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• W4210796756 abstract "Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Plant cells maintain a low luminal pH in the trans-Golgi-network/early endosome (TGN/EE), the organelle in which the secretory and endocytic pathways intersect. Impaired TGN/EE pH regulation translates into severe plant growth defects. The identity of the proton pump and proton/ion antiporters that regulate TGN/EE pH have been determined, but an essential component required to complete the TGN/EE membrane transport circuit remains unidentified − a pathway for cation and anion efflux. Here, we have used complementation, genetically encoded fluorescent sensors, and pharmacological treatments to demonstrate that Arabidopsis cation chloride cotransporter (CCC1) is this missing component necessary for regulating TGN/EE pH and function. Loss of CCC1 function leads to alterations in TGN/EE-mediated processes including endocytic trafficking, exocytosis, and response to abiotic stress, consistent with the multitude of phenotypic defects observed in ccc1 knockout plants. This discovery places CCC1 as a central component of plant cellular function. Editor's evaluation This paper reports compelling evidence that the single isoform of cation chloride cotransporter (CCC1) encoded in Arabidopsis thaliana provides a cation and anion efflux mechanism to regulate pH in the trans-Golgi/Early Endosome Network. https://doi.org/10.7554/eLife.70701.sa0 Decision letter eLife's review process Introduction The transport of ions across biological membranes is fundamental to diverse cellular functions such as energy production, nutrient uptake and distribution, growth, signalling, and osmoregulation. Ion influx and efflux must be coordinated to regulate osmotic potential, and to maintain charge balance within and across cellular compartments. Ion efflux out of cells contributes towards processes as varied as neuronal function in animals, heavy metal tolerance in bacteria, and salinity tolerance in plants. Ion efflux out of subcellular compartments is also critical for core cellular functions such as chloroplastic photosynthetic efficiency in plants (Correa Galvis et al., 2020). Cation chloride cotransporters (CCCs) are cation and anion symporters that are present in all domains of life, whether archaea, animal, yeast, or plant. Chloride (Cl-) is transported by CCCs electroneutrally across membranes with the cations of sodium (Na+) and/or potassium (K+). A single CCC exists in the archeaon, Methanosarcina acetivorans, which does not share a high degree of similarity to eukaryotic CCCs (Hartmann and Nothwang, 2015). In animals, CCCs have been separated into two subfamilies: the Cl- and K+ efflux symporters (KCCs), and a second subfamily with two clades of CCC, the Cl- and Na+/K+ influx symporters (NKCCs), and the Cl- and Na+ influx symporters (NCCs). Vertebrates typically harbour several CCC isoforms from each subfamily. For example, humans (Homo sapiens) have seven CCC isoforms (4 KCC, 3 NKCC/NCCs) and zebrafish (Danio rerio) have 12 CCCs in total (Hartmann et al., 2014). In non-vascular plants there are two clades of CCC, CCC1 and CCC2, with some bryophytes, such as the moss Physcomitrium patens (formerly Physcomitrella patens) with up to seven isoforms (Henderson et al., 2018). In contrast, in flowering plants (angiosperms), CCCs are represented by only the CCC1 clade, with very few isoforms per plant. For instance, rice (Oryza sativa), contains two CCC1s, and Arabidopsis (Arabidopsis thaliana) has only one CCC1. In vertebrates, CCCs are involved in diverse but specific cellular processes including osmoregulation in kidneys and neurons (regulation of cell volume), potassium secretion in muscles and regulation of Cl– concentrations in neural tissue (Blaesse et al., 2009; Hartmann and Nothwang, 2015). NKCCs and NCCs drive influx of ions across the cell membrane, often by utilising large extracellular Na+ concentrations to drive transport of Cl- and K+, or just Cl- in the case of NCC, whereas KCCs utilise K+ gradients to mediate Cl- efflux out of the cytosol (Blaesse et al., 2009; Arroyo et al., 2013). The importance of Cl- management and osmoregulation for cell and organ function is highlighted by the large number of human diseases and conditions associated with CCC malfunction including schizophrenia, deafness, muscle spasticity, epilepsy, Andermans syndrome (neuropathy), and, Bartter and Gitelman syndrome (both characterised by kidney disfunction) (Simon et al., 1996; Kunchaparty et al., 1999; Howard et al., 2002; Boettger et al., 2002; Rivera et al., 2002; Boulenguez et al., 2010; Kim et al., 2012). The single CCC1 protein in the model plant Arabidopsis also appears to have a crucial, but unexplained, role in plant function. When Arabidopsis CCC1 is genetically ablated, ccc1 plants exhibit complex and severe phenotypic defects which include a large reduction in overall plant size, a bushy appearance characterised by increased axillary shoot outgrowth, frequent stem necrosis, very low fertility, alterations in pathogen response, changes in cell wall composition, and changes in seed ion concentrations (Johnson et al., 2004; Colmenero-Flores et al., 2007; McDowell et al., 2013; Henderson et al., 2018; Han et al., 2020). Arabidopsis ccc1 also displays changes in ion distribution. Hydroponically grown ccc1 plants accumulate more Cl- in shoots under both control and 50 mM NaCl conditions while soil-grown ccc1 plants accumulate more Cl- in shoots when exposed to 50 mM Cl- salts (Colmenero-Flores et al., 2007; Henderson et al., 2015). Similarly, roots of rice Osccc1.1 plants also had altered ion accumulation profiles under both control and saline conditions (Chen et al., 2016). While rice harbours two CCC1 proteins, Arabidopsis is the ideal system for investigating the role of CCCs in higher plants due to the presence of only a single CCC homologue, eliminating the possibility for functional redundancy (Henderson et al., 2018). Plant, fungi, and animal CCC subcellular localisations are different. While many of the animals’ CCCs have been found on the cell membrane (plasma membrane [PM]), the yeast (Saccharomyces cerevisiae) CCC homologue, VHC1, is localised not on the exterior membrane of the cell, but on the yeast vacuolar membrane (Petrezselyova et al., 2013). Independent proteomic studies in Arabidopsis have revealed that the Arabidopsis CCC1 is highly abundant in the trans-Golgi network/early endosome (TGN/EE) (Sadowski et al., 2008; Drakakaki et al., 2012; Groen et al., 2014; Nikolovski et al., 2014), with the latest study defining CCC1 as a TGN/EE marker protein (Parsons et al., 2019). Heterologous expression studies in tobacco (Nicotiana benthamiana) epidermal cells also suggested an endomembrane localisation of the Arabidopsis CCC1 using a translational fluorescent protein fusion (Henderson et al., 2015). This raises the question of the role of CCC1 in this organelle. Several transport proteins in the TGN/EE have been identified, with mutants of the transport proteins involved in TGN/EE pH regulation exhibiting similar phenotypic aspects to ccc1 knockouts. CCC1 is therefore a candidate ion transporter that might contribute to TGN/EE pH regulation. The plant TGN/EE is an organelle with a complex cellular role. One of its key roles is sorting and delivering proteins to the apoplast, PM, and vacuole (Dettmer et al., 2006; Viotti et al., 2010; Sze and Chanroj, 2018). Four TGN/EE Cl- and K+ transporters have previously been identified: the H+/K+ antiporters NHX5 and NHX6, and the H+/Cl- antiporters CLCd and CLCf. These antiporters import K+ and Cl- into the TGN/EE in exchange for luminal protons and work in conjunction with the TGN/EE V-ATPase complex to regulate luminal pH (Bassil et al., 2011; Reguera et al., 2015; Scholl et al., 2021). Finely tuned luminal pH is required for the cellular function of the TGN/EE (Martinière et al., 2013; Luo et al., 2015; Reguera et al., 2015). Plants with reduced V-H+-ATPase activity (e.g. det3 mutants) have an elevated TGN/EE pH and exhibit severe developmental growth defects, which is accompanied by alterations in exocytosis of PM proteins, such as BRI1, defects in cell division and expansion and hypersensitivity to salt (Schumacher et al., 1999; Dettmer et al., 2006; Brüx et al., 2008; Krebs et al., 2010; Luo et al., 2015). Double nhx5/nhx6 mutants have similar, but not identical, growth defects to det3, with reduced cell length, a large decrease in overall plant size, and increased salt sensitivity of root growth and seed germination (Bassil et al., 2011; Reguera et al., 2015). As in det3, trafficking of BRI1 is altered in nhx5/nhx6; however, only recycling is affected while secretion to the PM is not (Dragwidge et al., 2019). In addition, the PM abundance of the membrane integrated proteins, PIN1 and PIN2, are reduced in nhx5/nhx6 (Dragwidge et al., 2018). However, in contrast to det3, the nhx5/nhx6 TGN/EE lumen is hyperacidic, consistent with the proposed role of NHX in exporting protons out of the TGN/EE (Luo et al., 2015; Reguera et al., 2015). The hyperacidity of nhx5/nhx6 TGN/EE lumen results in mis-sorting of vacuolar proteins due to altered binding of targets by the vacuolar sorting receptor VSR1;1 (Reguera et al., 2015). A clcld single knockout has a mild increase in pathogen sensitivity. The less severe phenotype of this knockout might be a result of the two TGN/EE resident CLC proteins, which are likely to exhibit functional redundancy similar to the two TGN/EE NHXs. Collectively, these observations demonstrate that a typical consequence of alterations in TGN/EE lumen pH regulation are defects in protein trafficking, particularly in protein exocytosis, changes in salt tolerance, and defects in cell elongation and division, resulting in severe impacts on plant growth. The current model of TGN/EE pH regulation, as outlined above, is incomplete; it currently includes a proton pump (V-H+-ATPase) and anion- and cation-proton exchangers (CLC, NHX) (Sze and Chanroj, 2018), but a transport protein that mediates either cation or anion efflux has not yet been identified. Both import and export of ions are crucial components of dynamic cellular and organellar ion regulation. If both do not occur, cation and anion import into the TGN/EE would cease once the gradient becomes too high, which would inhibit the function of the antiporters. Plant CCC1s, with their cation and anion symport function and their residency in the TGN/EE, are ideal candidates to fill this missing role and complete the circuit through export of Cl- and K+ from the TGN/EE. Here, we characterised the role of Arabidopsis CCC1 in the TGN/EE. We demonstrate that TGN/EE-localised CCC1 rescues cellular defects of ccc1 knockouts in Arabidopsis, and that CCC1 impacts TGN/EE luminal pH and osmotic regulation. Genetic ablation of CCC1 function leads to defects in TGN/EE-dependent processes, specifically, ccc1 plants exhibit reduced rates of exocytosis and endocytic trafficking, typical of altered TGN/EE pH regulation. As such, we propose that CCC1 is a missing core component of the ion- and pH-regulating machinery of the TGN/EE. Results CCC1 is ubiquitously expressed Previous reports on CCC1 expression are contradictory. Promoter-GUS studies indicated that CCC1 expression was restricted to specific tissues, such as root stele or hydathodes and pollen, while RNA transcriptomic studies, including single-cell RNAseq, suggest expression occurs in a broader range of cell types (Colmenero-Flores et al., 2007; Wendrich et al., 2020). To clarify the tissue expression pattern of CCC1, we transformed wildtype Arabidopsis plants with a 2 kb genomic DNA sequence upstream of the CCC1 coding region driving the expression of nuclear-localised triple Venus (a bright variant of the yellow fluorescent protein) or β-glucuronidase (GUS), named CCC1prom::Venus and CCC1prom::GUS, respectively. Combined analysis of fluorescence and GUS staining revealed that CCC1 is expressed in all cell types, including all root cells, hypocotyl, leaf and stem epidermis, guard cells and trichomes, as well as mesophyll cells and all flower parts (Figure 1). CCC1 promoter activity reported by Venus fluorescence, or by GUS-activity, was slightly different despite use of the identical promoter sequence. For instance, fluorescence was detectable in root cortex and epidermis cells, including root hairs, and in the gynoecium, while GUS staining did not indicate expression in these cells. This is likely due to the greater sensitivity of the Venus fluorescence method. Figure 1 Download asset Open asset CCC1 is expressed ubiquitously. CCC1 promoter-driven expression of either NLS-Venus (yellow, bright YFP variant with a nuclear localisation signal) or ẞ-glucuronidase (blue GUS staining). (A–H) NLS-Venus expression indicating CCC1 promoter activity in all root cells, including (A) the root tip, (B) root epidermal cells; (C) hypocotyl; all leaf cells including (D) trichomes, (E) guard cells, and (F) leaf epidermal and mesophyll cells; and reproductive organs (G) gynoecium, and (H) stamen tissues. (I–M) GUS staining indicating promoter activity predominantly in (I) younger leaves, (J) pollen, (K) root stele, (L) floral stem, and (M) stamen. Scale bars are 50 µm (images A, B, G), 100 µm (images C, D, H, J), 5 µm (image E), 20 µm (image F), 5 mm (image I), 200 µm (images K, M) and 1000 µm (image L). Loss of CCC1 results in growth defects in root cells ccc1 knockout plants are severely affected in their growth, including a reduced shoot size and shorter primary roots (Figure 2A, and previously quantified by Colmenero-Flores et al., 2007; Henderson et al., 2015). We investigated the origin of the root phenotype of ccc1 at a cellular level and found that CCC1 function is required for cell elongation, contributing to the reduced root length in knockout mutants. ccc1 develop both shorter root epidermal cells, and shorter root hairs (Figure 2B–E) and a complete lack of collet hairs (Figure 2F). Collet hairs are epidermal root hairs formed in some plant species in the transition zone between the root and the hypocotyl (Sliwinska et al., 2015). In addition, ccc1 root hairs displayed branching and bulging (Figure 2—figure supplement 1), although at an extremely low frequency; while ruptured root hairs were never observed. Figure 2 with 1 supplement see all Download asset Open asset ccc1 plants show defects in cell elongation. (A) Top image, ccc1 (right) have smaller shoots and deformed leaves compared to wildtype (left) plants. Plants grown 26 days in short day, scale bar is 2 cm. Bottom image, ccc1 (right) have shorter primary roots compared to wildtype (left) plants. Plants grown 14 days in long day, scale bar is 2 cm. (B–C) Root epidermal cells are shorter in ccc1. n > 13 plants. Images are maximum intensity projections of cell wall autofluorescence. Scale bars are 50 µm. (D–E) ccc1 plants have shorter root hairs. n > 900 root hairs of >30 plants. Scale bars are 200 µm. (F) ccc1 plants do not develop collet hairs. Scale bars are 500 µm. Boxplots show range; median, first and third quartile are indicated. One-way ANOVA used to determine p. * indicates p < 0.05, **** indicates p < 0.0001. Figure 2—source data 1 Data analysed and presented in Figure 2 and supplement. https://cdn.elifesciences.org/articles/70701/elife-70701-fig2-data1-v3.xlsx Download elife-70701-fig2-data1-v3.xlsx Independent of the defect of root hair elongation, ccc1 plants frequently developed root hairs in cell files that usually exclusively contain atrichoblasts (non-root hair cells) in the wildtype. These ectopic root hairs are formed in cells that have trichoblast identity, as they express the trichoblast marker PRP3::H2B-2× mCherry (Marquès-Bueno et al., 2016). Fluorescence of this nuclear-localised marker occurred in multiple adjacent cells in ccc1, which did not occur in wildtype plants (Figure 2—figure supplement 1). Functional GFP-CCC1 localises to the endomembrane system We had previously localised Arabidopsis CCC1-GFP to the Golgi and TGN/EE in transient expression assays in N. benthamiana (Henderson et al., 2015). In addition, several proteomic studies corroborate that CCC1 is present in this organelle (Sadowski et al., 2008; Drakakaki et al., 2012; Groen et al., 2014; Nikolovski et al., 2014), with one study identifying CCC1 as a high-confidence TGN/EE resident protein (Parsons et al., 2019). Therefore, we sought to investigate the role of CCC1 in this organelle. We generated an N-terminally tagged GFP-CCC1 construct, which, when stably expressed in ccc1 plants, was able to complement the root hair phenotype. To express this construct, we used the EXP7 (Expansin7) trichoblast-specific promoter, which drives expression within root epidermal cells. This approach was adopted after many attempts to generate plants that express the CCC1 protein with different tags and different fusion orientations using the native promoter (Supplementary file 1a). Approaches included the use of different protein linkers between CCC1 and the different tags, green and red fluorescent proteins, the smaller FLAG tag, and multiple tag locations, including both termini and internal placements. No transformants were recovered with a terminally tagged protein expressed using the native promoter. This difficulty in obtaining transformed plants might suggest that terminal tagging interferes with CCC1 function in embryonic or meristematic tissue where CCC1 is active. Generation of an antibody was also not successful. Internal tagging might have disrupted protein folding, despite the selection of tagging sites according to a protein model made using Swissport since transformants were recovered, but no fluorescence could be detected and the ccc1 knockout phenotype was not complemented in those plants. We therefore decided to express GFP-CCC1 in a mature cell type, in which we had identified a clear phenotypic defect in ccc1 − trichoblasts. GFP-CCC1 expression in these epidermal cells was successful and complemented the short root hair phenotype of ccc1 knockout plants (Figure 3). GFP-CCC1 expression driven by the EXP7 promoter did not rescue the altered trichoblast patterning of ccc1 roots (Figure 3—figure supplement 1). This is consistent with the EXP7 promoter driving expression after cell fate determination, but before final cell length is achieved. Interestingly, expression of GFP-CCC1 on the EXP7 promoter did not rescue collet hair formation, which highlights the difference between root hair and collet hair development and indicates that different gene networks are important for collet hair formation compared to that of root hairs. Figure 3 with 1 supplement see all Download asset Open asset Stably expressed GFP-CCC1 is functional and localised to the trans-Golgi-network/early endosome (TGN/EE). (A) GFP-CCC1 (green) and VHAa1-RFP (magenta) colocalise. Colocalisation was calculated using DiAna object-based colocalisation plugin in ImageJ; Pearson’s coefficient was also calculated as 0.86 ± 0.055. Error is standard deviation. n = 15 cells of five plants. Scale bars are 10 µm. Images are single representative optical sections from a stack. (B–C) Expression of GFP-CCC1 rescues ccc1 root hair length defects. n > 1300 root hairs. Scale bars are 200 µm. Boxplot shows range; median, first and third quartile are indicated. One-way ANOVA used to determine p. **** indicates p < 0.0001. Figure 3—source data 1 Data analysed and presented in Figure 3 and supplement. https://cdn.elifesciences.org/articles/70701/elife-70701-fig3-data1-v3.xlsx Download elife-70701-fig3-data1-v3.xlsx The stable expression of GFP-CCC1 in Arabidopsis revealed a similar subcellular localisation pattern to what we previously observed in N. benthamiana (Henderson et al., 2015), showing that CCC1 is localised to internal organelles in a native cell type. Time-lapse imaging of GFP-CCC1 movement was consistent with what could be expected for the Golgi or TGN/EE, however, GFP-CCC1 labelled organelles did not resemble the Golgi (Videos 1 and 2). To identify the GFP-CCC1 labelled compartments, we crossed the stably expressed marker, VHAa1-RFP, into plants expressing GFP-CCC1 (Figure 3A). Colocalisation of GFP-CCC1 and VHAa1-RFP was measured using object-based colocalisation analysis, with the ImageJ plugin DiAna, which revealed that 73% ± 9% of VHAa1-RFP colocalised with GFP-CCC1, while 58% ± 11% of GFP-CCC1 colocalised with VHAa1-RFP. The asymmetrical colocalisation indicates that, in addition to the TGN/EE, CCC1 might localise to additional organelles of the endomembrane system (Figure 3A), which is similar to NHX5 and NHX6 (Reguera et al., 2015). In addition, the Pearson’s correlation coefficient of pixel signal intensity for RFP and GFP channels was calculated, which gave a correlation value of 0.86 ± 0.055. Pharmacological treatment further confirmed the endosomal localisation. Treatment with the trafficking inhibitor, brefeldin A (BFA), caused the GFP signal to accumulate in the centre of BFA bodies, consistent with a TGN/EE localisation (Figure 3—figure supplement 1). Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse showing movement of GFP-CCC1 labelled subcellular compartments in a root hair. GFP-CCC1 localises to motile intracellular organelles, expression driven with EXP7 promoter. Time series of the root hair was taken through the centre plane of the root hair. 20 s shown per second at 10 frames per second. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse showing movement of GFP-CCC1 labelled subcellular compartments in a root epidermal cell. GFP-CCC1 localises to motile intracellular organelles, expression driven with EXP7 promoter. Time series was imaged below the radial plasma membrane (PM). 20 s shown per second at 10 frames per second. We then investigated if CCC1 might cycle between the TGN/EE and PM. Cases like this have been observed for other ion transporters that localise mainly in endosomes but function at the PM, such as the iron transporter, IRT1. PM localisation of IRT1 can be visualised using pharmacological treatment with tyrphostin A23 (TyrA23), which acidifies the cytosol resulting in, among other effects, an inhibition in endocytosis and subsequent accumulation of cycling proteins at the PM (Barberon et al., 2011; Dejonghe et al., 2016). For CCC1, no change in the subcellular localisation was observed after treatment with TyrA23 (Figure 3—figure supplement 1). Similarly, an osmotic shock treatment, which can sometimes induce a change in protein localisation (Boursiac et al., 2008; Hachez et al., 2014), did not lead to any observable changes in GFP-CCC1 localisation (Figure 3—figure supplement 1). Loss or knockdown of other TGN/EE-localised proteins, such as the H+-V-ATPase, can affect the TGN/EE morphology (Dettmer et al., 2006). Transmission electron microscopy (TEM) of high-pressure-frozen, freeze-substituted samples revealed that the lack of CCC1 did not lead to obvious morphological changes in the TGN/EE ultrastructure, with the appearance of all organelles being similar between ccc1 mutant and wildtype cells (Figure 3—figure supplement 1). This suggests that the defects observed in ccc1 knockouts do not manifest through gross morphological changes in organelle structure, and so may instead be connected to other properties of the TGN/EE such as their luminal conditions. Hyperosmotic stress rescues root hair elongation and improves seed germination of ccc1 Knockouts of the TGN/EE-localised ion transporters, NHX5 and NHX6, as well as the proton pump V-H+-ATPase are salt-sensitive, with salt-associated germination and root growth phenotypes (Krebs et al., 2010; Bassil et al., 2011). We therefore further investigated the response of ccc1 plants to salt stress. As rice OsCCC1.1 is implicated in maintaining cell sap osmolality (Chen et al., 2016), we also investigated the response to osmotic stress. Plants were grown on media with added salt (NaCl), or an isosmotic concentration of mannitol or sorbitol, to determine if any changes in tolerance were the result of ionic or osmotic stress (Figure 4A–B and Figure 4—figure supplement 1). Wildtype and ccc1 seeds were germinated on media containing 0–300 mM NaCl or 0–600 mM of mannitol or sorbitol. ccc1 seeds typically germinated slightly earlier than wildtype seeds, so germination of seeds was assessed 8 days after imbibition (=6 days after transfer to the growth chamber). Under control conditions, over 90% of wildtype, ccc1-1 and ccc1-2 seeds germinated. The germination rate of all genotypes decreased with higher salt, mannitol and sorbitol concentrations, yet, this decrease was smaller in the ccc1 knockouts than wildtype. On 450 mM mannitol, 30% ± 7% (SEM) of wildtype seeds germinated, while 79% ± 6% of ccc1-1 and 64% ± 7% of ccc1-2 seeds germinated. On 600 mM mannitol, 4% ± 2% of the wildtype seeds germinated, while 27% ± 6% of ccc1-1 and 20% ± 6% of ccc1-2 seeds were able to germinate in this condition. Similar results were obtained on sorbitol (Figure 4—figure supplement 1). A similar trend was also observable when plants were grown on media containing NaCl. Growth and germination of both wildtype and ccc1 plants on NaCl was lower than observed on isosmotic concentrations of mannitol and sorbitol. In addition, the increased tolerance of ccc1 seed germination to NaCl was less pronounced than on mannitol and sorbitol. At 150 mM NaCl, 67% ± 7% of wildtype seeds germinated compared with 85% ± 5% of ccc1-1 and 95% ± 2% of ccc1-2 seeds. Figure 4 with 1 supplement see all Download asset Open asset Increased external osmolarity rescues cell elongation defects in ccc1. (A, D) A higher percentage of ccc1 seeds germinate on media with a higher osmolarity, adjusted with increasing (A) mannitol or (D) NaCl concentrations. Number of germinated seeds assessed 6 days after imbibition. n > 90 seeds. (B–C, E–F) Collet hair formation in ccc1 is rescued on media with a higher osmolarity. n > 9 plants for 450 mM treatments, >30 plants for all other treatments. Scale bars are 300 µm. Germination and collet hair assays with mannitol, sorbitol, and NaCl were performed together and therefore share the same control (0 mM). (G–H) The slower rate of root hair elongation in ccc1 is rescued when grown in media with 150 mM mannitol (see Videos 3–6). Under control conditions, between 50 and 100 min after initiation, wildtype roots hairs elongated at a rate of 0.88 ± 0.27 µm min–1 compared with a rate of 0.47 ± 0.08 µm min–1 in ccc1. Figure 4—source data 1 Data analysed and presented in Figure 4 and supplement. https://cdn.elifesciences.org/articles/70701/elife-70701-fig4-data1-v3.xlsx Download elife-70701-fig4-data1-v3.xlsx In addition to the higher germination rate of ccc1 seeds on media with a high osmolarity, we observed that a striking phenotype of ccc1 – the absence of collet hair formation (Figure 2) – was rescued when ccc1 was grown on media with a high osmolarity. Under control conditions, over 90% of wildtype seedlings had a complete ring of collet hairs, while they were present in 0% of ccc1 seedlings of either knockout allele (Figure 4C–F and Figure 4—figure supplement 1). When grown on 150 mM mannitol, 86% of ccc1-1 seedlings and 75% of ccc1-2 seedlings had collet hairs present as either a partial or complete ring around the hypocotyl. This improved to 100% of ccc1-1% and 98% of ccc1-2 on media with 300 mM mannitol. As occurred for germination, little difference was seen between mannitol and sorbitol treatments (Figure 4—figure supplement 1). NaCl treatment also resulted in the presence of collet hairs in ccc1 knockouts, however, not to the same magnitude as observed in mannitol and sorbitol treatments. When treated with 150 mM NaCl, 98% of ccc1-1% and 63% of ccc1-2 had collet hairs. Inspection of the root-hypocotyl base region where collet hairs form under control conditions shows that collet hairs do initiate in ccc1 plants but fail to elongate (Figure 4—figure supplement 1). This suggests that the absence of collet hairs in ccc1 is due to cell elongation defects, with no obvious defects in cell identify (Figure 4—figure supplement 1). This further suggests that osmotic stress rescues cell elongation defects in ccc1 roots. To further investigate if raising external osmolarity leads to increased cell elongation in ccc1 roots, we investigated if the reduced speed of root hair growth of ccc1 plants is also rescued by increasing external osmolarity. To determine root hair elongation speed, root hairs below a final length of 50 µm were not measured, this consists of all root hairs that initiated, but did not elongate. We did this, as it was not possible to acquire an accurate measurement of elongation rate from such root hairs. Time-lapse microscopy revealed that ccc1 root hairs grew for the same duration as wildtype hairs, but at a reduced speed leading to a reduced final root hair length (Figure 4G, Videos 3–6). Between 50 and 100 min after elongation initiated, wildtype root hairs had an average elongation rate of 0.88 ± 0.27 µm min–1, while ccc1 root hairs elongated at half that speed, at 0.47 ± 0.08 µm min–1 (Figure 4G). Strikingl" @default.
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• W4210796756 title "Author response: Plant Trans-Golgi Network/Early Endosome pH regulation requires Cation Chloride Cotransporter (CCC1)" @default.
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