The acidity of oxalic acid induces regulated cell death in Arabidopsis

doi:10.21203/rs.3.rs-2064277/v1

Download PDF

Research Article

The acidity of oxalic acid induces regulated cell death in Arabidopsis

https://doi.org/10.21203/rs.3.rs-2064277/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Key message The acidity of oxalic acid (OA) induces an unknown type of cell death in Arabidopsis. Acid treatment and Sclerotinia sclerotiorum infection trigger some overlapping transcriptomic responses. The influx of calcium is likely required for OA-induced cell death in Arabidopsis. Abstract Numerous pathogens trigger host cell death that either favors or blocks infection. Many phytopathogens produce cytolytic compounds functioning as virulence determinants, like oxalic acid (OA). It was reported that oxalate itself but not the acidity of OA induced programmed cell death in plants. Accumulation of oxalate often reaches millimolar concentrations, and the pH value decreases to around 4.0 in the Sclerotinia sclerotium -infected plant tissues. However, the role of OA acidity in inducing cell death remains unknown. We used the approaches of Sytox green/fluorescein diacetate (FDA)/propidium iodide (PI)/Evan's blue staining, transcriptomic analysis, and dynamic gene expression analysis to investigate the role of OA acidity in inducing cell death and the regulatory mechanisms of OA-induced cell death. We found that the acidity of OA and HCl induced cell death in Arabidopsis with characteristic features including impaired plasma membrane and induction of cell-death related genes, but not DNA fragmentation. Moreover, calcium influx is required for OA-induced cell death. Our work reveals that acids induce an uncharacterized-type of cell death that is a regulated process, instead of a passive event.

oxalic acid

acidity

cell death

Arabidopsis

The manipulation of host homeostasis by a variety of pathogens often induces host cell death, which either favors or blocks infection (Blériot and Lecuit 2016). It has been reported that an array of phytopathogens trigger programmed cell death (PCD), necrosis, or other uncharacterized-type of cell death in plants during microbial infection (Qutob et al. 2006; Dodds and Rathjen 2010; Ma et al. 2015; Lonjon et al. 2020). The hypersensitive response (HR) is a plant-specific type of PCD, which occurs around the site of infection in host by incompatible pathogens, to present a barrier to pathogen movement and disease establishment (Dodds and Rathjen 2010). The type of cell death induced by phytopathogen infection was not always PCD. For example, AvrE1, an effector from Pseudomonas, induces cell death in Nicotiana benthamiana; interestingly, the AvrE1-induced cell death is not likely HR-type (Jin et al. 2016). In another instance, a xyloglucanase of the soybean (Glycine max) pathogen Phytophthora sojae, called XEG1, triggers an uncharacterized-type of cell death independent of its xyloglucanase activity (Ma et al. 2015).

Numerous phytopathogenic microorganisms produce a range of cytolytic compounds that function as key virulence determinants. Particularly, necrotrophic fungi release many host selective and nonselective toxins, thereby promoting host cell death (Wolpert et al. 2002; Glazebrook 2005). A well-known phytotoxin, fusicoccin induces strong cell death in sycamore (Acer pseudoplatanus L.) cultures, which shows features similar to apoptosis (the form of animal PCD), i.e., cell shrinkage, changes in nucleus morphology, and DNA fragmentation (Malerba et al. 2003). In another case, fusaric acid (FA), a fusarium mycotoxin, also triggers apoptotic-like cell death in Crocus sativus Linnaeus roots. The apoptotic-like features include generation of H₂O₂ and nucleosomal DNA fragmentation (Samadi and Shahsavan Behboodi 2006).

The phytotoxin oxalic acid (OA) is secreted by a wide range of necrotrophic plant fungi, such as Sclerotinia sclerotiorum (Kesarwani et al. 2000), Botrytis cinerea (Kumari et al. 2014), Septoria musiva (Liang et al. 2001), Sclerotium rolfsii (Bateman and Beer 1965), and Penicillium oxalicum (Ikotun 1984), etc. OA is a simple organic acid that serves as a non-host selective fungal toxin, and is considered to be a key factor in pathogenesis for these necrotrophic fungi. It was reported that oxalate itself but not the acidic nature (acidity) of OA induced PCD in tobacco leaf discs (Kim et al. 2008). When treated with oxalate (pH 5.8) at millimolar concentrations in the absence of living fungi, Arabidopsis suspension cells exhibit cell shrinkage and DNA fragmentation. Other features associated with PCD, such as mitochondrial depolarization, plasma membrane depolarization, and ethylene synthesis, were also observed after oxalate treatment (Errakhi et al. 2008).

The concentration of oxalate accumulated in the S. sclerotium-infected plant tissues often reaches millimolar levels (up to 10 mM), and pH decreases to around 4.0 (Bateman and Beer 1965; Marciano et al. 1983). Therefore, the role of acidity of OA in triggering plant cell death has to be considered. In this work, we found that acidity of OA induced cell death in Arabidopsis with characteristic features including impaired plasma membrane, influx of calcium, and induction of cell-death related genes, but not DNA fragmentation. Our work reveals that the acid nature of OA induces an uncharacterized-type of cell death in Arabidopsis.

Plant materials and growth conditions

Arabidopsis seeds were sterilized in 75% EtOH for 1 min and then in 50% bleach solution for 5 min, followed by extensive washing with sterile distilled water 6 times; and then the seeds were plated on ½MS medium supplemented with 0.5% sucrose and kept at 4°C for 24 h. The plates were then transferred to a growth chamber at 22°C with a 12 h-light/12 h-dark photoperiod.

Fungal materials and pH measurement

Sclerotinia sclerotiorum wild-type strain was maintained and cultured on the yeast-sugar-salt (YSS) medium as described (Zhang et al. 2015). A piece of solid YSS medium in the Petri dishes culturing S. sclerotiorum was cut and replaced with 4 ml methyl orange solution (MOS, 25µg ml^− 1 methyl orange in ddH₂O), which was used for pH measurement.

RNA extraction, cDNA synthesis and real-time quantitative PCR

Ten-day-old Col-0 seedlings were treated with OA, HCl, or KOX. Total RNA extraction, cDNA synthesis, and real-time quantitative PCR were performed as previously described (Zou et al. 2018). Gene expression levels were normalized to that of GAPC. The qPCR primers are listed in Table S1.

Transcriptome sequencing and gene expression analysis

Ten-day-old Col-0 seedlings were treated with OA, HCl, or KOX for 60 min. Total RNA was extracted as above. Library construction and transcriptome sequencing were performed by Novogene (Tianjin, China). Three biological replicates were carried out for the RNA-Seq analysis. The data analysis was performed as previously described (Liu et al. 2022). The differentially expressed genes (DEGs) were filtered based on thresholds of |log₂FoldChange| > 1 and P_adj < 0.05. Gene Ontology (GO) enrichment analysis of DEGs was performed with the cluster Profiler R package. GO terms with corrected p-value < 0.05 were considered significantly enriched. Gene Set Enrichment Analysis (GSEA) was performed using the local version of the GSEA analysis tool (http://www.broadinstitute.org/gsea/index.jsp).

Cell death assays

For cell death assay in Arabidopsis root tips, 6-day-old Arabidopsis seedlings were transferred to sterile distilled water. Then the seedlings were treated with OA, HCl, or KOX, incubated in 2 µM Sytox green (Invitrogen: S7020) solution for 10 min, and followed by confocal microscopy as described (Distéfano et al., 2017). Arabidopsis roots were stained with 10 µg ml^− 1 FDA (Sigma: F7378), treated with OA, HCl, or KOX, and followed by confocal microscopy as described (Errakhi et al. 2008). For PI (propidium iodide, Sigma: P4170) staining, 40 µg ml^− 1 PI was dipped on the microslide, then the Arabidopsis roots were immersed in the PI drop, and subjected to confocal microscopy.

For cell death assay in Arabidopsis seedlings, 10-d-old plants were incubated overnight in sterile distilled water (8 seedlings per well), and then treated with OA, HCl, or KOX. The seedlings were stained with 1.25 mg/ml Evan’s blue solution [1.25 mg/ml Evan’s blue (Solarbio: E8010), 10 mM CaCl₂, pH 5.6, modified from (Vijayaraghavareddy et al. 2017)] for 30 min, and then rinsed with 1.5 ml deionized water for at least 4 times. The stained Arabidopsis leaves were decolored in 95% ethanol, and the Evan’s blue was solubilized with 300 µl 1% (w/v) SDS in 50% (v/v) methanol at 50℃ for 2 h. Then the absorbance was measured at 600 nm wavelength (Romero-Puertas et al. 2004). A Standard curve for Evan’s blue absorbance was generated as described (Vijayaraghavareddy et al. 2017) to determine the concentration of Evan’s blue (C₁) in the SDS solution (300 µl). Then the Evan’s blue content of seedlings was calculated as below:

C (Evan’s blue) = 300 µl * C₁ /TW, (TW, the weight of seedlings after OA treatment). Therefore, the Evan’s blue content was defined as Evan’s blue (ng) per gram of Arabidopsis seedlings after OA treatment.

Statistical analysis

Data were analyzed using GraphPad Prism 7, and two-way ANOVA with Tukey’s test was performed using GraphPad Prism 7 to generate P values. Statistically significant differences are indicated with asterisks: *, P < 0.05; **, P < 0.01, or with different letters (significance set at P < 0.05).

Low concentration of oxalic acid induces cell death in Arabidopsis roots

High concentration of oxalate is accumulated in the S. sclerotium-infected plant tissues (Marciano et al. 1983). We found that culturing S. sclerotiorum on solid medium resulted in acidification of environments with pH value dropping below 3.0 (Fig. 1). Therefore, we have to consider the acid nature (acidity) of OA in triggering cell death in plants. We first treated 6-d-old Arabidopsis seedlings with OA for 30 min at concentrations ranging from 0.05 to 1.0 mM (pH values ranging from 4.3 to 3.0). The death of Arabidopsis root cells was assayed by staining with Sytox green, a dead cell specific nucleic acid fluorescent dye. Low concentrations of OA induced cell death in Arabidopsis roots in a dose-dependent manner (Fig. 2a). Similar results were obtained when the OA-treated Arabidopsis roots were stained with fluorescein diacetate (FDA), which fluoresces in living cells but not in dead cells (Fig. 2b). Moreover, the dead cells in Arabidopsis roots treated with OA extended bidirectionally over time (Fig. S1).

It is known that propidium iodide (PI) can only be taken up by cells that have a compromised membrane (Autheman et al. 2013). So, it was widely used to stain the necrotic animal cells. However, for plant cells, PI also binds to pectin which forms the primary component of the cell wall (Rieger et al. 2011). So, the use of PI as a dead cell indicator was limited in plants. In our work, we used PI/FDA double staining method to check the OA-induced cell death. Arabidopsis roots were treated with 0.05 mM OA. After 30 min of OA treatment, the dead cells that could not be stained by FDA exhibited strong PI-staining inside the root cells, while in living regions, only the cell walls were stained (Fig. 2c). Consistently, the dead cell region extended bidirectionally in Arabidopsis roots in a time course of 45 min of OA treatment as assayed by PI/FDA double staining (Fig. S2). These results suggest that OA treatment results in emergence of compromised plasma membrane (PM) in Arabidopsis root cells.

Low concentration of OA induces cell death in Arabidopsis seedlings

Then we detected cell death in whole Arabidopsis seedlings upon OA treatment. We found that, after exposure to low concentrations of OA, both Arabidopsis roots and leaves could be stained with Evan's blue, a dead cell specific dye with a maximum absorbance at 600 nm wavelength (Fig. 3a, b). Meanwhile, the leaves became gradually wilting during cell death after OA treatment (Fig. 3a). So, we measured the rate of weight loss (RWL) of Arabidopsis seedlings after OA treatment and found that RWL is positively correlated with the results obtained from Evan's blue staining (Fig. 3b, c), suggesting that RWL could also be used as a readout to quantify the OA-induced cell death in Arabidopsis seedlings.

The acidity of OA induces cell death in Arabidopsis

Next, we sought to determine, between acidity and oxalate of OA, which induces cell death in Arabidopsis at a relatively low concentration (0.5 mM) for a short time of treatment. We found that just like OA (0.5 mM, pH 3.2), hydrochloric acid (HCl, pH 3.2) but not KOX (0.5 mM) induced cell death in Arabidopsis roots as assayed by Sytox green/FDA staining, or in seedlings as assayed by Evan's blue staining or determined by measuring RWL (Fig. 4a-e). These results suggest that the acidic nature of OA induced cell death in Arabidopsis.

The acidity of OA induces an unknown type of cell death in Arabidopsis

DNA-cleavage is a characteristic feature of PCD, which results in a ladder of internucleosomal fragments (Ryerson and Heath 1996). To further characterize the OA-induced cell death, we examined whether OA treatment results in DNA fragmentation. DNA extracted from Arabidopsis seedlings was separated with agarose gel. Consistent with a previous work (Errakhi et al. 2008), DNA isolated from 20 mM KOX-treated seedlings (24h) displayed DNA laddering. However, no DNA fragmentation was detected when seedlings were treated with OA (up to 1.0 mM) for 30 min (Fig. 4f). These results suggest that the type of OA-induced cell death is probably not PCD.

The global gene expression in Arabidopsis upon OA treatment

To further confirm the premise that the acidic nature of OA induces cell death and to further characterize its features, we performed RNA sequencing (RNA-seq) to analyze the genes differentially expressed upon treatment with OA (0.5 mM, pH 3.2), HCl (pH 3.2), KOX (0.5 mM), or H₂O (the control) for 60 min. A total of 3074 and 3525 DEGs (differentially expressed genes, |log₂FoldChange| > 1 and P_adj < 0.05) were identified by comparing transcriptomes of OA- and HCl-treated Arabidopsis to the control, respectively (HCl- or OA-treated DEGs are provided in Dataset S1). However, there were no significant DEGs obtained by comparing KOX transcriptome with the control (Fig. 5a). The expression of a number of DEGs upon OA and HCl treatments determined by RNA-seq was confirmed by RT-qPCR (Fig. S3). Further hierarchical clustering analysis of DEGs upon treatment with OA, HCl, KOX, or H₂O showed a high degree of gene coexpression between OA-treated versus HCl-treated transcriptomes, and between KOX-treated versus H₂O-treated transcriptomes (Fig. 5b), indicating that OA and HCl have a comparable effect on global gene expression in Arabidopsis.

Furthermore, 2042 genes are overlapping between the 2462 genes (82.9%) induced by HCl and 2284 genes (89.4%) induced by OA, and 526 genes are overlapping between the 1063 genes (49.5%) downregulated by HCl and 790 genes (66.6%) by OA (Fig. 5c, d; Dataset S1). Furthermore, gene ontology (GO) analysis showed that genes up-regulated by both OA and HCl were primarily enriched in the defense and stress related biological process (BP) categories, such as immune system process, defense response to bacterium, defense response to fungus, regulation of defense response, as well as response to chitin, wounding, and jasmonic acid (JA) (Fig. 5e). Moreover, gene set enrichment analysis (GSEA) demonstrates that "defense response", "defense response to bacterium", "response to fungus", "calcium ion binding", and "cell death" gene sets are enriched in both HCl- and OA upregulated genes (Fig. S4).

Interestingly, of the 2042 overlapping genes induced by both HCl and OA treatment, 1468 ones are also up-regulated by S. sclerotiorum infection (Badet et al. 2017) (Fig. 5f). These results suggest that acid treatment, S. sclerotiorum infection, and other plant-biotic interactions trigger some overlapping transcriptomic responses, and OA likely plays a significant role in inducing reprogramming of gene expression in plants upon S. sclerotiorum infection.

The influx of calcium is likely required for the OA-induced cell death in Arabidopsis

Ca²⁺ influx is a prerequisite of HR-type cell death (Ma et al. 2008; Gao et al. 2013). Our transcriptome analysis also showed that the "calcium ion binding" gene set was enriched in both HCl- and OA-treated Arabidopsis transcriptomes (Fig. 6a, b). RT-qPCR analysis further confirmed the induction of "calcium ion binding" genes by both HCl and OA treatment (Fig. 6c, d). To examine whether calcium influx is required for OA-induced cell death in plants, we tested the effect of inhibiting calcium-channels by two calcium-channel inhibitors, LaCl₃ and GdCl₃ on OA-triggered cell death. The OA-induced cell death in Arabidopsis roots was obviously blocked by LaCl₃ or GdCl₃ pretreatment as assayed by staining with Sytox green or FDA (Fig. 7a, b). Similarly, although pretreatments with LaCl₃ and GdCl₃ resulted in a slight increase in cell death, the OA-induced cell death in Arabidopsis seedlings was significantly blocked by these two chemicals as assayed by Evan's blue staining or determined by measuring RWL (Fig. 7c-e). Moreover, the induction of cell death related genes, such as WRKY46, OXI1, and PAD4 by OA was significantly suppressed by LaCl₃ or GdCl₃ pretreatment (Fig. 8a-c). Together, these data suggest that the influx of calcium is likely required for the OA-induced cell death in plants.

It is known that an array of phytopathogens and cytolytic compounds they produced induce PCD, necrosis, or other uncharacterized-type of cell death in plants during microbial infection (Malerba et al. 2003; Qutob et al. 2006; Samadi and Shahsavan Behboodi 2006; Dodds and Rathjen 2010; Ma et al. 2015; Lonjon et al. 2020). Our results showed that the acid-induced cell death was a tightly regulated process, instead of a passive event, which exhibits impaired plasma membrane as examined by PI staining, calcium influx as assayed by chemical inhibition assay, and induction of a large variety of genes as analyzed by RNA-seq (Figs. 2c, 7, 8, 5). However, no DNA fragmentation was observed when plants were treated with OA (Fig. 4f). These results suggest that the OA-induced cell death in Arabidopsis is very likely not HR-type, but an uncharacterized-form of cell death in plants. In mammals, necrosis is induced by toxins, severe physical stimuli, and sometimes severe diseases. Necrosis had been thought as an uncontrolled and passive event exhibiting cell and organelle swelling, cell leakage, and plasma membrane rupture (Haunstetter and Izumo 1998). In recent years, there is growing evidence supporting that some forms of necrosis are tightly regulated and result from specific host cell-dependent biochemical pathways. This kind of necrosis is called as regulated necrosis, programmed necrosis, or necroptosis (Galluzzi et al. 2014). Microbial infection can trigger regulated necrosis in mammalian cells, which either favor infection or plays a role in controlling infection (Sridharan and Upton 2014). For example, a virulence factor of Clostridium perfringens, C. perfringens β-toxin (CPB), rapidly triggered cell death in mammals, with the features such as ATP depletion, a marked rise in Ca²⁺ influx, and PI uptake, but no obvious DNA fragmentation. Furthermore, chelation of intracellular Ca²⁺ also significantly inhibited CPB-induced cell death, suggesting that the CPB induced cell death is not a passive event caused by toxin-triggered plasma membrane disruption, but a regulated process executed by distinct biochemical pathways, which was regarded as necroptosis (Autheman et al. 2013). Although necroptosis is well characterized in mammals, report on this regulated cell death is still lacking in plants. It will be very interesting to examine whether there is regulated necrosis in plants and whether the acid-induced cell death is related with necroptosis.

We found that S. sclerotiorum cultured on solid medium triggered acidification of environments with pH value dropping below 3.0 (Fig. 1). And the acidity of OA induces cell death (Figs. 2, 3, 4). Moreover, a high degree of gene coexpression between OA-treated versus S. sclerotiorum-infected transcriptomes was also observed (Fig. 5f). These results suggest that the acidity of OA secreted by S. sclerotiorum might contribute to cell death, which likely favors the infection of plants by S. sclerotiorum. Interestingly, when tomato leaves were pretreated with KOX (20 mM, pH 5.0) and then inoculated with the S. sclerotiorum A-1 mutant, its pathogenicity was only partially restored (Kim et al., 2008), this strongly suggests that the acidity of OA is required for the successful infection of plants by S. sclerotiorum. It is still unknown whether pretreatment of plants with OA at lower pH could restore the pathogenicity of S. sclerotiorum A-1 mutant, when the plants are inoculated with this mutant strain.

Supplementary Information The data that supports the findings of this study are available in the supplementary material of this article.

Acknowledgements We thank Jianhang Sun for initiating this project Nana Cao for assisting gene expression analysis. This work was supported by the National Natural Science Foundation of China (Grant no. 31571260, 32070555), and the State Key Laboratory of Plant Genomics of China.

Author contributions DL and GH designed the research; GH, KC, XL, YZ, ZX and NC performed the experiments; GH and DL analyzed the data; and DL wrote the article with contributions of GH.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Autheman D, Wyder M, Popoff M, et al (2013) Clostridium perfringens beta-toxin induces necrostatin-inhibitable, calpain-dependent necrosis in Primary Porcine Endothelial Cells. PLoS One 8:. https://doi.org/10.1371/journal.pone.0064644
Badet T, Voisin D, Mbengue M, et al (2017) Parallel evolution of the POQR prolyl oligo peptidase gene conferring plant quantitative disease resistance. PLOS Genet 13:e1007143. https://doi.org/10.1371/journal.pgen.1007143
Bateman DF, Beer S V (1965) Simultaneous production and synergistic action of oxalic acid and polygalacturonase during pathogenesis by Sclerotium rolfsii. Phytopathology 55:204–211
Blériot C, Lecuit M (2016) The interplay between regulated necrosis and bacterial infection. Cell Mol Life Sci 73:2369–2378. https://doi.org/10.1007/s000 18-016-2206-1
Dodds PN, Rathjen JP (2010) Plant immunity: towards an integrated view of plant–pathogen interactions. Nat Rev Genet 11:539–548. https://doi.org/10.10 38/nrg2812
Errakhi R, Meimoun P, Lehner A, et al (2008) Anion channel activity is necessary to induce ethylene synthesis and programmed cell death in response to oxalic acid. J Exp Bot 59:3121–3129. https://doi.org/10.1093/jxb/ern166
Galluzzi L, Kepp O, Krautwald S, et al (2014) Molecular mechanisms of regulated necrosis. Semin Cell Dev Biol 35:24–32. https://doi.org/10.1016/j. semcdb.2014.02.006
Gao X, Chen X, Lin W, et al (2013) Bifurcation of Arabidopsis NLR Immune Signaling via Ca²⁺-Dependent Protein Kinases. PLoS Pathog 9:e1003127. https://doi.org/10.1371/journal.ppat.1003127
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu Rev Phytopathol 43:205–227. https://doi.org/10.1146/annurev.phyto.43.040204.135923
Haunstetter A, Izumo S (1998) Apoptosis: basic mechanisms and implications for cardiovascular disease. Circ Res 82:1111–1129. https://doi.org/10.1161/01. res.82.11.1111
Ikotun T (1984) Production of oxalic acid by Penicillium oxalicum in culture and in infected yam tissue and interaction with macerating enzyme. Mycopathologia 88:9–14. http://doi.org/10.1007/BF00439288
Jin L, Ham JH, Hage R, et al (2016) Direct and indirect targeting of PP2A by conserved bacterial type-III effector proteins. PLoS Pathog 12:1–33. https://doi.org/10.1371/journal.ppat.1005609
Kesarwani M, Azam M, Natarajan K, et al (2000) Oxalate Decarboxylase from Collybia velutipes: molecular cloning and its overexpression to confer resistance to fungal infection in transgenic tobacco and tomato. J Biol Chem 275:7230–7238. https://doi.org/https://doi.org/10.1074/jbc.275.10.7230
Kim KS, Min J-YY, Dickman MB, et al (2008) Oxalic acid is an elicitor of plant programmed cell death during Sclerotinia sclerotiorum disease development. Mol Plant-Microbe Interact 21:605–612. https://doi.org/10.1094/MPMI -21-5-0605
Kumari S, Tayal P, Sharma E, Kapoor R (2014) Analyses of genetic and pathogenic variability among Botrytis cinerea isolates. Microbiol Res 169:862–872. https://doi.org/https://doi.org/10.1016/j.micres.2014.02.012
Liang H, Maynard CA, Allen RD, Powell WA (2001) Increased Septoria musiva resistance in transgenic hybrid poplar leaves expressing a wheat oxalate oxidase gene. Plant Mol Biol 45:619–629. https://doi.org/10.1023/A:1010631318831
Liu X, Zhou Y, Du M, et al (2022) The calcium-dependent protein kinase CPK28 is targeted by the ubiquitin ligases ATL31 and ATL6 for proteasome-mediated degradation to fine-tune immune signaling in Arabidopsis. Plant Cell 34:679–697. https://doi.org/10.1093/plcell/koab242
Lonjon F, Rengel D, Roux F, et al (2020) HpaP sequesters HrpJ, an essential component of Ralstonia solanacearum virulence that triggers necrosis in Arabidopsis. Mol Plant Microbe Interact 33:200–211. https://doi.org/10.1094/ MPMI-05-19-0139-R
Ma W, Smigel A, Tsai Y-C, et al (2008) Innate immunity signaling: Cytosolic Ca²⁺ elevation is linked to downstream Nitric Oxide generation through the action of Calmodulin or a Calmodulin-like protein. Plant Physiol 148:8 18–828. https://doi.org/10.1104/pp.108.125104
Ma Z, Song T, Zhu L, et al (2015) A Phytophthora sojae Glycoside Hydrolase 12 Protein is a major virulence factor during Soybean infection and is recognized as a PAMP. Plant Cell 27:2057–2072. https://doi.org/10.1105/tpc. 15.00390
Malerba M, Cerana R, Crosti P (2003) Fusicoccin induces in plant cells a programmed cell death showing apoptotic features. Protoplasma 222:113–116. https://doi.org/10.1007/s00709-003-0023-5
Marciano P, Di Lenna P, Magro P (1983) Oxalic acid, cell wall-degrading enzymes and pH in pathogenesis and their significance in the virulence of two Sclerotinia sclerotiorum isolates on sunflower. Physiol Plant Pathol 22:339–345
Vijayaraghavareddy P, Adhinarayanreddy V, Vemanna R.S, et al (2017) Quantification of membrane damage/cell death using Evan’s blue staining technique. BIO-PROTOCOL 7:1–8. https://doi.org/10.21769/BioProtoc.2519
Qutob D, Kemmerling B, Brunner F, et al (2006) Phytotoxicity and innate immune responses induced by Nep1-Like Proteins. Plant Cell 18:3721–3744. https://doi.org/10.1105/tpc.106.044180
Rieger AM, Nelson KL, Konowalchuk JD, Barreda DR (2011) Modified Annexin V/Propidium iodide apoptosis assay for accurate assessment of cell death. J Vis Exp. https://doi.org/10.3791/2597
Romero-Puertas MC, Rodríguez-Serrano M, Corpas FJ, et al (2004) Cadmium-induced subcellular accumulation of O^2.- and H₂O₂ in pea leaves. Plant, Cell Environ 27:1122–1134. https://doi.org/10.1111/j.1365-3040.2004.01217.x
Ryerson DE, Heath MC (1996) Cleavage of nuclear DNA into oligonucleosomal fragments during cell death induced by fungal infection or by abiotic treatments. Plant Cell 8:393–402
Samadi L, Shahsavan Behboodi B (2006) Fusaric acid induces apoptosis in saffron root-tip cells: Roles of caspase-like activity, cytochrome c, and H₂O₂. Planta 225:223–234. https://doi.org/10.1007/s00425-006-0345-6
Sridharan H, Upton JW (2014) Programmed necrosis in microbial pathogenesis. Trends Microbiol 22:199–207. https://doi.org/10.1016/j.tim.2014.01.005
Wolpert TJ, Dunkle LD, Ciuffetti LM (2002) Host-selective toxins and avirulence determinants: what’s in a name? Annu Rev Phytopathol 40:251–285. https://doi.org/10.1146/annurev.phyto.40.011402.114210
Zhang C, He Y, Zhu P, et al (2015) Loss of bcbrn1 and bcpks13 in Botrytis cinerea not only blocks melanization but also increases vegetative growth and virulence. Mol Plant-Microbe Interact 28:1091–1101. https://doi.org/10.1094/mpmi-04-15-0085-r
Zou Y, Wang S, Zhou Y, et al (2018) Transcriptional regulation of the immune receptor FLS2 controls the ontogeny of plant innate immunity. Plant Cell 30:2779–2794. https://doi.org/10.1105/tpc.18.00297

Download PDF

Version 1

posted

You are reading this latest preprint version

The acidity of oxalic acid induces regulated cell death in Arabidopsis

Status:

Version 1

Abstract

Figures

Introduction

Methods And Materials

Plant materials and growth conditions

Fungal materials and pH measurement

RNA extraction, cDNA synthesis and real-time quantitative PCR

Transcriptome sequencing and gene expression analysis

Cell death assays

Statistical analysis

Results

Low concentration of oxalic acid induces cell death in Arabidopsis roots

Low concentration of OA induces cell death in Arabidopsis seedlings

The acidity of OA induces cell death in Arabidopsis

The acidity of OA induces an unknown type of cell death in Arabidopsis

The global gene expression in Arabidopsis upon OA treatment

The influx of calcium is likely required for the OA-induced cell death in Arabidopsis

Discussion

Declarations

References

Supplementary Files

Status:

Version 1