tomato using whole-genome bisulfite sequencing. We find the asymmetric division of the microspore gives its two daughter cells differential methylome. Compared with the generative cell (GC), the VC is hypomethylated at CG sites while hypermethylated at CHG and CHH sites, with the majority of differentially methylation regions targeted to transposable elements (TEs). SCs have a nearly identical DNA methylome to the GC, suggesting that the methylation landscape in SCs may be pre-established following the asymmetric division or inherited from the GC. The random forest classifier for predicting gene and TE expression shows that methylation within the gene body is a more powerful predictor for gene expression. Among all tested samples, gene and TE expression in the microspore may be more predictable by DNA methylation. Our results depict an intact DNA methylome landscape of SC lineage in higher plants, and reveal that the impact of DNA methylation on transcription is variant in different cell types.

DNA methylation dynamics of sperm cell lineage development in tomato.

Over 160 years ago, scientists made the first microscopic observations of angiosperm pollen. Unlike in animals, male meiosis in angiosperms produces a haploid microspore that undergoes one asymmetric division to form a vegetative cell and a generative cell. These two cells have distinct fates: the vegetative cell exits the cell cycle and elongates to form a tip-growing pollen tube; the generative cell divides once more in the pollen grain or within the growing pollen tube to form a pair of sperm cells. The concept that male germ cells are less active than the vegetative cell came from biochemical analyses and pollen structure anatomy early in the last century and is supported by the pollen transcriptome data of the last decade. However, the mechanism of how and when the transcriptional repression in male germ cells occurs is still not fully understood. In this review, we provide a brief account of the cytological and metabolic differentiation between the vegetative cell and male germ cells, with emphasis on the role of temporary callose walls, dynamic nuclear pore density, transcription repression, and histone variants. We further discuss the intercellular movement of small interfering RNA (siRNA) derived from transposable elements (TEs) and reexamine the function of TE expression in male germ cells.

Male gametophyte development in flowering plants: A story of quarantine and sacrifice.

Cell-type-specific markers are valuable tools to reveal developmental processes underlying cell differentiation during plant reproduction. Here we report the pollen vegetative cell marker gene ) of rice  was expressed specifically in late pollen and was predicted to encode a small FAF domain-containing protein of 205 amino acid residues (aa). The expression of reporter fusion proteins showed that VCS1 was exclusively targeted to the vegetative nucleus of pollen. Upon pollen germination, VCS1 lost vegetative nucleus localization, and appeared diffused in the vegetative cytoplasm of pollen grain but not in the pollen tube. T-DNA insertional mutation which disrupted the carboxyl-terminal 21 aa of VCS1 did not affect plant vegetative growth and pollen development, while destruction of  by CRISPR/Cas9 only moderately affect pollen viability. VCS1 is evolutionally conserved in monocots but appeared absent in dicotyledons. This study reveals a molecular tool for visualizing the vegetative cell of rice and possible other monocots, which has potential values in the genetic engineering of male-sterile lines.

The rice VCS1 is identified as a molecular tool to mark and visualize the vegetative cell of pollen.

Pollen germination is an excellent process to study cell polarity establishment. During this process, the tip-growing pollen tube will start elongating. The plasma membrane as the selectively permeable barrier that separates the inner and outer cell environment plays crucial roles in this process. This protocol described an efficient aqueous polymer two-phase system followed by alkaline solution washing to prepare  or  plasma membrane with high purity.

Plasma Membrane Preparation from  and  Mature and Germinated Pollen.

Pollen exine is composed of finely-organized nexine, bacula and tectum, and is crucial for pollen viability and function. Pollen exine development involves a complicated molecular network that coordinates the interaction between pollen and tapetal cells, as well as the biosynthesis, transport and assembly of sporopollenin precursors; however, our understanding of this network is very limited. Here, we report the roles of PEM1, a member of methyl-CpG-binding domain family, in rice pollen development. PEM1 expressed constitutively and, in anthers, its expression was detectable in tapetal cells and pollen. This predicted PEM1 protein of 240 kDa had multiple epigenetic-related domains. pem1 mutants exhibited abnormal Ubisch bodies, delayed exine occurrence and, finally, defective exine, including invisible bacula, amorphous and thickened nexine and tectum layer structures, and also had the phenotype of increased anther cuticle. The mutation in PEM1 did not affect the timely degradation of tapetum. Lipidomics revealed much higher wax and cutin contents in mutant anthers than in wild-type. Accordingly, this mutation up-regulated the expression of a set of genes implicated in transcriptional repression, signaling and diverse metabolic pathways. These results indicate that PEM1 mediates Ubisch body formation and pollen exine development mainly by negatively modulating the expression of genes. Thus, the PEM1-mediated molecular network represents a route for insights into mechanisms underlying pollen development. PEM1 may be a master regulator of pollen exine development.

Article	Year
Receptor-Like Kinase RUPO Interacts with Potassium Transporters to Regulate Pollen Tube Growth and Integrity in Rice. PLoS genetics\| PubMed ID: 27447945	2016
Transcriptomics analyses reveal the molecular roadmap and long non-coding RNA landscape of sperm cell lineage development. The Plant journal : for cell and molecular biology\| PubMed ID: 30047180	2018
Deficiency of very long chain alkanes biosynthesis causes humidity-sensitive male sterility via affecting pollen adhesion and hydration in rice. Plant, cell & environment\| PubMed ID: 31380565	2019
Pollen germination is impaired by disruption of a Shaker K channel OsAKT1.2 in rice. Journal of plant physiology\| PubMed ID: 32114250	2020
DNA methylation dynamics of sperm cell lineage development in tomato. The Plant journal : for cell and molecular biology\| PubMed ID: 33249677	2021
Male gametophyte development in flowering plants: A story of quarantine and sacrifice. Journal of plant physiology\| PubMed ID: 33548696	2021
The rice VCS1 is identified as a molecular tool to mark and visualize the vegetative cell of pollen. Plant signaling & behavior\| PubMed ID: 33982648	2021
Plasma Membrane Preparation from and Mature and Germinated Pollen. Bio-protocol\| PubMed ID: 34541066	2017
The methyl-CpG-binding domain family member PEM1 is essential for Ubisch body formation and pollen exine development in rice. The Plant journal : for cell and molecular biology\| PubMed ID: 35765221	2022

Lingtong Liu

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