Plant Protoplasts Research Articles

Extracellular malate induces stomatal closure via direct activation of guard-cell anion channel SLAC1 and stimulation of Ca2+ signalling.

Plants secrete malate from guard cells to apoplast under stress conditions and exogenous malate induces stomatal closure. Malate is considered an extracellular chemical signal of stomatal closure. However, the molecular mechanism of malate-induced stomatal closure is not fully elucidated. We investigated responses of stomatal aperture, ion channels, and cytosolic Ca2+ to malate. A treatment with malate induced stomatal closure in Arabidopsis thaliana wild-type plants, but not in the mutants deficient in the slow (S-type) anion channel gene SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1). The treatment with malate increased S-type anion currents in guard-cell protoplasts of wild-type plants but not in the slac1 mutant. In addition, extracellular rather than intracellular application of malate increased the S-type currents of constitutively active mutants of SLAC1, which have kinase-independent activities, in a heterologous expression system using Xenopus oocytes. The treatment with malate transiently increased cytosolic Ca2+ concentration in the wild-type Arabidopsis guard cells and the malate-induced stomatal closure was inhibited by the Ca2+ channel blocker and the Ca2+ chelator. These results indicate that extracellular malate directly activates SLAC1 and simultaneously stimulates Ca2+ signalling in guard cells, resulting in steady and solid activation of SLAC1 for stomatal closure.

Protoplasts: small cells with big roles in plant biology.

Compared with Agrobacterium-mediated stable transformation, which is often expensive, time-consuming, and technically challenging, plant protoplasts provide a versatile cell system for conducting plant biology research by taking advantage of its low cost, high speed, high flexibility, and convenience. So far, techniques for protoplast isolation, transfection, and incubation have been well-established in many plant species, such as arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and maize (Zea mays).

Building the Plant SynBio Toolbox through Combinatorial Analysis of DNA Regulatory Elements.

While the installation of complex genetic circuits in microorganisms is relatively routine, the synthetic biology toolbox is severely limited in plants. Of particular concern is the absence of combinatorial analysis of regulatory elements, the long design-build-test cycles associated with transgenic plant analysis, and a lack of naming standardization for cloning parts. Here, we use previously described plant regulatory elements to design, build, and test 91 transgene cassettes for relative expression strength. Constructs were transiently transfected into Nicotiana benthamiana leaves and expression of a fluorescent reporter was measured from plant canopies, leaves, and protoplasts isolated from transfected plants. As anticipated, a dynamic level of expression was achieved from the library, ranging from near undetectable for the weakest cassette to a ∼200-fold increase for the strongest. Analysis of expression levels in plant canopies, individual leaves, and protoplasts were correlated, indicating that any of the methods could be used to evaluate regulatory elements in plants. Through this effort, a well-curated 37-member part library of plant regulatory elements was characterized, providing the necessary data to standardize construct design for precision metabolic engineering in plants.

Advances in protoplast transfection promote efficient CRISPR/Cas9-mediated genome editing in tetraploid potato.

An efficient method of DNA-free gene-editing in potato protoplasts was developed using linearized DNA fragments, UBIQUITIN10 promoters of several plant species, kanamycin selection, and transient overexpression of the BABYBOOM transcription factor. Plant protoplasts represent a reliable experimental system for the genetic manipulation of desired traits using gene editing. Nevertheless, the selection and regeneration of mutated protoplasts are challenging and subsequent recovery of successfully edited plants is a significant bottleneck in advanced plant breeding technologies. In an effort to alleviate the obstacles related to protoplasts' transgene expression and protoplasts' regeneration, a new method was developed. In so doing, it was shown that linearized DNA could efficiently transfect potato protoplasts and that UBIQUITIN10 promoters from various plants could direct transgene expression in an effective manner. Also, the inhibitory concentration of kanamycin was standardized for transfected protoplasts, and the NEOMYCIN PHOSPHOTRANSFERASE2 (NPT2) gene could be used as a potent selection marker for the enrichment of transfected protoplasts. Furthermore, transient expression of the BABYBOOM (BBM) transcription factor promoted the regeneration of protoplast-derived calli. Together, these methods significantly increased the selection for protoplasts that displayed high transgene expression, and thereby significantly increased the rate of gene editing events in protoplast-derived calli to 95%. The method developed in this study facilitated gene-editing in tetraploid potato plants and opened the way to sophisticated genetic manipulation in polyploid organisms.

Effects of light-emitting diodes on protoplast regeneration from gametophytic cells of the commercial kelp <italic>Undaria pinnatifida</italic> (Laminariales, Phaeophyceae)

Light-emitting-diodes (LEDs) are a lighting source useful for the precise evaluation of light quality effect on biological systems. Despite the importance of light spectra on the regeneration of land plant protoplasts (“naked cells”), this factor has not been tested yet on protoplasts from multicellular algae. This study reports on the effects of pure primary colors (red, blue, and green), dichromatic (red plus blue, RB, 1 : 2) and white LEDs on protoplast regeneration from male and female Undaria pinnatifida gametophytes. We also evaluated the effect of different light spectra on pigment composition (chlorophyll a, chlorophyll c, and fucoxanthine), and the light intensities under the best condition on the regeneration process. In the early stages, blue or RB LEDs increased the percentage of dividing female protoplasts, whereas red, blue, and RB LEDs enhanced that of dividing male protoplasts. In the later stages, RB LEDs showed a positive effect only on the percentage of multiple rhizoid-like protrusions (male gametophyte). They also increased the final area of both regenerated gametophytes. The LEDs did not affect pigment composition in female gametophytes. In male gametophytes, in contrast, they reduced chlorophyll c, while blue, RB, and green LEDs decreased fucoxanthin. Under RB LEDs, the optimal light intensity was 80 μmol photons m-2 s-1 for female gametophytes and 40 to 60 μmol photons m-2 s-1 for male gametophytes. Our results suggest that dichromatic LED illumination (red–blue) improves regeneration of U. pinnatifida gametophyte-isolated protoplasts. Thus, dichromatic LEDs might a suitable light source for enhancing protoplast regeneration in brown seaweeds.

CRISPR-Cas9 Targeting of the eIF4E1 Gene Extends the Potato Virus Y Resistance Spectrum of the Solanum tuberosum L. cv. Desirée.

Translation initiation factors and, in particular, the eIF4E family are the primary source of recessive resistance to potyviruses in many plant species. However, no eIF4E-mediated resistance to this virus genus has been identified in potato (Solanum tuberosum L.) germplasm. As in tomato, the potato eIF4E gene family consists of eIF4E1, its paralog eIF4E2, eIF(iso)4E, and nCBP. In tomato, eIF4E1 knockout (KO) confers resistance to a subset of potyviruses, while the eIF4E1/2 double KO, although conferring a broader spectrum of resistance, leads to plant developmental defects. Here, the tetraploid potato cv. Desirée owning the dominant Ny gene conferring resistance to potato virus Y (PVY) strain O but not NTN was used to evaluate the possibility to expand its PVY resistance spectrum by CRISPR-Cas9-mediated KO of the eIF4E1 susceptibility gene. After a double process of plant protoplast transfection-regeneration, eIF4E1 KO potatoes were obtained. The knockout was specific for the eIF4E1, and no mutations were identified in its eIF4E2 paralog. Expression analysis of the eIF4E family shows that the disruption of the eIF4E1 does not alter the RNA steady-state level of the other family members. The eIF4E1 KO lines challenged with a PVYNTN isolate showed a reduced viral accumulation and amelioration of virus-induced symptoms suggesting that the eIF4E1 gene was required but not essential for its multiplication. Our data show that eIF4E1 editing can be usefully exploited to broaden the PVY resistance spectrum of elite potato cultivars, such as Desirée, by pyramiding eIF4E-mediated recessive resistance.

Laser microirradiation as a versatile system for probing protein recruitment and protein–protein interactions at DNA lesions in plants

Plant protoplasts are generated by treatment with digestion enzymes, producing plant cells devoid of the cell wall and competent for efficient polyethylene glycol mediated transformation. This way fluorescently tagged proteins can be introduced to the protoplasts creating an excellent system to probe the localization and function of uncharacterized plant proteins in vivo. We implement the method of laser microirradiation to generate DNA lesions in Arabidopsis thaliana, which enables monitoring the recruitment and dynamics of the DNA repair factors as well as bimolecular fluorescence complementation assay to test transient, conditional interactions of proteins directly at sites of DNA damage. We demonstrate that laser microirradiation in protoplasts yields a physiological cellular response to DNA lesions, based on proliferating cell nuclear antigen (PCNA) redistribution in the nucleus and show that factors involved in DNA repair, such as MRE11 or PCNA are recruited to induced DNA lesions. This technique is relatively easy to adopt by other laboratories and extends the current toolkit of methods aimed to understand the details of DNA damage response in plants. The presented method is fast, flexible and facilitates work with different mutant backgrounds or even different species, extending the utility of the system.

Highly Efficient Genome Editing in Plant Protoplasts by Ribonucleoprotein Delivery of CRISPR-Cas12a Nucleases.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) mediated genome editing is a powerful approach for crop improvement. Traditional transformation methods based on plasmid delivery pose concerns associated with transgene integration and off-target effects. CRISPR delivered as ribonucleoproteins (RNPs) can prevent exogenous DNA integration, minimize off-target effects, and reduce cellular toxicity. Although RNP delivered CRISPR genome editing has been demonstrated in many plant species, optimization strategies that yield high editing efficiencies have not been thoroughly investigated. Using rice and citrus protoplast systems we demonstrated highly efficient genome editing using Cas12a delivered as RNPs. Four Cas12a variants, including LbCas12a, LbCas12a-E795L, AsCas12a, and AsCas12a Ultra, were investigated. Nearly 100% editing efficiency was observed for three out of four target sites by LbCas12a, LbCas12a-E795L, and AsCas12a Ultra, as measured by restriction fragment length polymorphism (RFLP) and verified by next generation sequencing of PCR amplicons. RNP delivery resulted in higher editing efficiencies than plasmid delivery at 32°C and 25°C. LbCas12a and LbCas12a-E795L demonstrated increased editing efficiencies in comparison to AsCas12a and AsCas12a Ultra, especially when used at lower RNP concentrations. In addition, we discovered that a 1:1 Cas12a:crRNA molar ratio is sufficient to achieve efficient genome editing. Nuclear localization signals (NLSs) are essential for efficient RNP-based genome editing. However, the different crRNA modifications tested did not significantly improve genome editing efficiency. Finally, we applied the Cas12a RNP system in citrus protoplasts and obtained similarly high editing efficiencies at the target site. Our study provides a comprehensive guideline for Cas12a-mediated genome editing using RNP delivery in plant cells, setting the foundation for the generation of transgene-free genome edited plants.

The Use of Fluorescent Protein Fusions to Monitor the Unfolded Protein Response and Protein Foldase-Substrate Interactions in Plant Protoplasts.

Endoplasmic reticulum (ER) stress and the resulting unfolded protein response (UPR) are critical stress response pathways in eukaryotes. To study these types of interactions in plants, a wide range of methods have been used, including generation of transgenic plants, subcellular immunolocalization of protein foldases, and co-immunoprecipitation (co-IP) assays. Although these more time-consuming methods have been successfully implemented, there is a need for a versatile and rapid in vivo system to investigate ER stress and UPR. Here, we describe a transient expression system that uses plant protoplasts to define in vivo subcellular localizations and protein-protein interactions of protein foldases and their substrates fused to fluorescent protein reporters. This accurate and robust assay utilizes a variety of analyses, such as subcellular localization, FLIM-FRET, co-IP, mutagenesis, and RT-PCR in the genetically amenable Arabidopsis model system. We demonstrate the methodology by using the representative protein foldase, protein disulfide isomerase-9 (PDI9), as well as subcellular markers, secretory proteins, and dithiothreitol (DTT)-mediated induction of the UPR as monitored by RT-PCR. Together, these methods yield reliable high output results for investigating subcellular localization and protein-protein interactions in plants to decipher the UPR pathways.

A Versatile and Efficient Plant Protoplast Platform for Genome Editing by Cas9 RNPs.

The ultimate goal of technology development in genome editing is to enable precisely targeted genomic changes in any cells or organisms. Here we describe protoplast systems for precise and efficient DNA sequence changes with preassembled Cas9 ribonucleoprotein (RNP) complexes in Arabidopsis thaliana, Nicotiana benthamiana, Brassica rapa, and Camelina sativa. Cas9 RNP-mediated gene disruption with dual gRNAs could reach ∼90% indels in Arabidopsis protoplasts. To facilitate facile testing of any Cas9 RNP designs, we developed two GFP reporter genes, which led to sensitive detection of nonhomologous end joining (NHEJ) and homology-directed repair (HDR), with editing efficiency up to 85 and 50%, respectively. When co-transfected with an optimal single-stranded oligodeoxynucleotide (ssODN) donor, precise editing of the AtALS gene via HDR reached 7% by RNPs. Significantly, precise mutagenesis mediated by preassembled primer editor (PE) RNPs led to 50% GFP reporter gene recovery in protoplasts and up to 4.6% editing frequency for the specific AtPDS mutation in the genome. The rapid, versatile and efficient gene editing by CRISPR RNP variants in protoplasts provides a valuable platform for development, evaluation and optimization of new designs and tools in gene and genomic manipulation and is applicable in diverse plant species.

Isolation and Transfection of Citrus Protoplasts with Citrus Exocortis Viroid.

Viroids are RNA-based infectious agents that are single-stranded, covalently closed circular, non-coding, and naked. Unlike RNA viruses, which at least encode proteins for replication, encapsidation, and movement, lack of protein-coding capacity of viroids makes them completely reliant on host for replication and movement. The high genetic diversity in viroids is believed to be due to the absence of proof-reading activity of the host RNA polymerases, the large population size, and the rapid rate of replication. Protoplasts are viable plant cells that are prepared by enzymatic removal of cell walls. Plant protoplasts provide a synchronous single-cell system for studying early events of viroid infection such as replication and genetic diversity at the cellular level. A simple and efficient method to isolate and transfect citrus protoplasts with transcript RNA of citrus exocortis viroid is described in this chapter.

Multiplex Site-Directed Gene Editing Using Polyethylene Glycol-Mediated Delivery of CRISPR gRNA:Cas9 Ribonucleoprotein (RNP) Complexes to Carrot Protoplasts.

The aim of this work was to show an efficient, recombinant DNA-free, multiplex gene-editing method using gRNA:Cas9 ribonucleoprotein (RNP) complexes delivered directly to plant protoplasts. For this purpose, three RNPs were formed in the tube, their activity was confirmed by DNA cleavage in vitro, and then they were delivered to carrot protoplasts incubated with polyethylene glycol (PEG). After 48 h of incubation, single nucleotide deletions and insertions and small deletions at target DNA sites were identified by using fluorescent-PCR capillary electrophoresis and sequencing. When two or three RNPs were delivered simultaneously, long deletions of 33–152 nt between the gRNA target sites were generated. Such mutations occurred with an efficiency of up to 12%, while the overall editing effectiveness was very high, reaching 71%. This highly efficient multiplex gene-editing method, without the need for recombinant DNA technology, can be adapted to other plants for which protoplast culture methods have been established.

Protoplast Regeneration and Its Use in New Plant Breeding Technologies.

The development of gene-editing technology holds tremendous potential for accelerating crop trait improvement to help us address the need to feed a growing global population. However, the delivery and access of gene-editing tools to the host genome and subsequent recovery of successfully edited plants form significant bottlenecks in the application of new plant breeding technologies. Moreover, the methods most suited to achieve a desired outcome vary substantially, depending on species' genotype and the targeted genetic changes. Hence, it is of importance to develop and improve multiple strategies for delivery and regeneration in order to be able to approach each application from various angles. The use of transient transformation and regeneration of plant protoplasts is one such strategy that carries unique advantages and challenges. Here, we will discuss the use of protoplast regeneration in the application of new plant breeding technologies and review pertinent literature on successful protoplast regeneration.

Protoplast Isolation, Fusion, Culture and Transformation in the Woody Plant Jasminum spp.

Plant protoplasts are significant for plant cell culture, somatic cell fusion, genetics, and breeding studies. In addition, in vitro plant regeneration has great importance for developmental biology, manifesting potential applications in agriculture and biotechnology. In this regard, we present a well-established protocol regarding protoplast isolation, cell culture and protoplast fusion of Jasminum spp. In particular, different tissues of Jasminum samab L. and Jasminum mesnyi were employed for protoplast isolation, and stem explants provided a high callus induction rate in a short period of time. The best source for protoplast isolation was calli tissues. The optimized isolation protocol consisted of digesting callus in an enzyme solution containing 0.4 M mannitol, 0.2 M MES, 1 M CaCl2, 0.2 M KCL and 1 M NaH2PO4, 1.5% Cellulases onozuka R-10, 0.4% Macerozyme R-10 and 0.8% Pectinase for 4 h at 26 °C in the dark, providing a yield of 23.8 × 106 Protoplast/gFW with 88% viability. Protoplasts were cultured both in liquid and agarose medium under optimum conditions, leading to microcalli formation after eight weeks. A 5% protoplast-fusion rate can be achieved when cultured in 40% (w/v) PEG-MW6000 supplemented with 0.1 M CaCl2, 0.1 M sorbitol and 1 M Tris for 20 min. Furthermore, we developed an efficient PEG-mediated transformation protocol for jasmine protoplasts. The best results regarding protoplast transformation were obtained when the protoplast concentration was 4 × 105 cells/mL and the exogenous plasmid DNA added had a concentration of 10 µg DNA/100 µL protoplast solution, followed by the application of 40% PEG-4000 for 10 min.

TSA Promotes CRISPR/Cas9 Editing Efficiency and Expression of Cell Division-Related Genes from Plant Protoplasts.

Trichostatin A (TSA) is a representative histone deacetylase (HDAC) inhibitor that modulates epigenetic gene expression by regulation of chromatin remodeling in cells. To investigate whether the regulation of chromatin de-condensation by TSA can affect the increase in the efficiency of Cas9 protein-gRNA ribonucleoprotein (RNP) indel formation from plant cells, genome editing efficiency using lettuce and tobacco protoplasts was examined after several concentrations of TSA treatments (0, 0.1, 1 and 10 μM). RNP delivery from protoplasts was conducted by conventional polyethylene glycol (PEG) transfection protocols. Interestingly, the indel frequency of the SOC1 gene from TSA treatments was about 3.3 to 3.8 times higher than DMSO treatment in lettuce protoplasts. The TSA-mediated increase of indel frequency of the SOC1 gene in lettuce protoplasts occurred in a concentration-dependent manner, although there was not much difference. Similar to lettuce, TSA also increased the indel frequency by 1.5 to 1.8 times in a concentration-dependent manner during PDS genome editing using tobacco protoplasts. The MNase test clearly showed that chromatin accessibility with TSA treatments was higher than that of DMSO treatment. Additionally, TSA treatment significantly increased the level of histone H3 and H4 acetylation from lettuce protoplasts. The qRT-PCR analysis showed that expression of cell division-related genes (LsCYCD1-1, LsCYCD3-2, LsCYCD6-1, and LsCYCU4-1) was increased by TSA treatment. These findings could contribute to increasing the efficiency of CRISPR/Cas9-mediated genome editing. Furthermore, this could be applied for the development of useful genome-edited crops using the CRISPR/Cas9 system with plant protoplasts.

Phosphorylation of a reinitiation supporting protein, RISP, determines its function in translation reinitiation.

Reinitiation supporting protein, RISP, interacts with 60S (60S ribosomal subunit) and eIF3 (eukaryotic initiation factor 3) in plants. TOR (target-of-rapamycin) mediates RISP phosphorylation at residue Ser267, favoring its binding to eL24 (60S ribosomal protein L24). In a viral context, RISP, when phosphorylated, binds the CaMV transactivator/ viroplasmin, TAV, to assist in an exceptional mechanism of reinitiation after long ORF translation. Moreover, we show here that RISP interacts with eIF2 via eIF2β and TOR downstream target 40S ribosomal protein eS6. A RISP phosphorylation knockout, RISP-S267A, binds preferentially eIF2β, and both form a ternary complex with eIF3a in vitro. Accordingly, transient overexpression in plant protoplasts of RISP-S267A, but not a RISP phosphorylation mimic, RISP-S267D, favors translation initiation. In contrast, RISP-S267D preferentially binds eS6, and, when bound to the C-terminus of eS6, can capture 60S in a highly specific manner in vitro, suggesting that it mediates 60S loading during reinitiation. Indeed, eS6-deficient plants are highly resistant to CaMV due to their reduced reinitiation capacity. Strikingly, an eS6 phosphomimic, when stably expressed in eS6-deficient plants, can fully restore the reinitiation deficiency of these plants in cellular and viral contexts. These results suggest that RISP function in translation (re)initiation is regulated by phosphorylation at Ser267.

Cell size: a key determinant of meristematic potential in plant protoplasts.

Metabolic pathway reconstruction and gene edits for native natural product synthesis in single plant cells are considered to be less complicated when compared to the production of non-native metabolites. Being an efficient eukaryotic system, plants encompass suitable post-translational modifications. However, slow cell division rate and heterogeneous nature is an impediment for consistent product retrieval from plant cells. Plant cell synchrony can be attained in cultures developed in vitro. Isolated plant protoplasts capable of division, can potentially enhance the unimpaired yield of target bioactives, similar to microbes and unicellular eukaryotes. Evidence from yeast experiments suggests that 'critical cell size' and division rates for enhancement machinery, primarily depend on culture conditions and nutrient availability. The cell size control mechanisms in Arabidopsis shoot apical meristem is analogous to yeast notably, fission yeast. If protoplasts isolated from plants are subjected to cell size studies and cell cycle progression in culture, it will answer the underlying molecular mechanisms such as, unicellular to multicellular transition states, longevity, senescence, 'cell-size resetting' during organogenesis, and adaptation to external cues.

A simplified method to isolate rice mitochondria

BackgroundMitochondria play critical roles in plant growth, development and stress tolerance. Numerous researchers have carried out studies on the plant mitochondrial genome structure, mitochondrial metabolism and nuclear-cytoplasmic interactions. However, classical plant mitochondria extraction methods are time-consuming and consist of a complicated ultracentrifugation procedure with expensive reagents. To develop a more rapid and convenient method for the isolation of plant mitochondria, in this study, we established a simplified method to isolate rice mitochondria efficiently for subsequent studies.ResultsTo isolate rice mitochondria, the cell wall was first disrupted by enzymolysis to obtain the protoplast, which is similar to animal mitochondria. Rice mitochondria were then isolated with a modified method based on the animal mitochondria isolation protocol. The extracted mitochondria were next assessed according to DNA and protein levels to rule out contamination by the nucleus and chloroplasts. Furthermore, we examined the physiological status and characteristics of the isolated mitochondria, including the integrity of mitochondria, the mitochondrial membrane potential, and the activity of inner membrane complexes. Our results demonstrated that the extracted mitochondria remained intact for use in subsequent studies.ConclusionThe combination of plant protoplast isolation and animal mitochondria extraction methods facilitates the extraction of plant mitochondria without ultracentrifugation. Consequently, this improved method is cheap and time-saving with good operability and can be broadly applied in studies on plant mitochondria.

Isolation of Plant Root Nuclei for Single Cell RNA Sequencing.

The characterization of the transcriptional similarities and differences existing between plant cells and cell types is important to better understand the biology of each cell composing the plant, to reveal new molecular mechanisms controlling gene activity, and to ultimately implement meaningful strategies to enhance plant cell biology. To gain a deeper understanding of the regulation of plant gene activity, the individual transcriptome of each plant cell needs to be established. Until recently, single cell approaches were mostly limited to bulk transcriptomic studies on selected cell types. Accessing specific cell types required the development of labor-intensive strategies. Recently, single cell sequencing strategies were successfully applied on isolated Arabidopsis thaliana root protoplasts. However, this strategy relies on the successful isolation of viable protoplasts upon the optimization of the enzymatic cocktails required to digest the cell wall and on the compatibility of fragile plant protoplasts with the use of microfluidic systems to generate single cell transcriptomic libraries. To overcome these difficulties, we present a simple and fast alternative strategy: the isolation and use of plant nuclei to access meaningful transcriptomic information from plant cells. This protocol was specifically developed to enable the use of the plant nuclei with 10× Genomics' Chromium technology partitions technology. Briefly, the plant nuclei are released from the root by chopping into a nuclei isolation buffer before purification by filtration then nuclei sorting. Upon sorting, the nuclei are resuspended in a low divalent ion buffer compatible with the Chromium technology in order to create single nuclei ribonucleic acid-sequencing libraries (sNucRNA-seq). © 2020 Wiley Periodicals LLC. Basic Protocol 1: Arabidopsis seed sterilization and planting Basic Protocol 2: Nuclei isolation from Arabidopsis roots Basic Protocol 3: Fluorescent-activated nuclei sorting (FANS) purification Support Protocol: Estimation of nuclei density using Countess II automated cell counter Alternate Protocol 1: Proper growth conditions for Medicago truncatula and Sorghum bicolor Alternate Protocol 2: Estimation of nuclei density using sNucRNA-seq technology.

Potassium Efflux and Cytosol Acidification as Primary Anoxia-Induced Events in Wheat and Rice Seedlings.

Both ion fluxes and changes of cytosolic pH take an active part in the signal transduction of different environmental stimuli. Here we studied the anoxia-induced alteration of cytosolic K+ concentration, [K+]cyt, and cytosolic pH, pHcyt, in rice and wheat, plants with different tolerances to hypoxia. The [K+]cyt and pHcyt were measured by fluorescence microscopy in single leaf mesophyll protoplasts loaded with the fluorescent potassium-binding dye PBFI-AM and the pH-sensitive probe BCECF-AM, respectively. Anoxic treatment caused an efflux of K+ from protoplasts of both plants after a lag-period of 300–450 s. The [K+]cyt decrease was blocked by tetraethylammonium (1 mM, 30 min pre-treatment) suggesting the involvement of plasma membrane voltage-gated K+ channels. The protoplasts of rice (a hypoxia-tolerant plant) reacted upon anoxia with a higher amplitude of the [K+]cyt drop. There was a simultaneous anoxia-dependent cytosolic acidification of protoplasts of both plants. The decrease of pHcyt was slower in wheat (a hypoxia-sensitive plant) while in rice protoplasts it was rapid and partially reversible. Ion fluxes between the roots of intact seedlings and nutrient solutions were monitored by ion-selective electrodes and revealed significant anoxia-induced acidification and potassium leakage that were inhibited by tetraethylammonium. The K+ efflux from rice was more distinct and reversible upon reoxygenation when compared with wheat seedlings.