Whole-genome landscape of histone H3K4me3 modification during sperm cell lineage development in tomato.

BACKGROUND: During male gametogenesis of flowering plants, sperm cell lineage (microspores, generative cells, and sperm cells) differentiated from somatic cells and acquired different cell fates. Trimethylation of histone H3 on lysine 4 (H3K4me3) epigenetically contributes to this process, however, it remained unclear how H3K4me3 influences the gene expression in each cell type. Here, we conducted chromatin immunoprecipitation sequencing (ChIP-seq) to obtain a genome-wide landscape of H3K4me3 during sperm cell lineage development in tomato (Solanum lycopersicum). RESULTS: We show that H3K4me3 peaks were mainly enriched in the promoter regions, and intergenic H3K4me3 peaks expanded as sperm cell lineage differentiated from somatic cells. H3K4me3 was generally positively associated with transcript abundance and served as a better indicator of gene expression in somatic and vegetative cells, compared to sperm cell lineage. H3K4me3 was mutually exclusive with DNA methylation at 3' proximal of the transcription start sites. The microspore maintained the H3K4me3 features of somatic cells, while generative cells and sperm cells shared an almost identical H3K4me3 pattern which differed from that of the vegetative cell. After microspore division, significant loss of H3K4me3 in genes related to brassinosteroid and cytokinin signaling was observed in generative cells and vegetative cells, respectively. CONCLUSIONS: Our results suggest the asymmetric division of the microspore significantly reshapes the genome-wide distribution of H3K4me3. Selective loss of H3K4me3 in genes related to hormone signaling may contribute to functional differentiation of sperm cell lineage. This work provides new resource data for the epigenetic studies of gametogenesis in plants.

Cysteine and glycine-rich protein 2 is crucial for maintaining the malignant phenotypes of gliomas through its action on Notch signalling cascade.

Cysteine and glycine-rich protein 2 (CSRP2) is expressed differently in numerous cancers and plays a key role in carcinogenesis. However, the role of CSRP2 in glioma is unknown. This study sought to determine the expression profile and clinical significance of CSRP2 in glioma and explore its biological functions and mechanisms via lentivirus-mediated CSRP2 silencing experiments. Increased CSRP2 was frequently observed in gliomas, which was associated with clinicopathological characteristics and an unfavourable prognosis. Decreasing CSRP2 led to the suppression of malignant proliferation, metastasis and stemness in glioma cells while causing hypersensitivity to chemotherapeutic drugs. Mechanistic investigations revealed that CSRP2 plays a role in mediating the Notch signalling cascade. Silencing CSRP2 decreased the levels of Notch1, cleaved Notch1, HES1 and HEY1, suppressing the Notch signalling cascade. Reactivation of Notch markedly diminished the tumour-inhibiting effects of CSRP2 silencing on the malignant phenotypes of glioma cells. Notably, CSRP2-silencing glioma cells exhibited reduced potential in the formation of xenografts in nude mice in vivo, which was associated with an impaired Notch signalling cascade. These results showed that CSRP2 is overexpressed in glioma and has a crucial role in sustaining the malignant phenotypes of glioma, suggesting that targeting CSRP2 could be a promising strategy for glioma treatment.

The methyl-CpG-binding domain family member PEM1 is essential for Ubisch body formation and pollen exine development in rice.

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.

DNA methylation dynamics of sperm cell lineage development in tomato.

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.

miR-1297 sensitizes glioma cells to temozolomide (TMZ) treatment through targeting adrenomedullin (ADM).

BACKGROUND: Gliomas account for about 80% of all malignant brain and other central nervous system (CNS) tumors. Temozolomide (TMZ) resistance represents a major treatment hurdle. Adrenomedullin (ADM) has been reported to induce glioblastoma cell growth. METHODS: Cell viability was measured using the CCK-8 assay. The apoptosis analysis was performed using the Annexin V-FITC Apoptosis Detection Kit. The mitochondrial membrane potential was determined by JC-1 staining. A nude mouse tumor assay was used to detect tumor formation. Hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining were performed in tissue sections. Activation of Akt and Erk and expression of apoptosis-related proteins were determined by immunoblotting. RESULTS: ADM expression has been found upregulated in TMZ -resistant glioma samples based on bioinformatics and experimental analyses. Knocking down ADM in glioma cells enhanced the suppressive effects of TMZ on glioma cell viability, promotive effects on cell apoptosis, and inhibitory effects on mitochondrial membrane potential. Moreover, ADM knockdown also enhanced TMZ effects on Bax/Bcl-2, Akt phosphorylation, and Erk1/2 phosphorylation. Bioinformatics and experimental investigation indicated that miR-1297 directly targeted ADM and inhibited ADM expression. miR-1297 overexpression exerted similar effects to ADM knockdown on TMZ-treated glioma cells. More importantly, under TMZ treatment, inhibition of miR-1297 attenuated TMZ treatment on glioma cells; ADM knockdown partially attenuated the effects of miR-1297 inhibition on TMZ-treated glioma cells. CONCLUSIONS: miR-1297 sensitizes glioma cells to TMZ treatment through targeting ADM. The Bax/Bcl-2, Akt, and Erk1/2 signaling pathways, as well as mitochondrial functions might be involved.

Mobility-Aware Privacy-Preserving Mobile Crowdsourcing.

The increasing popularity of smartphones and location-based service (LBS) has brought us a new experience of mobile crowdsourcing marked by the characteristics of network-interconnection and information-sharing. However, these mobile crowdsourcing applications suffer from various inferential attacks based on mobile behavioral factors, such as location semantic, spatiotemporal correlation, etc. Unfortunately, most of the existing techniques protect the participant's location-privacy according to actual trajectories. Once the protection fails, data leakage will directly threaten the participant's location-related private information. It open the issue of participating in mobile crowdsourcing service without actual locations. In this paper, we propose a mobility-aware trajectory-prediction solution, TMarkov, for achieving privacy-preserving mobile crowdsourcing. Specifically, we introduce a time-partitioning concept into the Markov model to overcome its traditional limitations. A new transfer model is constructed to record the mobile user's time-varying behavioral patterns. Then, an unbiased estimation is conducted according to Gibbs Sampling method, because of the data incompleteness. Finally, we have the TMarkov model which characterizes the participant's dynamic mobile behaviors. With TMarkov in place, a mobility-aware spatiotemporal trajectory is predicted for the mobile user to participate in the crowdsourcing application. Extensive experiments with real-world dataset demonstrate that TMarkov well balances the trade-off between privacy preservation and data usability.

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

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.

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

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.

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

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.

Deferoxamine Mitigates Ferroptosis and Inflammation in Hippocampal Neurons After Subarachnoid Hemorrhage by Activating the Nrf2/TXNRD1 Axis.

Ferroptosis is a distinct peroxidation-driven form of cell death tightly involved in subarachnoid hemorrhage (SAH). This study delved into the mechanism of deferoxamine (DFO, an iron chelator) in SAH-induced ferroptosis and inflammation. SAH mouse models were established by endovascular perforation method and injected intraperitoneally with DFO, or intraventricularly injected with the Nrf2 pathway inhibitor ML385 before SAH, followed by detection of neurological function, blood-brain barrier (BBB) permeability, and brain water content. Apoptotic level of hippocampal neurons, symbolic changes of ferroptosis, and levels of pro-inflammatory cytokines were assessed using TUNEL staining, Western blotting, colorimetry, and ELISA. The localization and expression of nuclear factor-erythroid 2-related factor 2 (Nrf2) were detected. HT22 cells were exposed to Hemin as in vitro SAH models and treated with FIN56 to induce ferroptosis, followed by evaluation of the effects of DFO on FIN56-treated HT22 cells. The regulation of Nrf2 in thioredoxin reductase 1 (TXNRD1) was analyzed by co-immunoprecipitation and Western blotting. Moreover, HT22 cells were treated with DFO and ML385 to identify the role of DFO in the Nrf2/TXNRD1 axis. DFO extenuated brain injury, and ferroptosis and inflammation in hippocampal neurons of SAH mice. Nrf2 localized at the CA1 region of hippocampal neurons, and DFO stimulated nuclear translocation of Nrf2 protein in hippocampal neurons of SAH mice. Additionally, DFO inhibited ferroptosis and inflammatory responses in FIN56-induced HT22 cells. Nrf2 positively regulated TXNRD1 protein expression. Indeed, DFO alleviated FIN56-induced ferroptosis and inflammation via activation of the Nrf2/TXNRD1 axis. DFO alleviated neurological deficits, BBB disruption, brain edema, and brain injury in mice after SAH by inhibiting hippocampal neuron ferroptosis via the Nrf2/TXNRD1 axis. DFO ameliorates SAH-induced ferroptosis and inflammatory responses in hippocampal neurons by activating the Nrf2/TXNRD1 axis.

Cerebellar cavernous malformations with and without associated developmental venous anomalies.

BACKGROUND: The clinical profiles of cerebellar cavernous malformations (CCMs) with and without associated developmental venous anomalies (DVAs) are not well known. The aims of this study were to analyze the clinical and radiological characteristics of CCMs and to assess the various therapeutic strategies. METHODS: A consecutive series of 41 patients with identified CCMs were retrospectively reviewed. Of these, 11 patients (26.8%) were found to have associated DVAs. We compared the clinical profile of the two groups of patients (CCMs with and without DVAs). The CCMs with DVAs cases underwent radical resection of the CCMs, and the distal radicles of the DVAs that directly drain from the CCMs were coagulated and dissected at the length of the CCMs. RESULTS: There were no statistically significant differences between the two groups with regard to age, sex, location and size of lesions, multiplicity, and surgical prognosis. The patients with CCMs with DVAs did not experience any brain swelling or hemorrhagic tendency intraoperatively. The postoperative course was uneventful for all of the 36 surgical patients with the exception of two of the patients with CCMs with associated DVAs, who suffered from serious cerebellar edema, and one of these two patients underwent an emergency suboccipital decompression craniotomy. With the exception of three patients who were lost to follow-up (mean, 22.3 months), all of the CCMs patients exhibited good long-term prognosis (modified Rankin scale values of 0-2) and no reoccurrence. CONCLUSIONS: It is not rare that associated DVAs occur in CCMs. The total removal of the CCM combined with the coagulation and dissection of the distal radicles of DVA at the length of the associated CCM may result in good long-term prognosis in patients.

Sylvian fissure arteriovenous malformations: long-term prognosis and risk factors.

BACKGROUND AND OBJECTIVE: Sylvian fissure arteriovenous malformations (AVMs) are among the most challenging AVMs to manage surgically. The estimates of their risk factors and prediction of their long-term prognosis are crucial for clinical decision-making. The authors conducted a retrospective review to patients with sylvian AVMs treated microsurgically to evaluate the risk factors associated with long-term prognosis. METHODS: Forty-one patients with sylvian fissure AVMs treated microsurgically between June 2009 and December 2011 were retrospectively reviewed with a mean follow-up time of 23 months (range 6-35 months). Chi-square test was utilized to compare proportions and rank-sum test to compare ordinal materials. Odds ratios (ORs) were used to assess risk factors associated with postoperative short-term outcome and long-term adverse outcome (mRS scores 3-6). RESULTS: One patient died in 1 month after surgery. Eighteen (43.9%) patients had postoperative transient neurological deterioration. Good outcomes (mRS scores 0-2) were observed in 29 (72.5%) patients in the follow-up. From the multiple logistic regression, a history of AVM bleeding and deep venous drainage increase the risk of postoperative transient neurological deterioration, with OR = 8.211 and OR = 4.869, respectively. A history of AVM bleeding was a risk factor for long-term adverse outcome, with OR = 7. CONCLUSIONS: Despite different Sugita classification subtypes, sylvian fissure AVMs' long-term results with microsurgical resection are better than expected; a history of AVM bleeding is a risk factor for postoperative temporary neurological deterioration and for long-term adverse outcome, while the AVM deep venous drainage is a risk factor only for temporary neurological deterioration.

Endovascular intervention vs. microsurgery on the prognosis of anterior circulation blood blister-like aneurysm: A cohort study.

Background: There are no universally acknowledged standardized treatment strategies for blood blister-like aneurysms (BBAs). This study compared the prognosis of patients with BBA who underwent craniotomy microsurgery vs. endovascular intervention. Methods: This retrospective cohort study included patients with BBA treated between September 2009 and August 2020 at Sichuan Provincial People's Hospital affiliated to the Sichuan Academy of Medical Science. Patients were divided into the microsurgery and endovascular groups. The preoperative Hunt-Hess grade and modified Fisher grade were collected. The intraoperative and postoperative complications (including intraoperative aneurysm rupture and hemorrhage, postoperative cerebral hemorrhage, and BBA recurrence) were recorded. Results: Seventy-two patients were included: 28 and 44 in the microsurgery and endovascular groups, respectively. Only the preoperative Fisher grade was different between the two groups (P = 0.041). The proportion of patients with good outcomes was lower in the microsurgery group (28.6%) than in the endovascular group (72.7%), and the mortality rate was higher in the microsurgery group (32.1%) than in the endovascular group (11.4%) (P < 0.05). After adjustment for the modified Fisher grade, the multivariable analysis showed that compared with craniotomy microsurgery, an endovascular intervention was associated with the prognosis of patients with BBA (OR = 0.128, 95%CI: 0.040-0.415, P < 0.001). The rate of complications (intraoperative hemorrhage, cerebral infarction, and recurrence) was higher in the microsurgery group than in the endovascular group. Conclusion: In patients with BBA, an endovascular intervention appears to be associated with a better prognosis compared with craniotomy microsurgery.

Primary intracranial epithelioid hemangioendothelioma: a low-proliferation tumor exhibiting clinically malignant behavior.

Epithelioid hemangioendothelioma is an extremely rare intracranial tumor and is regarded as a low-proliferation tumor. We present two cases of primary intracranial epithelioid hemangioendothelioma and give an overview of the English literature pertaining to this disease. We described two new cases of primary intracranial epithelioid hemangioendothelioma and performed a search of MEDLINE (PubMed) using the words "epithelioid hemangioendothelioma". Only cases in the English language that were intracranially located and contained clinical information pertinent to the analysis were included. The tumor in case 1 originated from the right temporal bone and invaded the surrounding cranium, dura and temple muscles. The tumor in case 2 was located in the petroclival bone and had also invaded the surrounding cranium. Both tumors were well vascularized. The tumors were totally (case 1) or subtotally (case 2) removed with moderate blood loss. A total of 36 cases of intracranial epithelioid hemangioendothelioma were found in the literature. The tumor was typically diagnosed in young adults and infants. There was no sex predominance in adult patients, while in children, males were more frequently affected (M/F ratio, 3.5:1). Surgical removal was the main therapeutic protocol, and adjuvant therapy included radiotherapy or chemotherapy. Including the 2 patients presented here, a total of 38 patients were analyzed: 32 % showed local invasion, 24 % suffered a recurrence, 15 % had metastases, and the mortality rate was 15 %. Intracranial epithelioid hemangioendothelioma is a rare, low-proliferation tumor, but it exhibits some clinically malignant behaviors, such as local invasion, recurrence and metastasis. Total resection is mandatory where possible, and radiotherapy and/or chemotherapy are otherwise required. Preoperative feeding-artery embolization is recommended.

An Efficient Clearing Protocol for the Study of Seed Development in Tomato (Solanum lycopersicum 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 biology during plant reproduction. Visualization of finer embryonic structure within a tomato seed is often hampered by seed coat mucilage, multi-cell-layered integument, and a thick-walled endosperm, which needs to be resolved by laborious embedding-sectioning. A simpler alternative is to employ tissue clearing techniques that turn the seed almost transparent using chemical agents. Although conventional clearing procedures allow deep insight into smaller seeds with a thinner seed coat, clearing tomato seeds continues to be technically challenging, especially in the late developmental stages. Presented here is a rapid and labor-saving clearing protocol to observe tomato seed development from 3 to 23 days after flowering when embryonic morphology is nearly complete. This method combines chloral hydrate-based clearing solution widely used in Arabidopsis with other modifications, including the omission of Formalin-Aceto-Alcohol (FAA) fixation, the addition of sodium hypochlorite treatment of seeds, removal of the softened seed coat mucilage, and washing and vacuum treatment. This method can be applied for efficient clearing of tomato seeds at different developmental stages and is useful in full monitoring of the developmental process of mutant seeds with good spatial resolution. This clearing protocol may also be applied to deep imaging of other commercially important species in the Solanaceae.

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

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.

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

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 VCS1 (Vegetative Cell Specific 1) of rice (Oryza sativa japonica). VCS1 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 VCS1 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.

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

Plasma Membrane Preparation from Lilium davidii and Oryza sativa Mature and Germinated 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 Lilium davidii or Oryza sativa plasma membrane with high purity.

Cortical electrical stimulation promotes neuronal plasticity in the peri-ischemic cortex and contralesional anterior horn of cervical spinal cord in a rat model of focal cerebral ischemia.

PURPOSE: This study evaluated the effect of cortical electrical stimulation (CES) on function recovery, dendritic plasticity, astrogliosis, and neuron recruitment in the peri-ischemic cortex (PIC) and contralesional anterior horn of cervical spinal cord (CSC) in a rat model of focal cerebral ischemia. MATERIALS AND METHODS: Rats were pre-trained on single pellet retrieval task, then received focal ischemic lesions and electrodes implantation. Seven days after surgery, rats received CES (CES group) or no stimulation (NS group) during 18 days of training. Behavior data on stimulation days 2, 4, 6, 8, 10, 12, 14, 16 and 18 were pooled for use. Immunohistochemical investigations for microtubule-associated protein 2 (MAP-2), glial fibrillary acidic protein (GFAP) and neuronal nuclei antigen (NeuN) were performed. RESULTS: Rats in CES group showed greater functional recovery of the impaired forelimb compared to the NS group. Moreover, the functional improvement coincided with a significant increase in MAP-2-immunoreactive dendritic surface density in PIC and CSC (P=0.011; P=0.005, respectively). CES group had a significant decrease in GFAP-immunoreactive astrocytic surface density in PIC and CSC (P=0.039; P=0.013, respectively). In the immunoassaying of NeuN, there was no significant difference between the two groups in PIC and CSC (P=0.834, P=0.782, respectively). CONCLUSION: CES can promote dendritic plasticity and reduce astrogliosis in the PIC and CSC in a rat model of focal cerebral ischemia. CES is still an appealing method for post-stroke rehabilitation provided that viability of pathways is evaluated presurgically.