Monodehydroascorbate reductase (MDAR or MDHAR), which is responsible for growth, development and stress response in plants, is a key enzyme in the maintenance of the ascorbate acid (AsA) pool through the AsA–glutathione (AsA–GSH) cycle. High temperature affects a broad spectrum of cellular components and metabolism including AsA–GSH cycle in plants. In rice, however, the detailed roles of OsMDHAR4 in resistance against heat stress remains unclear.
Here, we report that OsMDHAR4 protein was localized to the chloroplasts. OsMDHAR4 expression was detected in all tissues surveyed and peaked in leaf blade. OsMDHAR4 was responsive to multiple stresses and was relatively strongly induced by heat treatment. In comparison with wild type, the osmdhar4 mutant exhibited improved tolerance to heat stress, whereas OsMDHAR4 overexpression lines exhibited enhanced sensitivity to heat stress. Moreover, we found that suppression of OsMDHAR4 promoted stomatal closure and hydrogen peroxide accumulation, and overexpression of OsMDHAR4 increased stomatal opening and decreased hydrogen peroxide content in rice leaves.
Taken together, these results indicated that OsMDHAR4 negatively regulates tolerance to heat stress by mediating H2O2-induced stomatal closure in rice.
Extreme temperature is one of the serious threats affecting crop production and distribution worldwide (Yokotani et al., 2008). In plants, a transient increase of 10–15 °C above ambient, is generally considered as heat shock or heat stress, which negatively affects plant growth, seed germination, photosynthesis, respiration, water relation, and membrane stability in plants (Wahid et al., 2007), and is often accompanied by the generation of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), hydroxyl radical, superoxide anion radicals, and singlet oxygen (Liu and Huang, 2000; Mittler, 2002; Apel and Hirt, 2004; Song et al., 2014). The understanding of plant responses to heat stress in physiology, genetics, and molecular biology will be greatly helpful in improving the heat tolerance of plants through genetic engineering.
Compared with other ROS, H2O2 is more stable, more diffusive, and have a long half-life (approximately 1 ms) and high permeability across membranes (Levine et al., 1994), so it can readily escape from the organelle where it was produced to the cytosol. Previous research has shown that H2O2 plays a dual role in plants: at low concentrations, it acts as a secondary messenger involved in triggering tolerance to various biotic and abiotic stresses; but at high concentrations, it leads to programmed cell death (Quan et al., 2008). H2O2 also acts as a key regulator in a broad range of physiological processes, such as photorespiration and photosynthesis (Noctor and Foyer, 1998), growth and development (Foreman et al., 2003), the cell cycle (Mittler et al., 2004), senescence (Peng et al., 2005), and stomatal movement (Bright et al., 2006).
Stomatal pores that are located in the epidermis of plant leaves control the uptake of CO2 for photosynthesis and the water loss during transpiration, and play a crucial role in abiotic stress tolerance (Schroeder et al., 2001; Hetherington and Woodward, 2003). In the process of ABA-dependent stomatal closure, H2O2 plays a vital role as a signal molecule by elevating calcium levels in guard cells through the activation of plasma membrane calcium channels (Pei et al., 2000; Wang and Song, 2008). OsHTAS, a RING finger ubiquitin E3 ligase, functions in rice heat tolerance through H2O2-induced stomatal closure and is mainly involved in the ABA-dependent pathway (Liu et al., 2016). The ABA-activated SnRK2 protein kinase OPEN STOMATA1 phosphorylates NADP (NADPH) oxidase (AtrbohF), which functions to produce ABA-induced ROS in guard cells (Sirichandra et al., 2009). There's another way of ABA-independent stomatal closure. DROUGHT AND SALT TOLERANCE (DST), a zinc finger transcription factor, negatively regulates H2O2-induced stomatal closure by directly regulating the expression of genes related to H2O2 scavenging (Huang et al., 2009). OsSRO1c suppresses DST to positively regulate H2O2-induced stomatal closure (You et al., 2013).
ROS is normally scavenged by an enzymatic anti-oxidative system containing catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GPX) and superoxide dismutase (SOD), and by a non-enzymatic anti-oxidative system including ascorbic acid (AsA), glutathione (GSH), tocopherols (TOCs) and phenolic compounds to protect plant cells. The monodehydroascorbate reductase (MDAR or MDHAR), well-known as flavin adenine dinucleotide (FAD) enzyme, is involved in the ascorbate–glutathione cycle, and plays an important role in directly reducing monodehydroascorbate (oxidized ascorbate) to ascorbate using NAD(P)H as an electron donor (Apel and Hirt 2004). MDARs were found in many eukaryotes, including cucumbers, potatoes, soybean root nodules, and rot fungus (Gill and Tuteja, 2010), and were localized in chloroplasts, mitochondria, peroxisomes, and the cytosol in plants (Omoto et al., 2013), and in microsomes, mitochondria, the Golgi apparatus, and erythrocytes in animals (Sakihama et al., 2000).
The roles of MDAR have been extensively reported under abiotic and biotic stress. Overexpression of AtMDAR1 in tobacco confers enhanced tolerance to ozone, salt and polyethylene glycol stresses (Eltayeb et al., 2007). Overexpression of LeMDAR from tomato (Lycopersicon esculentum Mill.) enhanced tolerance to temperature and methylviologen-mediated oxidative stresses (Li et al., 2010). OsMDHAR-expressing yeast cells displayed enhanced tolerance to H2O2 (Kim et al., 2016). Silencing OsMDHAR3 gene increased salt sensitivity in rice (Kim et al., 2017). Knockdown of TaMDHAR4 and TaMDAR6 through virus-induced gene silencing (VIGS) enhanced the wheat resistance to Puccinia striiformis f. sp. tritici (Pst) (Feng et al., 2014; Abou-Attia et al., 2016). However, the roles of rice MDAR in heat stress response remain unclear.
In this study, a new gene encoding the chloroplastic MDHAR (OsMDHAR4) from the rice plant was cloned, and its function was analyzed. The osmdhar4 mutant displayed significantly improved heat tolerance with increased stomatal closure and reduced water loss by promoting H2O2 accumulation. OsMDHAR4-overexpressing transgenic plants showed obviously enhanced sensitivity to heat stress with decreased stomatal closure and accelerated water loss speed. Our results suggest that the OsMDHAR4 gene negatively regulates resistance to heat stress in rice.
The coding sequence (CDS) of OsMDHAR4 gene spans 1431 bp and encodes a protein of 476 amino acids and has a predicted molecular weight of 51.86 kDa. The prediction of protein domains using NCBI and InterProScan databases indicated that OsMDHAR4 protein contains pyridine nucleotide-disulfide oxidoreductase domains (Pyr_redox_2 and NAD(P)-binding domain) and a FAD/NAD-linked reductase, dimerisation domain. The multiple amino acid sequence alignment of OsMDHAR4 and other plant MDHARs was performed using the BLAST tool at the NCBI database. OsMDHAR4 showed 87, 88, 86, 43, 69, 43, 70 and 53% identity and 92, 93, 93, 61, 80, 60, 82 and 69% similarity to ZmMDAR (Zea mays), BdMDAR (Brachypodium distachyon), TaMDHAR4, TaMDAR6 (Triticum aestivum), AtMDAR4, AtMDAR6 (Arabidopsis thaliana), GmMDAR (Glycine max), and SlMDAR (Solanum lycopersicum), respectively (Additional file 1: Figure S1).
To study the subcellular localization of OsMDHAR4 protein, we fused the CDS of OsMDHAR4 to the N-terminal of yellow fluorescent protein (YFP) driven by the cauliflower mosaic virus 35S promoter. The empty and recombinant vectors were transiently transformed into the epidermis of Nicotiana benthamiana by agroinfiltration. Fluorescence microscopic analysis revealed that OsMDHAR4-YFP was mainly detected in chloroplast, partially colocalized with the red autofluorescence of chlorophyll. On the contrary, 35S-YFP was widely distributed in the nucleus and cytoplasm and was not colocalized with the red autofluorescence of chloroplasts (Fig. 1). This result indicated that OsMDHAR4 protein was localized to the chloroplasts.
A total of 6 representative tissues, including root, shoot, stem, sheath and blade of flag leaf, and panicle, were prepared for spatio-temporal expression analysis of OsMDHAR4 in rice Zhonghua11 (ZH11). The mRNA abundance of OsMDHAR4 was detected using quantitative RT-PCR (qRT-PCR). OsMDHAR4 transcripts were detected in all of the tissues surveyed, and the expression level was relatively lowest in root and peaked in the blade of the flag leaf (Fig. 2a).
To investigate the physiological and functional relevance of the OsMDHAR4 gene, we further examined the expression level of OsMDHAR4 under heat stress and other treatments, including polyethylene glycol (PEG), abscisic acid (ABA), H2O2, salt and cold, at the seedling stage. It was found that, after heat treatment, the OsMDHAR4 transcripts increased rapidly at 0.5 h (1.8-fold), after which a slight decrease occurred at 3 h, and then they continued to rise and peaked at 24 h (2.8-fold). Under the PEG treatment, the transcript abundance of OsMDHAR4 was nearly unchanged at most time points except for 3 h and 24 h, where two induced peaks (1.7 and 2.2-fold) appeared. In the ABA treatment, OsMDHAR4 was induced quickly at 0.5 h and 3 h, after which a dramatic decrease appeared at 6 h and 12 h, and recovered to initial level until 24 h. When treated with H2O2, the OsMDHAR4 expression showed a significant trend of rising followed by falling. The OsMDHAR4 transcript level was slightly suppressed by salt and cold treatments at 6 h and 12 h (Fig. 2b). These results demonstrated that OsMDHAR4 is responsive to multiple stresses.
As shown in Fig. 2b, the expression of OsMDHAR4 was significantly induced by heat treatment, and the positive roles of MDHAR have been extensively reported under abiotic stress such as ozone, salt, PEG (Sharma and Davis 1997; Eltayeb et al.,2007) and drought (Sharma and Dubey 2005). However, the role of the MDHAR gene in rice remains largely unknown. To evaluate the function of OsMDHAR4 in heat stress response, we firstly searched for the Rice Mutant Database (http://rmd.ncpgr.cn/). Then, the osmdhar4 mutant (RMD_03Z11DA36), a transfer DNA (T-DNA) insertion line in the japonica rice ZH11 background was obtained (Wu et al., 2003; Zhang et al., 2006). DNA sequencing and genotyping revealed that the T-DNA was inserted in the first intron of the OsMDHAR4 gene, 254 bp downstream of the translation initiation site (Fig. 3a and Additional file 2: Figure S2). A qRT-PCR assay showed that OsMDHAR4 was expressed at a very low level in the osmdhar4 plants (Fig. 3b). Under normal growth conditions, the osmdhar4 mutant had no obvious differences compared with the wild type (WT, ZH11) (Fig. 3c, left). osmdhar4 and WT seedlings (5.5- to 6.5-leaf stage) were treated at high temperature (45 °C) for 72 h and subsequently returned to 26 °C for recovery. Survival rates were used as a measure of heat tolerance. After the heat treatment and recovery, 80.5% of the osmdhar4 seedlings survived compared with 45.6% of the WT seedlings (Fig. 3c, d). Subsequently, we measured the water loss rates of detached leaves from osmdhar4 and WT. The results showed that the detached leaves of osmdhar4 lost water more slowly than the WT leaves (Fig. 3e). This decreased water loss of the osmdhar4 mutant might lead to an increased tolerance to high temperature.
To further validate the functions of OsMDHAR4 in rice, we constructed an overexpression vector of OsMDHAR4 driven by 35S promoter. The OsMDHAR4-overexpressing vector was transformed into rice ZH11. PCR analysis using OsMDHAR4-specific primers (OsMDHAR4-OE-F and R) and vector-specific primers (Hyg-F and R) confirmed the presence of the transgene in the two independent overexpression lines OsMDHAR4-OE-1 and OsMDHAR4-OE-5 (Additional file 3: Figure S3). Both the two transgenic lines, in which the OsMDHAR4 expression level was significantly increased approximately triple and twice of that in WT (Fig. 4a), were selected for heat tolerance testing (45 °C for 48 h at the 5.5- to 6.5-leaf stage). Under normal conditions, we did not observe any phenotypic differences between these two overexpression lines and the WT (Fig. 4b, top). Under the heat stress treatment, both OsMDHAR4-OE-1 and OE-5 were more sensitive than the WT, which was consistent with the behavior of the osmdhar4 mutant in heat treatment. The survival rates ranged from 46.9 to 60.0% for the OsMDHAR4-overexpressing lines, and 90.0 to 90.6% for the WT plants after recovery, but there was no significant difference between OsMDHAR4-OE-1 and OE-5 (Fig. 4b, c). Consistent with this result, detached leaves of OsMDHAR4-overexpressing lines lost water more quickly than the WT leaves (Fig. 4d). Taken together, all of these results suggested that OsMDHAR4 plays a negative role in heat tolerance in rice.
The function of OsMDHAR4 in the control of water loss prompted us to investigate the stomatal aperture status, the major factor affecting the water-holding capacity in rice leaves. The stomatal apertures of the osmdhar4 mutant, OsMDHAR4-OE-1 and the WT plants were checked by scanning electron microscopy. No significant differences in the percentages of the three types of stomata and stomatal conductance between the osmdhar4 mutant and the WT were observed before heat stress. However, after heat treatment (45 °C for 24 h at 5.5- to 6.5-leaf stage), the results showed that 56.8% of stomata were completely closed in the osmdhar4 mutant, whereas only 25.0% were completely closed in the WT plants. On the other hand, only 10.5% of stomata were completely open in the osmdhar4 mutant, but 46% were completely open in WT. The percentage of partially open stomata didn't differ significantly from each other (Fig. 5a, b). Furthermore, the stomatal conductance was dramatically decreased in the osmdhar4 mutant compared the WT plants after heat treatment (Fig. 5c). These results were in agreement with the slower water loss of detached leaves from the osmdhar4 mutant. Either before or after heat treatment, OsMDHAR4-OE-1 plants had lower percentages of completely closed stomata than WT (before, 32.2% for OsMDHAR4-OE-1, 40.4% for the WT; after, 17.2% for OsMDHAR4-OE-1, 30.4% for the WT), while the percentages of completely open were higher than the WT (before, 24.6% for OsMDHAR4-OE-1, 20.1% for the WT; after, 36.6% for OsMDHAR4-OE-1, 25.1% for the WT; Fig. 5d). Moreover, compared with the WT, OsMDHAR4-OE-1 had significantly higher stomatal conductance after heat treatment (Fig. 5e). These results were also in agreement with the faster water loss of the OsMDHAR4-OE-1 plants.
ROS are continuously produced as the byproducts of various metabolic pathways and are scavenged by different antioxidative defense components in plants. The equilibrium between ROS production and scavenging may be perturbed by adverse environmental factors, such as heat stress (Apel and Hirt, 2004). As H2O2 is a very stable ROS with a long half-life (Levine et al., 1994) and a signal molecular that induces stomatal closure (McAinsh et al., 1996), so we measured the H2O2 content in the rice seedling leaves. A higher level of H2O2 accumulation was detected in the osmdhar4 mutant either before or after heat treatment (45 °C for 24 h at 5.5- to 6.5-leaf stage, Fig. 6a). The DAB staining results also support the quantitative analysis (Fig. 6b). In addition, more H2O2 accumulation was detected in the guard cells of the osmdhar4 mutant under normal conditions, as indicated by the ROS indicator, H2DCFDA (Additional file 4: Figure S4). Thus, ROS homeostasis in osmdhar4 seedlings was perturbed. These results indicated that the increased stomatal closure in the osmdhar4 mutant was probably due to accumulation of H2O2 in guard cells. As expected, in the OsMDHAR4-OE-1 seedling leaves, visibly less H2O2 accumulation was detected under normal conditions or after heat stress through the quantitative analysis and DAB staining (Fig. 6c, d). Taken together, our results indicated that OsMDHAR4 may negatively regulate H2O2-induced stomatal closure.
MDAR has been reported to be distributed widely across kingdoms pointing towards a universal role. In plants, it has been found to localize in chloroplasts, glyoxysomes, mitochondria, and cytosol. Chloroplastic MDAR localizes on the stroma of the chloroplast (Sano et al., 2005) and catalyzes the conversion of monodehydroascorbate (MDA) to AsA. AsA is a major antioxidant molecule that directly neutralizes ROS. Previous research has indicated that overexpression of chloroplastic MDAR from tomato enhanced tolerance to temperature and methyl viologen-mediated oxidative stress (Li et al., 2010). However, the physiological role of the chloroplastic MDAR in rice in response to heat stress is not reported.
In this study, OsMDHAR4, one new gene encoding monodehydroascorbate reductase was isolated from rice, and its amino acid sequence showed high identity and similarity to the MDAR sequences from several other plant species (Additional file 1: Figure S1). Bioinformatics studies predicted that ZmMDAR and BdMDAR, the two most homologous MDARs from Zea mays and Brachypodium distachyon, were located in the cytoplasm. However, intracellular localization studies using YFP fusion confirmed chloroplast localization of OsMDHAR4 (Fig. 1). Plant chloroplasts are the most significant generators of ROS and proposed to be heat sensors. A large number of chloroplasts are distributed in mesophyll cells (Ishikawa and Shigeoka, 2008; Maruta et al., 2012). OsMDHAR4 transcripts peaked in the blade of flag leaf, in which a lot of mesophyll cells were distributed, while it showed the lowest transcript level in the root (almost with no chloroplasts) (Fig. 2a), the results further confirmed the finding that OsMDHAR4 protein was located in chloroplast. Since 1995, several studies have reported that MDAR genes are regulated by abiotic stresses (Leterrier et al., 2005; Grantz et al., 1995; Eltelib et al., 2011). We found that OsMDHAR4 transcripts were induced by heat and PEG (2.8-fold and 2.2-fold). Under both ABA and H2O2 treatment, the OsMDHAR4 expression levels were increased in first and decreased at last, but salt and cold treatments had almost no effect on these (Fig. 2b). So, we assumed that OsMDHAR4 may function in rice heat resistance.
Many scholars have focused on conferring abiotic stress tolerance through the overexpression of MDAR genes. Overexpression of LeMDAR gene alleviated photoinhibition of PSI and PSII and enhanced the tolerance to various abiotic stresses by elevating AsA level (Li et al., 2010). Overexpressing AeMDHAR gene increased MDHAR enzyme activity compared to untransformed plants under both NaCl and control conditions (Sultana et al., 2012). OsMDHAR-expressing yeast cells displayed enhanced tolerance to H2O2 by maintaining redox homoeostasis, proteostasis, and the ascorbate (AsA)-like pool (Kim et al., 2016). In the above studies, we can find that all the MDAR genes play a positive role in response to different abiotic stresses. In our study, we found that disruption of OsMDHAR4 reduced the water loss rates and increased heat tolerance (Fig. 3), and overexpression of OsMDHAR4 accelerated the water loss rates and decreased heat tolerance (Fig. 4). Unlike previous researches, these results showed that OsMDHAR4 plays a negative role in heat tolerance in rice.
The importance of maintaining higher levels of AsA over DHA has been reported in previous studies. In transgenic tobacco plants overexpressing cucumber ascorbate oxidase, decreased AsA/DHA ratio increased ozone sensitivity (Sanmartin et al., 2003), whereas a higher AsA/DHA brought about by overexpression of DHAR and MDAR in cytosol resulted in enhanced tolerance to salt stress (Eltayeb et al., 2006). Higher AsA/DHA ratio, higher photosynthetic activity and lower H2O2 contents were reported in transgenic tobacco plants expressing ascorbate oxidase gene (Yamamoto et al., 2005). According to this, we measured the AsA and DHA contents in leaves. Consistently with previous study (Chen et al., 2003), the osmdhar4 mutant showed lower AsA contents and AsA/DHA ratio than the WT before or after heat treatment,and the OsMDHAR4-OE-1 plants showed higher AsA contents and AsA/DHA ratio than the WT before or after treatment (Additional file 5: Figure S5). However, the changes of ascorbate redox state were not be able to explain the increased heat tolerance in the osmdhar4 mutant and incremental heat sensitivity in OsMDHAR4 overexpression lines.
Stomata control the exchange of gases-most importantly water vapor and CO2-between the interior of the leaf and the atmosphere, and are involved in responses to abiotic stresses (Hetherington and Woodward, 2003). Stomatal closure degree makes major contribution to the ability of the plant to hold water in rice leaves. H2O2 is an essential signaling molecule involved in the regulation of stomatal movement (Wang and Song, 2008; Yao et al., 2013). Mutation of DST results in the down regulation of peroxidase 24 precursor (Prx-24, a scavenger of H2O2), might lead to the accumulation of H2O2 in guard cells and trigger stomatal closure, and enhances drought and salt tolerance (Huang et al., 2009). Arabidopsis mutants lacking either or both a cytosolic and chloroplastic ascorbate peroxidase (APX), which were responsible for H2O2 removal, were found to be more tolerant to salinity stress (Miller et al. 2007). Recently,one study showed that overexpression of Prx-24 (the target gene of DST) enhanced the sensitivity to drought and salt stress in rice (Cui et al., 2015). In our case, OsMDHAR4 might also be a target regulated by some transcript factors in response to heat stress, more research will be required to address this hypothesis. Liu et al. (2016) found that the oshtas mutant (gain-of-function) showed a strongly enhanced tolerance to heat stress with lower water loss rates, more closed stomata and significantly higher H2O2 contents after the treatment as compared with the WT. Our data also showed that the osmdhar4 mutant had more closed stomata and significantly reduced stomatal conductance after heat treatment. Meanwhile, the osmdhar4 mutant accumulated more H2O2 as compared with the WT before or after heat treatment. All of the above was found to be opposite in the OsMDHAR4 overexpression line (OsMDHAR4-OE-1, Figs. 5, 6). These findings suggested that OsMDHAR4 might play a negative regulation role in H2O2-induced stomatal closure.
Our results demonstrated that OsMDHAR4 negatively regulates tolerance to heat stress in rice by promoting H2O2-induced stomatal closure. This study increases our insights into the molecular mechanisms of rice responses to heat stress and may ultimately be helpful for enhancing heat-tolerance via genetic engineering in crop breeding programs.
Overexpression transgenic plants and the osmdhar4 mutant were all based on the Oryza sativa L. ssp. Japonica Zhonghua 11 rice background.
To check the expression levels of the OsMDHAR4 gene under various abiotic stresses or phytohormone treatment, Zhonghua 11 rice plants were grown in Yoshida solution for approximately 3 weeks under normal conditions. The seedlings at the 3.5- to 4.5-leaf stage were treated with abiotic stresses, including heat stress (exposing plants to 45 °C), simulated drought stress (treated with 20% [w/v] PEG 6000), ABA treatment (100 μM ABA), oxidative stress (treated with 100 mM H2O2), salt stress (treated with 250 mM NaCl), and cold stress (seedlings were transferred to a growth chamber at 4 °C), followed by sampling at the designated time points (0 h, 0.5 h, 3 h, 6 h, 12 h and 24 h). We prepared each RNA sample with shoots from at least four seedlings and ensured that every sampled seedling was of the same growth stage.
Before treatment, OsMDHAR4-overexpressing transgenic plants were firstly selected by germinated seeds on 1/2 MS medium containing 50 mg L− 1 hygromycin, WT and the osmdhar4 mutant were grown on normal 1/2 MS medium 1 d later. For heat stress testing, the uniformly germinated seeds were sown in the same barrels filled with a well-mixed soil, which was then cultured in a growth chamber with a 13-h-light (28 °C)/11-h-dark (25 °C) photoperiod, 70% humidity. The 5.5- to 6.5-leaf stage seedlings were transferred to a growth chamber with a 13-h-light (45 °C)/11-h-dark (45 °C) photoperiod, 70% humidity for 48 h or 72 h. The treated seedlings were transferred back into the previous growth conditions for recovery, and seedlings with newly growing leaf blades were then counted as surviving plants.
To generate the overexpression construct, the full-length CDS of OsMDHAR4 was amplified from the cDNA of Zhonghua 11 by PCR. The PCR fragment was inserted into vector pEXT06/g (target gene driven by 35S promoter). The resulting construct was then confirmed by sequencing and transformed into Zhonghua 11, a japonica rice that can be easily transformed, by the Agrobacterium tumefaciens-mediated co-cultivation method. Transformed calli were selected on hygromycin medium. The primers used in this work are listed in Additional file 6: Table S1.
Coding sequence of the OsMDHAR4 gene was amplified by PCR and directionally inserted into pCAMBIA1300-35S:YFP-NOS vector. The cultures of the Agrobacterium tumefaciens strain EHA105 harboring empty and recombinant vector were used to infect the healthy leaves of Nicotiana benthamiana (4 weeks old). The fluorescence signals were observed using a confocal microscope (Leica TCS SP5 or Zeiss LSM710). The primers used in this work are listed in Additional file 6: Table S1.
The TRIZol reagent (Invitrogen, USA) was used according to the manufacturer's instructions to extract total RNA. Before reverse transcription, total RNA was treated with gDNA Eraser (TaKaRa, Japan) for 5 min at 42 °C to degrade possibly contaminated residual genomic DNA. The cDNA templates were synthesized using PrimeScript™ reagent Kit (TaKaRa, Japan) according to the manufacturer's instructions. Quantitative real-time PCR was performed on an optical 96-well plate with a CFX96 Real-time PCR Detection System (Bio-Rad, USA) using SYBR Premix Ex Taq (TaKaRa, Japan). The PCR thermal cycling protocol was as follows: 95 °C for 10 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. Gene-specific primers for OsMDHAR4 were designed using the Roche Web site (Roche Applied Science). The rice Actin1 was used as the internal reference (Zhang et al., 2012), and data analyses with the 2–ddCt method were performed as described (Livak and Schmittgen, 2001). The primers used in this work are listed in Additional file 6: Table S1.
Leaves harvested from 3.5- to 4.5-leaf stage seedlings, with or without heat treatment, were used to measure H2O2 contents. The contents were measured spectrophotometrically after reaction with potassium iodide (KI). The reaction mixture consisted of 0.5 ml of 0.1% trichloroacetic acid (TCA), leaf extract supernatant, 0.5 ml of 100 mM potassium phosphate buffer (pH 7.8) and 1 ml reagent (1 M KI, w/v in fresh double-distilled water). The blank control consisted of 1 ml 0.1% TCA and 1 ml KI in the absence of leaf extract. After 1 h of reaction in darkness, the absorbance was measured at 390 nm. The amount of H2O2 was calculated using a standard curve prepared with known concentrations of H2O2.
3,3′-Diaminobenzidine (DAB) staining was performed following a published method with some modifications (Liu et al., 2014). Rice seedlings at 5.5- to 6.5-leaf stage were treated with a 45 °C high temperature for 24 h or not treated, then the leaves (3–5 mm in width) at the same position were detached and immersed in 1% 3,3′-diaminobenzidine (DAB) solution in HCl-acidifed (pH 3.8). After 30 min under vacuum the samples were incubated at room temperature for 24 h in the dark. The samples were then bleached by boiling in ethanol in order to remove the chlorophyll and reveal the brown spots, which are indicative of the reaction of DAB with H2O2. The samples were observed and imaged under a scanner.
H2O2 production in guard cells was detected using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes) as described previously (Huang et al., 2009) with small modifications. Leaves of 5.5- to 6.5-leaf stage seedlings were immerged in 0.01% Tween-20 and vacuum-infiltrated for 10 min. After rinsing twice with distilled water, leaves were incubated in 3% (w/v) cellulase RS (Yakult Honsha, Japan) for 5 h at 40 °C without shaking to facilitate peeling off the epidermal strips. The epidermal strips were peeled off from leaves using tissue forceps. After washing with loading buffer (10 mM Tris-HCl, 50 mM KCl at pH 7.2), the epidermal strips were incubated in staining buffer (loading buffer containing 50 mM H2DCFDA) for 10 min at room temperature in the dark. The epidermal strips were washed with distilled water to remove the excess dye. The fluorescence was examined using a confocal laser-scanning microscope (Leica TCS SP5, Germany). All confocal images were taken under identical conditions and the guard cell region was selected to quantify the mean grey value of guard cells.
Leaves of 3.5- to 4.5-leaf stage plants with or without heat treatment for 24 h were fixed with 2.5% (v/v) glutaraldehyde, and stomatal images (20 kV, 2000×) were obtained using scanning electron microscopy (KYKY-EM3200).
Plants germinated under normal conditions for 4 weeks. The leaves were detached from various lines with same age and position, and weighed immediately as the initial fresh weight. They were then placed in clean filter papers, and incubated at 25 °C. The decreases in fresh weight were recorded at every 30 min for 6.5 h or 7 h. Water loss was presented as percentage of fresh weight loss versus the initial fresh weight.
Leaves harvested from 3.5- to 4.5-leaf stage seedlings, with or without heat treatment, were used to measure the total AsA (tAsA), AsA and DHA contents determined by the spectrophotometric method described previously (Gillespie and Ainsworth, 2007), with minor modifications. The frozen leaf samples were ground with inert sand and 10% trichloroacetic acid (TCA) solution using a mortar and pestle. The homogenate was centrifuged at 12,000 rpm for 20 min. The tAsA contents were determined in a reaction mixture consisting of crude extract and 150 mM KH2PO4 buffer (pH 7.4) containing 5 mM EDTA and 10 mM dithiothreitol (DTT) for the reduction of DHA to AsA. The reaction mixtures were incubated at room temperature for 10 min and 0.5% N-ethylmaleimide (NEM) was added. AsA was assayed in a similar manner, except that deionized H2O was substituted for DTT and NEM. The color was developed in both the reaction mixtures by the addition of 10% TCA, 44% o-phosphoric acid, α,ά-dipyridyl in 70% ethanol, and 30% FeCl3. The reaction mixtures were incubated at 37 °C for 1 h and quantified spectrophotometrically at 525 nm.
This work was supported by the National Natural Science Foundation of China (Grant number: 31601232, 31761130073), the Natural Science Foundation of Fujian Province (Grant number: 2017 J05046), China Postdoctoral Science Foundation (Grant number: 2017 M612108) and Research Grant of Fujian Agriculture and Forestry University (Grant number: KXGH17005). The authors would like to thank Prof. Jumin Tu (Zhejiang University) for providing the plasmid for subcellular localization.
This study was supported by the National Natural Science Foundation of China (Grant number: 31601232, 31761130073), the Natural Science Foundation of Fujian Province (Grant number: 2017 J05046), China Postdoctoral Science Foundation (Grant number: 2017 M612108) and Research Grant of Fujian Agriculture and Forestry University (Grant number: KXGH17005).
LJP, XWF and ZJH contributed to the experimental design. LJP, SXJ, XFY, ZYJ, ZQ and MR contributed to experiment performance and data analysis. LJP, XWF and ZJH drafted the manuscript. LJS contributed to good advice on designing the experiment. All authors read and approved the final manuscript.


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Football Club United of Manchester is a semi-professional football club based in Moston, Manchester, England, that competes in the , the seventh tier of the English football league system, and plays home matches at Broadhurst Park.

Founded in 2005 by Manchester United supporters opposed to American businessman Malcolm Glazer's takeover, F.C. United entered Division Two of the North West Counties Football League, earned three consecutive promotions and were promoted for a fourth time to National League North for the 2015–16 season. In cup competitions, F.C. United reached the second round of the FA Cup in 2010–11 and the fourth round of the FA Trophy in 2014–15. In 2019 they were relegated back to the Northern Premier League.

After ground-sharing between 2005 and 2014 with Bury at Gigg Lane, F.C. United opened their own ground, Broadhurst Park in north-east Manchester, in May 2015. The team was managed by Karl Marginson from its formation in 2005 until October 2017. The current manager is Neil Reynolds, who took over as manager in October 2018 from David Chadwick who acted as temporary manager following Tom Greaves's resignation in August 2018. The club's regular kit colours are red shirts, white shorts and black socks. Their badge is based on the Manchester coat of arms and features a ship at sea and three stripes for the three rivers that flow through Manchester.

After Heart of Midlothian and Exeter City, United is the third-largest fan-owned football club in the United Kingdom by number of members, and has one of the highest home attendances in English non-league football. The club is democratically run by its members who have equal voting rights and own one share each in the club.

History

Formation

The club was founded in 2005 by disaffected supporters of Manchester United. Although there were varying reasons for their dissatisfaction, the catalyst for F.C. United's formation was the 12 May 2005 takeover of Manchester United by American businessman Malcolm Glazer. Supporters first considered forming a breakaway club in 1998 during an attempted takeover of Manchester United by BSkyB. The creation of F.C. United in the event of a Glazer buyout was first proposed in February 2005 by Manchester United fanzine Red Issue.

Public meetings for fans were held on 19 May 2005 at the Central Methodist Hall in Manchester's Northern Quarter and on 30 May at the Apollo Theatre. Subsequently, a steering group was created to set up the new club. After the name "F.C. United" was rejected by The Football Association for being too generic, those who had pledged money to the club were asked to vote on a name. On 14 June 2005, it was announced that "F.C. United of Manchester" had been chosen, beating "A.F.C. Manchester 1878", "Manchester Central", and "Newton Heath United", and F.C. United were officially registered with the Manchester County Football Association on the same day.

Karl Marginson was appointed as the club's manager on 22 June, and the club held trials for players four days later. Around 900 players applied to take part in the trials, of whom 200 were chosen to take part and 17 selected to play for F.C. United. Jonathan Mitten, great-nephew of Manchester United forward Charlie Mitten, was the club's first signing.

F.C. United's inaugural members' meeting was held on 5 July 2005 at the Methodist Central Hall. Members voted on the club's constitution, badge, and core principles and elected the 11-member board. By 6 July, over 4,000 people had pledged money to F.C. United and the club had over £100,000 in the bank. During F.C. United's formation, the owners of Leigh RMI offered to merge the two clubs, but the parties decided against the idea. F.C. United later arranged to play their first ever game against Leigh RMI on 16 July 2005; the match ended 0–0.

North West Counties years (2005–07)
For the 2005–06 season, F.C. United played in the Second Division of the North West Counties Football League (NWCFL), which is level ten of the English football league system and nine levels below the Premier League. The club played its home matches at Bury's Gigg Lane. F.C. United were ineligible to play in the FA Vase for their first season because the club was formed after the deadline to enter the competition. They were, however, able to play in the North West Counties League Challenge Cup.

F.C. United's first competitive match was a 5–2 away victory against Leek County School Old Boys on 13 August 2005. 2,590 people attended the game. In its home debut on 20 August, the club defeated Padiham 3–2 with two goals from Rory Patterson. During their first season, F.C. United consistently broke NWCFL attendance records. A crowd of 6,032 watched the team during their final home league match of the season against Great Harwood Town, which remains an NWCFL record . Despite losing that game 0–1, the team were promoted to Division One.

F.C. United's successful 2006/07 season in Division One of the NWCFL earned the club a promotion to Division One North of the Northern Premier League. They secured their second successive league title with a 7–1 win over Atherton Laburnum Rovers on 18 April 2007. They then won their second cup of the season by beating Curzon Ashton 2–1 in the NWCFL's Challenge Cup final. In the third round of the FA Vase competition, the club was eliminated when it lost 2–3 in the last minute of extra-time to Quorn.

Northern Premier League years (2007–15)
In 2007–08, F.C. United played in the inaugural season of the Northern Premier League (NPL) Division One North. They made their debut in the FA Cup that season, but lost 1–2 to Fleetwood Town in the first qualifying round. They advanced to the final of the 2007–08 NPL President's Cup, in which they beat Radcliffe Borough 2–0 to pick up their fourth trophy in the three years following the club's formation. The club finished the season second in the league, trailing champions Bradford Park Avenue by one point, and entered the play-offs for the other promotion place. After beating Bamber Bridge 3–2 in the semi-finals, F.C. United faced Skelmersdale United in the promotion play-off final, coming back from a goal down to win 4–1 and earn their third successive promotion to play in the Northern Premier League Premier Division.

In 2008–09, F.C. United made their debut in the FA Trophy, reaching the third qualifying round, and missed out on a play-off place on the last day of the regular season. In the 2009–10 season, they finished 13th in the league, their lowest league position in the first five seasons, before progressing to the play-offs in 2010–11. They beat Bradford Park Avenue 2–0 in the semi-finals but lost the final 0–1 to Colwyn Bay. Earlier that season, F.C. United reached the first round of the FA Cup for the first time, recording victories over Radcliffe Borough, Gainsborough Trinity, Norton & Stockton Ancients and Barrow to play League One side Rochdale. They defeated Rochdale 3–2 after a late winner from Mike Norton, and played eventual 2010–11 League One champions Brighton & Hove Albion in the second round. After a 1–1 away draw at Withdean Stadium, F.C. United lost the replay at Gigg Lane 0–4, in front of their highest home attendance of 6,731.

In the 2011–12 season, F.C. United reached the first round of the FA Trophy for the first time after knocking out Frickley Athletic, Durham City and Altrincham. In the league, they qualified for the end of season play-offs, despite finishing 6th, due to the demotion of Northwich Victoria for breaching of financial rules. They beat Chorley 2–0 in the play-off semi-final to qualify for consecutive play-off finals but lost 0–1 to Bradford Park Avenue in the penultimate minute of extra time.

F.C. United finished third in the 2012–13 season to book a place in the play-offs. They won 3–1 against Witton Albion in the semi-final, but lost the final for the third consecutive time with a 1–2 defeat to Hednesford Town. The following season, the club finished second in the league but lost in the play-offs in the semi-finals. During the 2014–15 campaign, F.C. United reached the fourth round of the FA Trophy and recorded a streak of 21 league games without a loss from December to April, including 16 victories. They secured their promotion to the National League North following a 1–0 win against Stourbridge on 21 April 2015; after seven years of trying. The club's top scorer for this championship winning season was Tom Greaves, the same player who scored the winning goal for Bradford Park Avenue against F.C. United in the 2012 play-off final.

National League years (2015–2019)
F.C. United recorded their first victory in the National League North in a 3–2 home win over Brackley Town on 22 August 2015, which was the club's first competitive win at Broadhurst Park. In October, for the second time in the club's history, they secured a place in the first round of the FA Cup with a 3–1 away win over Sporting Khalsa, but were eliminated from the competition after a 4–1 home loss against Chesterfield. In November, the club ended their cup runs for the 2015–16 season after two successive home defeats to Stalybridge Celtic 3–4 in the Manchester Premier Cup and to A.F.C. Telford United 1–2 in the FA Trophy. Between September and November 2015, F.C. United lost seven consecutive league matches; their worst run ever . They eventually finished the season in 13th place.

The following 2016–17 season, the team again finished 13th and won the Manchester FA county cup for the first time in their history, beating Stalybridge Celtic 1–0 in the final. After a poor start to the 2017–18 season, Karl Marginson, the team's first and only manager, left the club by mutual consent in October 2017. He was replaced by forward Tom Greaves who took over on a temporary basis and was appointed permanent player-manager the next month. F.C. United finished the season in 16th place and retained the Manchester FA county cup with a penalty shoot-out victory against Trafford F.C. Greaves, who became the club's all-time record goal-scorer in December 2017, saw his contract extended until the end of the 2018–19 season.

After a poor start to the 2018–19 season, Greaves left the club at the end of August 2018, with long-serving former player David Chadwick acting as interim manager. In late October, the club appointed Neil Reynolds as first team manager. On 22 April 2019, Easter Monday, FC United were relegated back to the Northern Premier League following a 1–2 home defeat to Blyth Spartans.

Colours and badge

F.C. United's club colours are red, white and black — the same colours worn by Manchester United. The shirt bears no sponsorship logo, as it was written into the club's constitution that the club should not have a shirt sponsor. The club's first kit was a plain red shirt, white shorts and plain black socks. The club introduced a new home shirt for the 2007–08 season with a striped collar and striped ends on the sleeves which lasted until 2009. This was changed for the 2009–11 seasons to a red shirt with a black and white stripe down the left side, manufactured by Admiral Sportswear. For seasons 2011–13 the club reverted to a plain red shirt, manufactured by O'Neills. At the start of the 2018–19 season, Errea replaced O'Neill's as kit supplier.

The club's second kit, worn when playing away against a team with a predominantly red kit, is a white shirt, white shorts and socks. The club has an alternative plain blue kit with the clenched fist logo and the words "Our Club Our Rules" on the shirt front for playing against a team with a red and white kit. In past seasons white shirts with diagonal black or red stripes and a white shirt with a red trim, black shorts and white socks have all been used as the second kits.

The club's badge is red, white, black and yellow, and incorporates elements from the coat of arms of Manchester City Council; a ship representing Manchester's industry and three stripes representing the three rivers that flow through Manchester: the Irk, the Irwell and the Medlock.

Stadium

F.C. United play at the 4,400 capacity Broadhurst Park, which opened in May 2015. The ground was built at a cost of £6.3 million, using £2 million from a Community Share Scheme and the remainder from a variety of governmental and charity grants. The ground is surrounded on all sides by covered stands: the St. Mary's Road End (east), the North Stand, the Lightbowne Road End (west) and the Main Stand (south), the latter of which has seating sections. Within the Main Stand is a clubhouse with a bar and catering facilities, club offices, changing rooms, a medical suite and a classroom. The stadium is shared with a local junior team, Moston Juniors F.C.

From their foundation in 2005 until 2014, F.C. United were based at Bury F.C.'s Gigg Lane stadium. F.C. United's first proposed stadium was announced in 2010 for Newton Heath, the original home of Manchester United. The development was planned to be located on the site of the Ten Acres Lane sports centre and would have cost £3.5 million, to be financed by public donations, a Community Shares issue and grant funding. However a year later, in March 2011, Manchester City Council backed out from funding the stadium. The Broadhurst Park site in Moston, north-east Manchester, was announced in April 2011. Detailed information about the new facility was released in June 2011 and Manchester City Council approved the planning permission for the site on 27 October 2011. F.C. United had to overcome some obstacles including funding agreements, contractor and lease negotiations and a legal challenge from local residents which caused a further two-year delay before building commenced in November 2013.

During their time at Bury, fixture clashes meant that F.C. United used a further six stadia for home fixtures: Altrincham's Moss Lane; Radcliffe Borough's Stainton Park; Hyde United's Ewen Fields; Stalybridge Celtic's Bower Fold; Curzon Ashton's Tameside Stadium; and Flixton's Valley Road.

For the 2014–15 season, F.C. United ended their groundsharing agreement with Bury after nine seasons in preparation for their move to Broadhurst Park. As the ground was not ready at the start of the season, they used Bower Fold as a temporary home. Delays with Broadhurst Park meant that F.C. United were unable to move in until May 2015, and following fixture clashes with Stalybridge Celtic the club moved in December 2014 to the Tameside Stadium for the remainder of the season. F.C. United hosted a test event at Broadhurst Park on 16 May 2015, staging a short match between their first team and an Invitational XI made up of past players. The official opening game was a friendly against Benfica B on 29 May, the anniversary of Manchester United's victory over Benfica in the 1968 European Cup Final. Benfica won the opening game 1–0 in front of a crowd of 4,232.

Supporters
F.C. United are owned by 5,000 of their members and are the second  supporter-owned football club in the United Kingdom by number of members. Each member can vote on how the club is run, including voting for board members, kit designs and season ticket prices. Most F.C. United supporters still support Manchester United and many were previously season ticket holders at Old Trafford. F.C. United fans are known for the large range of songs that they sing at matches, and the atmosphere created by fans has been praised in the media.

During their first season (2005–06), F.C. United had the second-highest average attendance in English non-League football with an average gate of 3,059 and were the 87th best supported club across all divisions. Attendances fell in the next two seasons and they were the 92nd best supported club in 2006–07 and 100th best supported club by 2007–08. Their average league attendance then levelled out at approximately 2,000 per game, before rising to an average of 2,155 in 2014–15, the then-seventh highest attendance in non-League football. After moving to Broadhurst Park in May 2015, the club averaged a gate of 3,394 in 2015–16, a season-on-season increase of over 57% and the fourth highest attendance in non-League football.

Organisation
F.C. United operate as a community benefit society. Membership is obtained by paying an annual fee of £15 to the club (£3 for children) but each member receives only one share in the club and is entitled to a single vote at meetings, regardless of the amount donated. The board consists of up to 11 members who are elected by the members of the club. Day-to-day operations of the club are overseen by a Chief Executive Officer; Andy Walsh, a founding member of F.C. United, was appointed to the position in 2005. He stepped down from the role at the end of June 2016. Damian Chadwick, another founding member of the club, took up the position in November 2016. Chadwick stood down in 2018 and was replaced by Paul Smith.

The club's manifesto includes the following core principles:
 The Board will be democratically elected by its members;
 Decisions taken by the membership will be decided on a one-member, one vote basis;
 The club will develop strong links with the local community and strive to be accessible to all, discriminating against none;
 The club will endeavour to make admission prices as affordable as possible, to as wide a constituency as possible;
 The club will encourage young, local participation—playing and supporting—whenever possible;
 The Board will strive wherever possible to avoid outright commercialism;
 The club will remain a non-profit organisation.

The club accepts sponsorship but does not allow sponsors' logos to be displayed on the team's shirts. The club's main sponsor in its inaugural 2005–06 season was the Bhopal Medical Appeal and in the 2006–07 season it was the Williams BMW Group. Between 2011–2016, F.C. United were sponsored by mxData, a Manchester-based mobile app development company. In October 2014, F.C. United became the first football club in the United Kingdom to be accredited as a living wage employer by the Living Wage Foundation.

Criticism
F.C. United and their founders have been criticised and supported by fans of Manchester United and the media; some fans view those who chose to leave to follow F.C. United as "traitors". Manchester United manager Sir Alex Ferguson questioned the loyalty of fans who decided to form the club and the motivation behind the forming of F.C. United in a 2006 book:

Former Manchester United forward Eric Cantona has supported the club, describing them as "having a great idea" and expressing hope that F.C. United will "become a great club and win the European Cup in 50 years' time". In 2010, aged 43, he said that he would be prepared to play for the club. Steve Coppell, Manchester United winger between 1975 and 1983, joined the club as a co-owner in April 2016.

F.C. United have been criticised by their own members for abandoning their principles, including agreeing to a photo op with a Conservative Party government minister in October 2015, despite a club policy not to be used for political promotion, and a day after some club members took part in a march against government cuts in Manchester. F.C. United defended the visit as "an essential part" of the development of financial vehicles for football fans, arguing that it will help government representatives to better understand the benefits and challenges of social investment.

The club's programme editor resigned from his position in June 2015 over a one-off price rise of the programme for Broadhurst Park's opening game against Benfica the previous month; a Board decision that was described as breaching the club's founding principle of avoiding outright commercialism. F.C. United were also criticised at the time for a deficit in democracy, transparency and accountability between their officials and the membership.

In 2016, protests by supporters led to several board members stepping down and an Emergency General Meeting (EGM) to elect a new board was subsequently called. The protests culminated in several fans invading the pitch during the last home game of the 2015–16 season, calling for the remaining board members deemed responsible for the lack of democracy and transparency at the club to resign. Three board members resigned within a week of the on-pitch protest, along with the club's Press and Communications Officer. On 5 June, the EGM took place and a new board of 11 members were elected, leading to "a sense of progression and an air of optimism" among the club's members.

Statistics and records

The record for the most appearances for F.C. United is held by Jerome Wright, with 400 . Tom Greaves is the club's all-time record goal-scorer with 102 goals in all competitions, which includes 86 in the league and 16 in cup matches . Six other players, Rory Patterson, Mike Norton, Matthew Wolfenden, Jerome Wright, Simon Carden and Stuart Rudd have also scored more than 50 goals for the club. Rudd holds the record for most goals scored in a single season, having scored 45 goals in the 2006–07 season.

The largest number of points the team accrued is 112 in the 2006–07 season, and the highest number of goals scored in a season is 157, achieved in 42 matches in 2006–07. The club's best performance in the FA Cup was a second round appearance during the 2010–11 season. After a 3–2 first round win over Rochdale, F.C. United recorded a 1–1 away draw with Brighton & Hove Albion, forcing a home replay which they lost 0–4. The team has since advanced to the first round of the FA Cup twice, losing 1–4 in 2015–16 to Chesterfield and 1–5 in 2020–21 to Doncaster Rovers.

In the 2006–07 season, F.C. United reached the third round of the FA Vase, beating Padiham and Salford City in the first two rounds but lost 2–3 at home to Quorn after extra time. In the 2014–15 season, they progressed to the fourth round of the FA Trophy, defeating Harrogate Town, Chorley and AFC Fylde in the first three rounds before losing 0–1 away to Torquay United.

The club's record league victory was a 10–2 win over Castleton Gabriels on 10 December 2005 in the North West Counties Football League Division Two; Simon Carden scored five of the goals, which is the club record for the highest number of goals scored by a player in a single game. F.C. United achieved eight-goal victory margins on three further occasions, in 8–0 wins over Squires Gate, Glossop North End and Nelson, all during the 2006–07 season. The club's heaviest league defeat is 0–6 away to Harrogate Town on 10 March 2018 in the National League North.

F.C. United's highest home attendances are 6,731 against Brighton in the second round of the 2010–11 FA Cup on 8 December 2010 and 6,023 against Great Harwood Town in the North West Counties Football League Division Two on 22 April 2006, both at Gigg Lane.

Players

First team squad

Out on loan

Former players
See :Category:F.C. United of Manchester players to see a list of notable F.C. United players, past and present.

International representation
F.C. United have assisted in providing several players for the international football scene; the club's all-time record goal-scorer Rory Patterson went on to play and score for Northern Ireland at senior level and forward Matthew Walwyn debuted for Saint Kitts and Nevis in a friendly against Andorra in November 2015. F.C. United's youth team were represented in the England Schoolboys squad by Scott Cheetham in 2011 and in 2013, the club signed Pakistan international defender Amjad Iqbal from Bradford Park Avenue. Other F.C. United players to have played international football include Stephen O'Halloran (Republic of Ireland), Ludovic Quistin (Guadeloupe), Jason St Juste and Jacob Hazel (both St Kitts and Nevis).

Managerial history / Coaching staff

Upon its formation, F.C. United appointed Karl Marginson as its manager. He led the team to four promotions, three league titles, two league cups and a county cup. He left the club by mutual consent in October 2017 after a poor start to the 2017–18 season. He was replaced by forward Tom Greaves who took on the role of a caretaker player-manager, a decision that was made permanent on 21 November 2017. He was subsequently replaced by Dave Chadwick as Caretaker Manager in August 2018 when he resigned. Neil Reynolds is the current manager of the club having been in position since 18 October 2018, taking over from Chadwick.

List of managers
Information correct after match played on 12 March 2022. Only competitive matches are counted.
Key
 Names of caretaker managers are supplied where known, and the names of caretaker managers are highlighted in italics and marked with an asterisk (*).
 Names of player-managers are supplied where known, and are marked with a double-dagger ().

Current coaching staff

Honours

F.C. United have won three league titles, two league cups and two county cups in their history.

 Northern Premier League Premier Division
 Champions 2014–15
 Play-off runners-up 2010–11, 2011–12, 2012–13
 Northern Premier League Division One North
 Runners-up 2007–08
 Play-off winners 2007–08
 Northern Premier League President's Cup
 Winners 2007–08
 North West Counties League Division One
 Champions 2006–07
 North West Counties League Division Two
 Champions 2005–06
 North West Counties League Challenge Cup
 Winners 2006–07
 Fenix Trophy
 Winners 2021–22
 Manchester Premier Cup
 Winners 2016–17, 2017–18
 Supporters Direct Cup
 Winners 2006
 Brian Lomax Trophy
 Winners 2021

Records
Best FA Cup performance: Second round, 2010–11
Best FA Trophy performance: Quarter Final, 2014–15
Best FA Vase performance: Third round, 2006–07

In European competitions

Fenix Trophy

Women's team
F.C. United's women's team competed for the first time in the 2012–13 season. They finished second in the Greater Manchester Women's Football League behind Manchester City Ladies. They also reached the GMWFL League Cup final but lost 0–1 to Manchester City Ladies. After finishing runners-up again in 2013–14, the team won a league and cup double in 2015, gaining promotion to the North West Women's Regional Football League Division One South. They finished runners-up in the two following seasons, behind MSB Woolton Ladies in 2015–16 and Merseyrail Bootle in 2016–17, winning a league and county cup in the latter season. In 2017–18, the team won their first Treble, defending the league and county cups and securing a promotion to the North West Women's Regional Football League Premier Division after an unbeaten league campaign. After two seasons that were curtailed by the COVID-19 pandemic with no promotion or relegation, F.C. United were among various women's teams that gained promotion through an application process. As a result, the team reached the 4th division for the first time in their history.

# Shared with Middleton Athletic

See also

 List of fan-owned sports teams

Notes

References

External links

 F.C. United in Russia – statistics database

2005 establishments in England
Association football clubs established in 2005
Fan-owned football clubs in England
Football clubs in Manchester
United of Manchester
Northern Premier League clubs
Manchester United F.C.
North West Counties Football League clubs
Football clubs in England

redpajama_set_name: RedPajamaWikipedia


In preparation for Valentine's day, Spotify has released a list of the most-streamed love songs on its service.
Spotify also released a new feature called Love Notes, a way to send a kind of valentine to your loved one through Spotify. The tool sends someone a romantic playlist depending on your preferences.
The songs that appear most on "love" playlists on Spotify feature a strange mix from heartbreak ballads to heartfelt crooning — and even "No Diggity" by Blackstreet?


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Creating The Possibility of The Life You Love and Living it Powerfully
HomeMoreFunny22 Then & Now Pics Of The Internet's Favorite Meme Stars
22 Then & Now Pics Of The Internet's Favorite Meme Stars
At this point, the internet is full of all kinds of iconic memes featuring various people and animals from all around the world. Real-life people and animals with their very real lives. Yes, it's a simple and a pretty obvious fact but, to be completely honest, I rarely think about it while using these memes in my everyday life.
What are the names of these famous meme heroes? How did the photos of them go viral? Was it intentional? And most importantly—what do these internet legends look like now? Would we still recognize them if we were lucky enough to see them on the street? Today, let's discuss all of that. Without further ado, Bored Panda invites you to scroll down and find out what your favorite meme celebrities look like now. And as always, don't forget to vote for your favorites.
#1 Success Kid (Sam Griner)
The meme was born in 2007 after Laney Griner posted a photo of her son Sam trying to eat sand on Flickr. The photo first became famous as a meme captioned "I Hate Sandcastles," suggesting that the boy had just destroyed another child's sandcastle. Apparently, Laney Griner didn't like the context of the meme as she felt it made her son look like a bully, when in fact, he loved sandcastles. But then the "Success Kid" meme was created and the woman embraced it. Since then, the photo has been featured in various ads, appeared on Hot Topic shirts, Xbox screensavers, and many more.
Image credits: Sam Griner, Success Kid
#2 Confused Nick Young (Nick Young)
The photo featuring NBA basketball player Nick Young is a screenshot from a 2014 episode of the YouTube web series "Thru The Lens." There's a scene in which Nick makes a perplexed facial expression when his mother claims he was "a clown" in his younger years.
Image credits: Nick Young
#3 Disaster Girl (Zoe Roth)
The iconic photo originated back in 2004 when Dave Roth, the father of Zoe, captured her observing firefighters controlling the blaze two blocks away from their home in Mebane, North Carolina. In November 2007, Dave entered the shot into a competition run by a photography magazine and won it. After that, the photo was published in the magazine. Since then, Zoe's photo has been edited onto images of various famous events and has taken the internet by storm. "I'd love for the meme to help me get into or pay for college somehow," Zoe told Refinery 29 a few years ago. "But I ultimately want people to know me for me."
Image credits: zoeroth
#4 Bad Luck Brian (Kyle Craven)
This photo was turned into a meme after Ian Davies uploaded a yearbook photo of his friend Kyle Craven to Reddit with the caption "Takes driving test .?.?. gets first DUI" in January 2012. Apparently, before taking the photo, Kyle rubbed his face with a sweater to make it red and adopted an obnoxious smile. The meme is generally used to describe a variety of unlucky, embarrassing, and tragic events.
Image credits: mnhaverland
#5 Side-Eyeing Chloe (Chloe)
Chloe went viral on the interwebs back in 2013 after the YouTuber KAftC posted a video titled "Lily's Disneyland Surprise… AGAIN" capturing two sisters, Lily and Chloe, reacting to the news of a surprise trip to Disneyland on their way to school. While Lily is breaking in tears of joy, Chloe is captured looking straight into the camera with a disturbed look on her face as if she's not quite sure what's happening. If you'd like to see more of Chloe and how much she's changed, you can check out her personal Instagram account with over 580k followers.
Image credits: Lily & Chloe
#6 Trying To Hold A Fart Next To A Cute Girl In Class (Michael McGee)
The meme was born in 2014 when Reddit user aaduk_ala submitted a photograph titled "Trying to hold a fart next to a cute girl in class" to the /r/funny subreddit. "Well, the guy who took the picture has been a good friend of mine since I first moved out here to Texas, so he knew I was able to make the face anytime I wanted," Mike McGee told Unilad. "So he wanted to make a funny Twitter caption and told me to make the face. He posted it and like two or three months after he posted it, it took off!"
Image credits: aaduk_ala
#7 Scumbag Steve (Blake Boston)
The photo came from the cover of the album "Ma Gangsta" by the rap group Beantown Mafia and was first posted on Reddit in January 2011. The pic captures a guy named Blake Boston, also known as "Weezy B." In an interview with Know Your Meme, Blake revealed that the picture was originally taken by his mother. "I have no regrets in my life… no matter what I do. I can completely f*ck up on something and I still don't regret it. Because at the end of the day, that makes me who I am, so I wouldn't go back and erase anything, if I were to go back in time," the man told in an interview with RoughDraft TV.
Image credits: BlakeBoston617
#8 Blinking White Guy (Drew Scanlon)
The man starring in this iconic meme is named Drew Scanlon. The GIF comes from a live stream shot in 2013, when Drew Scanlon was a video producer at the popular gaming website called Giant Bomb. During one of the team's weekly "Unprofessional Fridays" shows, Scanlon watched as editor-in-chief Jeff Gerstmann played Starbound, a two-dimensional action-adventure game. "I've been doing some farming," Gerstmann narrated, "with my h*e." Scanlon reacted with the expression now known as the "Blinking White Guy."
Image credits: drewscanlon
#9 Roll Safe (Kayode Ewumi)
The meme comes from the 2016 BBC Three mini-documentary series "Hood Documentary" in which the character played by the actor Kayode Ewumi is shown pointing to his head and smiling after saying that he finds a woman "beautiful" because "she's got good brains." Kayode Ewumi is currently 27 years old.
Image credits: BuzzFeed UK
#10 Harold Hiding The Pain (András Arató)
The meme captures a senior stock photography model, András Arató, with a facial expression that appears to indicate suppressed pain. The meme was born in September 2011, when the stock photo with DreamTime's watermark was posted on the Facepunch forum. In September 2018, András Arató hosted a TedTalk in Kyiv, Ukraine, where he discussed the story of his life as a "meme-hero."
Image credits: BuzzFeedVideo
#11 First World Problems (Silvia Bottini)
Turns out, the woman from the "First-World Problems" meme is actually an award-winning Italian actress, Silvia Bottini.
Image credits: silviabottini_
#12 Overly Attached Girlfriend (Laina Morris)
The meme was born in 2012 after a YouTuber named Laina Morris posted a video in which she performs a parody version of Justin Bieber's "Boyfriend" with personalized lyrics that have been perceived as clingy. The video was submitted in response to Bieber's announcement of an online sing-off contest in promoting his celebrity perfume "Girlfriend." Unfortunately, after becoming a meme, Morris has dealt with some serious privacy issues, such as strangers finding her personal Facebook page and talking to her friends as well as people trying to access her college records. In 2019, Morris announced that she's quitting YouTube for good.
Image credits: Laina
#13 Doge (Kabosu)
The meme became popular in 2013 and was based on a 2010 photo of rescue-adopted Shiba Inu dog Kabosu that the owner uploaded to her personal blog. In the shot, Kabosu is captured sitting on a couch while glaring sideways at the camera with raised eyebrows. Kabosu is now 15 years old and she has an Instagram account.
Image credits: kabosumama
#14 Aliens Are To Blame For Everything (Giorgio Tsoukalos)
The guy behind the famous meme is a Swiss-born writer, ufologist, TV presenter and producer Giorgio Tsoukalos. The guy is mostly known for his appearances on the TV series called "Ancient Aliens." Fun fact—Giorgio is a proponent of the pseudoarchaeological theory that ancient alien astronauts interacted with ancient humans.
Image credits: Giorgio A. Tsoukalos
#15 Dramatic Dmitry (Vladimir Brest)
The "Dramatic Dmitry" photos are, in fact, images from the album Paranoia by Russian musician Vladimir Brest. The album was released in April 2011. "I myself am generally positive. However, I want my songs to be attached to these memes. It is also bad that most of the pictures are obscene. All this is seen by children. I don't want to be a person guilty of degradation and corruption of the young generation," the musician told Progorodsama back in 2013.
Image credits: Vladimir Brest
#16 Grumpy Cat (Tardar Sauce)
Grumpy Cat, whose real name was Tardar Sauce, first became famous after a few of her photos were posted on Reddit in late September 2012. The cat was known for her permanently grumpy appearance, which resulted from an underbite and feline dwarfism. Grumpy Cat's official Instagram account has over 2.5 million followers. Sadly, the cat passed away in May 2019 aged 7.
Image credits: realgrumpycat
#17 Good Luck Charlie (Mia Talerico)
The "Good Luck Charlie" meme came from a scene from the Disney Channel show "Good Luck Charlie." The photo and the GIF are often used as a synonym for "I don't know."
Image credits: Mia Talerico
#18 Ridiculously Photogenic Guy (Zeddie Smith)
The "Ridiculously Photogenic Guy" meme captures a man named Zeddie Smith running in the 2012 Cooper River Bridge Run. The shot gained quite a bit of popularity online after being posted on Reddit by computer programmer Will King, who took the photo, where users found the man to be incredibly handsome. The photo received over 40k upvotes.
Image credits: Zeddie Little
#19 Sweating Jordan Peele (Jordan Peele)
The famous gif/photo was born in September 2013, after the 3rd season premiere of "Key and Peele" aired in the US. The episode from which the meme originated includes a scene where a character played by Jordan Peele nervously sweats in response to his girlfriend confronting him about his internet habits.
Image credits: JordanPeele
#20 Hipster Barista (Dustin Mattson)
The meme originated in 2011 when someone uploaded it to a website called Quickmeme with a caption "I got this tattoo for my love of coffee, I got this one because it's ironic." "I do find it discouraging and disappointing that there was so much exposure brought to an attempt at making a joke of a culinary industry and the professional barista. To me, it's very telling on how we laud farm-to-table food, craft beer, cocktail mixology, but it's ok to have no respect for the specialty coffee world and the people who are committed to it," Dustin said in an interview with Eater.com.
Image credits: DustinColeMmm
#21 Ermahgerd (Maggie Goldenberger)
The "Ermahgerd" (which is a rhotacized pronunciation of "oh my god") meme first emerged on March 14th, 2012, as a post titled "Just a book owners smile…" The picture captures a girl named Maggie Goldenberger who, at the time, was around 11 years old. Maggie said that the picture was created in fourth or fifth grade when she and her friends seriously got into playing dress-up.
Image credits: Maggie Goldenberger
#22 A Schoolboy In A Swamp (Igor Nazarov)
16-year-old Russian schoolboy Igor Nazarov went viral on the interwebs back in 2016 after he did a photoshoot capturing him in the middle of a swamp behind a desk wearing a suit. The photo was taken in order to later be submitted to the most unusual photo contest in order to win a $277 prize.
Image credits: Igor Nazarov
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text:
Recent Premier League takeover looking to copy FSG and Liverpool success
The Clarets were taken over by ALK Capital on New Year's Eve
Dave PowellBusiness of Football Writer
New Burnley chairman Alan Pace. (Image: Burnley FC/PA Wire)
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When Michael Lewis penned his seminal book Moneyball: The Art of Winning an Unfair Game in 2003 the impact couldn't have been predicted.
Lewis' book, which looked at the revolutionary use of deep statistical analysis by Oakland Athletics general manager Billy Beane, shone a light on what is known as sabermetrics, the use of data aiding competitive performance through gains that others may not have been aware of.
The book was to be turned into a Hollywood movie with Brad Pitt playing Beane, and so the playing field in Major League Baseball started to become that little bit more level after Beane's trail blazing work.
Indeed, Liverpool owner John W. Henry, the chief of Fenway Sports Group, had long admired Beane's approach and sought to lure him to the Boston Red Sox in 2002, an offer that Beane declined despite big money on the table.
The Red Sox looked to someone else in Beane's mould, Theo Epstein, and then went about snapping an 86-year drought for a World Series title by implementing similar methods. Their 2004 success has been followed by a further three titles and their 10-year tenure at Liverpool has overseen a Champions League win and a first league championship in 30 years.
Beane and Henry could well get the chance to link up professionally later this year if, as anticipated, the reverse merger between FSG and Beane and financier Gerry Cardinale's RedBall Acquisition Corp goes ahead. It would be a deal that would likely see Beane, a minority shareholder in AZ Alkmaar and Barnsley, leave baseball to set about building a portfolio of European football clubs, similar to that of Red Bull and City Football Group.
Cardinale and Beane formed their special purchase acquisition company (SPAC) to enable them to enter such a market, to use Beane's knowledge of, and Cardinale's passion for, statistical analysis in sport. The addition of Dr Luke Bornn, a man who saw the true value of Mohamed Salah while in his role at Roma, is another nod to the direction that they see themselves heading.
FSG's approach at Liverpool has been similar, the Midas touch of Michael Edwards in the transfer market enabling the Reds to acquire elite talent who have yet to reach their ceiling at a lower price. The likes of Salah, Sadio Mane, Andrew Robertson and, most recently, Diogo Jota all fall into that bracket.
FSG and RedBall's potential deal
Trump loss could speed up merger
Cardinale offers insight
Reds could follow Man City and Leipzig
How it might affect transfer spend
FSG's success on the pitch, while not mirrored in the American ownership at Manchester United under the Glazers, has opened up the Premier League market to more US investors, keen to take a slice of the action.
The latest to arrive from across the Atlantic is ALK Capital, headed up by Wall Street veteran of some 20 years, Alan Pace.
Now, Burnley may not smack as being the most marketable or fashionable but Pace and ALK see value in the Clarets and have an interest themselves in statistical analysis having recently invested in player scouting software AiScout and Player LENS.
If you were to compare Burnley to the MLB then you would probably land at someone like Oakland, a small market team with one of the lowest budgets in the league who consistently punch above their weight owing to their approach to thinking outside the box.
Billy Beane has been linked with an investment in Liverpool (Image: Getty Images)
"There is a good chance for us to do things differently," Pace told the media upon his arrival at Turf Moor.
"What you'll learn from us is that we under-promise and over-deliver. The last thing we want to do is come here and say 'We'll spend £30million in the window, we'll be in the Champions League in five years'. We are here to work our tails off and there will be some cool stuff along the way.
"There is an evolution on the sports tech side and it's really time football started to go a bit further. When you look at data and analytics, they don't require someone needing to have done 30 years as a scout. There is a little bit of living in the past. (in football)."
Burnley do have appeal.
An average finish in the Premier League of 12th, previous participation in European competition, a Category 1 Academy and a fine manager used to conjuring rabbits out of hats each season mean that the recipe for further growth is there for ALK.
"There are going to be a few things that surprise people," Pace said.
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"We do think it's very different to what has gone before. I've been reading the fan forums and they say, 'Oh, they will bring amazing things from America'.
"We might bring some good ideas but you already have the best thing. You've already got the crown jewel, now we just have to put it into the crown.
"We don't want to rip out the heart of anything. It's nurturing what is already there. There should be announcements that people may say, 'Wow, they're doing that in a pandemic?'"
Will Burnley be challenging Liverpool's place at the top table? Probably not.
But there is likely to be more influence coming into the Premier League from the US, and more focus on data-driven analysis that could see changes to the traditional way that the transfer market has operated.
But should the RedBall/FSG deal come to fruition the Reds could have one of the pioneers in their corner in Beane. An intriguing 2021 lies in store both on and off the field.
Liverpool FCLiverpool transfer news - Mohamed Salah message, Erling Haaland latest, Diogo Jota returnLiverpool transfer news, rumours, gossip and speculation with the January transfer window open including the latest on Rodrigo De Paul, Ibrahima Konate, Kaide Gordon, Virgil van Dijk, Gini Wijnaldum and more.
Mohamed Salah sets record straight and sends message to Liverpool over future
Liverpool FCMohamed Salah has once again hinted that he would like to stay at Liverpool beyond his current contract
Dermot Gallagher says Paul Tierney got decisive Liverpool call wrong against Manchester United
Liverpool FCLiverpool and United took a point each as Sunday's Premier League showdown at Anfield ended in a goalless draw
Everton FCEverton transfer news - Tariq Lamptey linked, Joshua Zirkzee competition, Dele Alli exit claimEverton transfer news, rumours, gossip and speculation with the January transfer window open including the latest on Isco, Leon Bailey, Moise Kean, Joshua Zirkzee, Josh King, Bernard and more.
Liverpool transfer news - Mohamed Salah message, Erling Haaland latest, Diogo Jota return
Liverpool v Manchester United sets new Sky Sports TV record
Liverpool FCJurgen Klopp's men played out a goalless draw against Ole Gunnar Solskjaer's side in Sunday's crunch Premier League clash at Anfield
Liverpool might have stumbled across exciting new partnership against Manchester United
Liverpool FCLiverpool played out a frustrating draw with Manchester United on Sunday by Xherdan Shaqiri and Thiago Alcantara were shining lights in the game


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text:
Police expand search for man missing in freezing Victorian high country
Posted SunSunday 14 JulJuly 2019 at 12:46amSunSunday 14 JulJuly 2019 at 12:46am , updated SunSunday 14 JulJuly 2019 at 5:29amSunSunday 14 JulJuly 2019 at 5:29am
Poshik Sharma was wearing a beanie and red scarf when he was last seen. (Supplied: Victoria Police)
Police and emergency services have expanded their search for a 21-year-old man who has been missing since Thursday in "freezing cold" conditions in Victoria's high country.
Police say they have "grave concerns" for Poshik Sharma
The 21-year-old was last seen leaving a Marysville pub on Thursday afternoon
It has been cold, rainy and even snowing in the area since he disappeared
Poshik Sharma was last seen leaving the Duck Inn in Marysville, north-east of Melbourne, at about 3:50pm on Thursday.
Mr Sharma, an Indian national who lives in Werribee, in Melbourne's outer west, had been travelling with friends to the state's alpine region.
They were staying the night a short walk from the Duck Inn and had a disagreement before he left the pub.
"We think he may have been going back to his accommodation," Inspector David Ryan said.
"So in the space of about 50 metres this man has disappeared. He's walked off into the bush … or he's got a lift from someone. That's what we're hoping."
State Emergency Service (SES), Search and Rescue and police personnel have expanded their search to the bush, walking tracks and waterways surrounding the small town.
It has been a rainy and cold week for Marysville and its surrounds, with temperatures dipping to freezing overnight.
Emergency services workers have come from Melbourne and other parts of the state to join the search. (ABC News)
Inspector Ryan, who is overseeing police in the search for Mr Sharma, said it had snowed on Saturday and was "freezing cold".
"Our best hope is that Poshik has found some shelter. His condition, if he hasn't found some shelter, is likely to be very poor at this stage and that's why we hold really grave concerns for his safety," he said.
Dam drained as part of search
SES crews from other parts of Victoria have joined the search, alongside local officers, police dogs, and bush search and rescue experts.
Police began to drain a nearby dam on Sunday afternoon while search and rescue teams scoured the water.
"If he's tried to walk off, had a mishap and gone into the water — it's not a good outcome," Inspector Ryan said.
Julie Cowan, founder of Search and Rescue Dogs Australia, said her dogs had become "very interested" in the body of water.
The search was expanded on Sunday as fears grew for Mr Sharma's safety. (ABC News)
Ms Cowan described the conditions as "treacherous".
"Most of the tracks are wet and slippery, most of the trees have fallen over … I can't see him wandering off into this thick dense bush, given his history."
Disappearance is out of character, police say
Inspector Ryan said the disagreement between the friends was "nothing that would suggest he would do anything other than return to the accommodation".
"His disappearance in these circumstances is out of character, there is nothing to suggest that he is in any way suffering any psychological issues," Inspector Ryan said.
The friends had been drinking alcohol, but police did not believe intoxication was a factor in his disappearance.
Mr Sharma's social media and online banking accounts have not been touched since Thursday, and police were not able to trace his mobile phone.
He was wearing a red scarf, "distinctive" red shoes, black beanie and black puffer jacket when he was last seen, and police are urging anyone who may have seen him to come forward with information.
Family members joined the call for help on Sunday morning, telling reporters Mr Sharma and his four friends had stopped at Marysville on their way to the Mount Buller ski resort.
"I don't know where he is now, so please help me to find him," his sister Samta Sharma told Channel Nine news.
Inspector Ryan said they had not heard from anyone who had sighted Mr Sharma, and police were "desperate" for information from the public.
Marysville is a gateway from Melbourne to the popular Mount Buller and the state's alpine region. (ABC News: Patrick Rocca)
Posted 14 JulJuly 2019SunSunday 14 JulJuly 2019 at 12:46am , updated 14 JulJuly 2019SunSunday 14 JulJuly 2019 at 5:29am
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