The Class III Peroxidase Encoding Gene AtPrx62 Positively and Spatiotemporally Regulates the Low pH-Induced Cell Death in Arabidopsis thaliana Roots

Exogenous low pH stress causes cell death in root cells, limiting root development, and agricultural production. Different lines of evidence suggested a relationship with cell wall (CW) remodeling players. We investigated whether class III peroxidase (CIII Prx) total activity, CIII Prx candidate gene expression, and reactive oxygen species (ROS) could modify CW structure during low pH-induced cell death in Arabidopsis thaliana roots. Wild-type roots displayed a good spatio-temporal correlation between the low pH-induced cell death and total CIII Prx activity in the early elongation (EZs), transition (TZs), and meristematic (MZs) zones. In situ mRNA hybridization showed that AtPrx62 transcripts accumulated only in roots treated at pH 4.6 in the same zones where cell death was induced. Furthermore, roots of the atprx62-1 knockout mutant showed decreased cell mortality under low pH compared to wild-type roots. Among the ROS, there was a drastic decrease in O2●− levels in the MZs of wild-type and atprx62-1 roots upon low pH stress. Together, our data demonstrate that AtPrx62 expression is induced by low pH and that the produced protein could positively regulate cell death. Whether the decrease in O2●− level is related to cell death induced upon low pH treatment remains to be elucidated.


Introduction
Around 70% of arable soils are acidic (pH < 5.5) [1]. In these soils, a combination of unfavorable factors occurs for plant development, such as mineral toxicity and low nutrient level, especially for cations such as calcium [2]. These factors contribute to decreased root growth with a worldwide negative impact on crop productivity [3]. A major part of acidic soils is encountered in developing countries that are distributed in tropical and subtropical zones in which the economy is highly dependent on agricultural production [4]. Thus, besides the scientific issue, understanding how acidity affects plant growth is highly relevant for agriculture and food production safety.
In addition to the abovementioned factors present in acidic soils, exogenously applied low pH conditions, such as those achieved by the addition of HCl in plant growth solutions, are used in laboratory assays. It simulates soil stressful conditions, causing root growth inhibition in relevant scavengers [30,31]. Inhibition of total CIII Prx activity decreased ROS levels and sensitivity of barley roots to low pH and aluminum, the latter being toxic only in acidic conditions [32]. On the other hand, inhibition of NADPH oxidase and CIII Prx activity in S. lycopersicum greatly increased the sensitivity of root cells solely to low pH [5]. Taking these cited reports into account, it seems that depending on the type and magnitude of the acidic stress, ROS and CIII Prx activity can modulate cellular sensitivity to low pH.
Out of the 73 CIII Prxs predicted to be encoded in the genome of A. thaliana, 38 isoforms were detected in roots by proteomic analysis [33]. Hence, to identify which isoform(s) could be responsive to exogenous low pH stress and/or how they could regulate ROS homeostasis during this stressful seeming challenging. This issue has been tackled upon by acidic stress alone [5] or in combination with aluminum stress [32]. However, these studies were usually performed through pharmacological approaches, applying inhibitors of CIII Prxs or NAPDH oxidases [5,32]. These pharmacological studies are useful to quickly block enzymatic activities or ROS production. However, pharmacological inhibitors show two well-known disadvantages: cell toxicity or undesirable side-effects upon other metabolisms. So far, it is not clear in which root zones CIII Prx activity is induced by low pH or whether it has spatial coincidence with the low pH-induced cell death. Importantly, studies investigating the role of specific CIII Prx isoforms are lacking so far.
In this article, we show that CIII Prx activity and disruption in ROS balance are spatiotemporally correlated with low pH-induced cell death in roots of A. thaliana. Furthermore, by combining data mining of previously published transcriptomics data and reverse genetics tools, we were able to show that among the 73 CIII Prx encoding genes, AtPrx62 expression is induced in low pH-induced cell death zones and could be necessary for this low pH sensitive response.

Cell Death, CIII Prx Activity and ROS Distribution Colocalized in Wild-Type Roots Exposed to Low pH
First, we examined in wild-type roots (Col-0) exposed to low pH whether a spatial correlation occurred between (i) cell death (monitored through Evans blue staining), (ii) CIII Prx activity (visualized by guaiacol/H 2 O 2 ), and (iii) ROS (O 2 •− and H 2 O 2 ) distribution (stained with nitro blue tetrazolium chloride (NBT) and hydroxyphenyl fluorescein (HPF), respectively). The guaiacol/H 2 O 2 assay, used to visualize the CIII Prx activity, allows the detection of the total endogenous activity without distinction among the isoforms. In control roots treated at pH 5.8 for 2 or 3 h, cell death did not occur ( Figure 1A,I) and CIII Prx activity was detected along the entire root except in MZs ( Figure Figure S1C). Prominently, the CIII Prx activity increase in the TZ and MZ occurred before the death of cells in these zones ( Figure 1D; Supplementary Materials Figure S1).  Figure 1F,N). Thus, upon low pH, the O 2 •− distribution in the root tip was disrupted. This result could not be interpreted as a direct effect of low pH on NBT staining, since the reaction was performed after low pH treatment in phosphate buffer at pH 6.1 [27]. The H 2 O 2 distribution in roots was examined with hydroxyphenyl fluorescein (HPF) which becomes fluorescent after oxidation by H 2 O 2 and peroxidases [27]. Control roots treated at pH 5.8 or 4.6 for 2 or 3 h showed no detectable difference in H 2 O 2 distribution. The fluorescence was similar and pronounced in TZ, EZ, and root hairs ( Figure 1G,H,O,P), similarly to previous report [27]. The hydrogen peroxide level was also quantified by a highly sensitive fluorometric assay employing Amplex ® Red. Only a slight decrease in H 2 O 2 levels could be observed after a 4 h treatment in roots at pH 4.6 as compared to pH 5.8 (Supplementary Materials Figure S2). . Cell mortality was examined staining with Evans blue. Endogenous CIII Prx activity (total activity without distinction among the isoforms) was detected with a guaiacol/H2O2 assay. The detection of O2 •− was performed using nitro blue tetrazolium chloride (NBT). The detection of H2O2 was realized using hydroxyphenyl fluorescein (HPF). Scale bar: 200 μm. Three independent experiments (n = 10) were performed with similar results and representative images are shown. Details of root zones are presented in Supplementary Materials Figure S1.
The accumulation of superoxide ion (O2 •-) was visualized with NBT which forms a purple formazan precipitate after oxidation by O2 •- [27]. Control roots treated at pH 5.8 for 2 or 3 h accumulated O2 •-mostly in MZ, as compared to TZ and EZ ( Figure 1E,M; Supplementary Materials Figure S1A). This pattern was similar to the previously observed O2 •-distribution in A. thaliana root MZ [27]. In sharp contrast, roots treated at pH 4.6 for 2 or 3 h showed no signal for O2 •-accumulation in MZ ( Figure 1F,N). Thus, upon low pH, the O2 •-distribution in the root tip was disrupted. This result could not be interpreted as a direct effect of low pH on NBT staining, since the reaction was performed after low pH treatment in phosphate buffer at pH 6.1 [27].
The H2O2 distribution in roots was examined with hydroxyphenyl fluorescein (HPF) which becomes fluorescent after oxidation by H2O2 and peroxidases [27]. Control roots treated at pH 5.8 or 4.6 for 2 or 3 h showed no detectable difference in H2O2 distribution. The fluorescence was similar and pronounced in TZ, EZ, and root hairs ( Figure 1G,H,O,P), similarly to previous report [27]. The hydrogen peroxide level was also quantified by a highly sensitive fluorometric assay employing Amplex ® Red. Only a slight decrease in H2O2 levels could be observed after a 4 h treatment in roots at pH 4.6 as compared to pH 5.8 (Supplementary Materials Figure S2).

Data Mining of Published Transcriptomics Data Searching for CIII Prx Genes Expression upon Low pH Treatment: Identification of Candidate Genes of Interest
Our results showed a spatio-temporal correlation between the occurrence of cell death, the increase in CIII Prx activity, and the decrease of O 2 •− upon low pH stress. Thus, we took advantage of a publicly available low pH transcriptomic dataset from A. thaliana roots [18] to search among the 73 CIII Prx-encoding genes-those that were the most induced after 1 or 8 h of low pH treatment. Among them, AtPrx62 (At5g39580), encoding an A. thaliana CIII Prx from the phylogenetic group 2 [34], was the best candidate since its expression was induced 8.37 fold after 8 h of low pH treatment ( Figure 2A; Supplementary Materials Table S1 for full data and phylogenetic grouping). According to published tissue-specific transcriptomics [35], AtPrx62 is expressed at high levels in epidermal and stele cells at the beginning of the maturation zone in which root hairs start tip-growth ( Figure 2B) close to the TZ in which low pH-induced cell death occurred ( Figure 1I,J). A second-level candidate was AtPrx71 (At5g64120) encoding another group 2 CIII Prx closely phylogenetically related to AtPrx62 (Supplementary Materials Table S1) showing a 3.23 fold induction of expression, but with a lower expression level than AtPrx62 ( Figure 2A) and a more distal expression pattern ( Figure 2B). Finally, AtPrx42 (At4g21960) (phylogenetic group 1) was selected as a control considering its strong constitutive expression pattern (Figure 2; Supplementary Materials Table S1). Thus, we examined the sensitivity to low pH of mutants impaired in AtPrx62 or AtPrx71 [36].
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 6 of 20 Figure 2. Transcriptomic data mining enables selecting AtPrx62 and AtPrx71 CIII Prx candidate genes for involvement in low pH response, and AtPrx42 as a control gene. (A) NASC 470 low pH transcriptomic data [18] was downloaded and edited. The mean of the log2(expression values) for each condition (n = 3) was calculated as well as the ratio of absolute expression for low pH versus control pH. The 73 CIII Prxs [37] were searched within the transcriptomic data. An absolute heat map was drawn for the expression values (red to yellow to grey) with an arbitrary threshold value set as Figure 2. Transcriptomic data mining enables selecting AtPrx62 and AtPrx71 CIII Prx candidate genes for involvement in low pH response, and AtPrx42 as a control gene. (A) NASC 470 low pH transcriptomic data [18] was downloaded and edited. The mean of the log2(expression values) for each condition (n = 3) was calculated as well as the ratio of absolute expression for low pH versus control absolute heat map was also drawn for log2 (ratio) (blue to yellow). Note that AtPrx62 displayed the highest ratio among the 73 CIII Prxs. AtPrx71 was selected as a second candidate for its intermediate ratio despite its lower absolute expression values whereas AtPrx42 was chosen as a control considering its strong constitutive expression (see Supplementary Materials Table S1 for full data and phylogenetic grouping). (B) Tissue-specific expression map of the three selected genes from the electronic fluorescent pictographic (eFP) browser [38]. Note that the absolute maximum expression values are different for each gene.
2.3. Cell Viability and Total CIII Prx Activity in Atprx62 and Atprx71 Mutants Exposed to Low pH Although cell viability was not examined in Lager et al.'s [18] work, the low pH stress seemed to be less severe than in our conditions. Our treatment solution was based on a low ionic strength buffer and low calcium supply, important for a rapid imposition of low pH stress [2, 5,9]. The expression of AtPrx62 was markedly induced only after 8 h, rather than 1 h of stress treatment in Lager's work ( Figure 2A), when the stress exposure seemed to become critical. Thus, we extrapolated that our 2 to 3 h stress conditions ( Figure 1) that roughly corresponded to the transcriptomics data obtained after 8 h rather than 1 h of low pH treatment in the Lager et al. work [18].
As a first screening step, we applied our low pH stress conditions (3 h at pH 4.6) to seedlings of the atprx62 and atprx71 mutants. Then, Evans blue staining was performed ( Figure 3). Interestingly, only the atprx62-1 knockout (KO) mutant [36] showed a clear reduced cell mortality phenotype as compared to Col-0 ( Figure 3). Indeed, the atprx62-1 knockdown (KD) mutant, that displayed residual AtPrx62 expression (40-50%) [36], only displayed a tendency of reduction of cell mortality whereas both atprx71-1 KO and atprx71-2 KD mutants [36] displayed a pattern similar to that of Col-0 ( Figure 3). Therefore, we proceeded to further analyze the atprx62-1 KO mutant. The roots of the genotypes Col-0 and atprx62-1 treated at pH 5.8 repeatedly showed viable cells with negligible Evans blue staining ( Figures 4A,B and 5A). As expected, Col-0 roots treated at pH 4.6 showed increased cell mortality in TZs and MZs ( Figures 4J and 5A). However, atprx62-1 roots were significantly less sensitive to pH 4.6 than Col-0 roots as indicated by decreased Evans blue uptake ( Figures 4K and 5A).
The CIII Prx activity showed similar patterns in the roots of Col-0 and atprx62-1 treated at pH 5.8 ( Figures 4C,D and 5B). However, Col-0 roots showed an increase in CIII Prx activity when treated at pH 4.6 for 2 h ( Figure 5B), with higher staining in the region that seemed to be stele cells ( Figure 4L, Supplementary Materials Figure S3). In the atprx62-1 KO mutant, there was also a slight increase in CIII Prx activity in MZ and TZ compared to roots treated at pH 5.8 ( Figures  μm. This initial screening was performed with 10 biological replicates (roots from individual seedlings). From at least 13 roots, five representative images are shown for each genotype upon pH 4.6. Note that only atprx62-1 displayed a clear reduced cell mortality phenotype. For simplification, one image is shown for each genotype upon pH 5.8 (control), but in none of the genotypes was there cell death in these control root repeats.
Therefore, we proceeded to further analyze the atprx62-1 KO mutant. The roots of the genotypes Col-0 and atprx62-1 treated at pH 5.8 repeatedly showed viable cells with negligible Evans blue staining ( Figure 4A,B and Figure 5A). As expected, Col-0 roots treated at pH 4.6 showed increased cell mortality in TZs and MZs ( Figure 4J and Figure 5A). However, atprx62-1 roots were significantly less sensitive to pH 4.6 than Col-0 roots as indicated by decreased Evans blue uptake ( Figure 4K and Figure 5A). This initial screening was performed with 10 biological replicates (roots from individual seedlings). From at least 13 roots, five representative images are shown for each genotype upon pH 4.6. Note that only atprx62-1 displayed a clear reduced cell mortality phenotype. For simplification, one image is shown for each genotype upon pH 5.8 (control), but in none of the genotypes was there cell death in these control root repeats. The CIII Prx activity showed similar patterns in the roots of Col-0 and atprx62-1 treated at pH 5.8 ( Figure 4C,D and Figure 5B). However, Col-0 roots showed an increase in CIII Prx activity when treated at pH 4.6 for 2 h ( Figure 5B), with higher staining in the region that seemed to be stele cells ( Figure 4L, Supplementary Materials Figure S3). In the atprx62-1 KO mutant, there was also a slight increase in CIII Prx activity in MZ and TZ compared to roots treated at pH 5.8 ( Figure 4M, Figure 5B, Supplementary Materials Figure S3). No difference in O2 •− distribution was observed in atprx62-1 roots as compared to Col-0 roots. Both genotypes displayed similar patterns of reduced O2 •− labeling in MZs with NBT following low pH treatment (Supplementary Materials Figure S4). The increase in pixels intensity indicates an increase in CIII Prx activity. The bars are the standard error of three independent experiments. Different letters (a, b and c) indicate significant differences Statistical analysis was performed by Duncan's test. Note the good correlation between low pH-induced cell death and CIII Prx activity. Note that the decreased cell death observed in atprx62-1 as compared to WT was not followed by a reduction in CIII Prx activity.

Tissue-Specific Expression Patterns of AtPrx62 and AtPrx42 in Roots upon Low pH Treatment
We next localized AtPrx62 expression through whole mount in situ hybridization. No significant specific signal was detected in Col-0 roots treated at pH 5.8 and hybridized with the AtPrx62 antisense probe ( Figure 4E; Supplementary Materials Figure S5) when compared with hybridization with the AtPrx62 sense probe used as a negative control ( Figure 4F; Supplementary Materials Figure S5). The lack of specific signal was also observed in atprx62-1 treated at pH 5.8 and hybridized with the AtPrx62 antisense probe ( Figure 4G; Supplementary Materials Figure S5). The increase in pixels intensity indicates an increase in CIII Prx activity. The bars are the standard error of three independent experiments. Different letters (a, b and c) indicate significant differences Statistical analysis was performed by Duncan's test. Note the good correlation between low pH-induced cell death and CIII Prx activity. Note that the decreased cell death observed in atprx62-1 as compared to WT was not followed by a reduction in CIII Prx activity.

Tissue-Specific Expression Patterns of AtPrx62 and AtPrx42 in Roots upon Low pH Treatment
We next localized AtPrx62 expression through whole mount in situ hybridization. No significant specific signal was detected in Col-0 roots treated at pH 5.8 and hybridized with the AtPrx62 antisense probe ( Figure 4E; Supplementary Materials Figure S5) when compared with hybridization with the AtPrx62 sense probe used as a negative control ( Figure 4F; Supplementary Materials Figure S5). The lack of specific signal was also observed in atprx62-1 treated at pH 5.8 and hybridized with the AtPrx62 antisense probe ( Figure 4G; Supplementary Materials Figure S5).
Interestingly, Col-0 roots treated at pH 4.6 and hybridized with AtPrx62 antisense probe showed a significant signal in TZ, MZ, and early EZ ( Figure 4N; Supplementary Materials Figure S5) when compared with the sense probe hybridization ( Figure 4O; Supplementary Materials Figure S5). This signal was coincident with the zone of cell death ( Figure 4J; Supplementary Materials Figure S5). The lack of signal was also confirmed in roots of atprx62-1 treated at pH 4.6 and hybridized with the antisense probe for AtPrx62 ( Figure 4P; Supplementary Materials Figure S5). The specificity of the AtPrx62 expression pattern at pH 4.6 was strengthened by the observation of a constitutive expression pattern for AtPrx42 ( Figure 4H,I,Q,R; Supplementary Materials Figure S5). These results corroborated, from a spatiotemporal point of view, the transcriptomics values and ratios (Figure 2A). Importantly, the in situ hybridization has allowed demonstrating the spatiotemporal correlation between the low pH-induced AtPrx62 expression and cell death zone (MZ, TZ, and early EZ) upon low pH treatment, thus suggesting that AtPrx62 was involved in low pH-induced cell death.

Discussion
The cell wallremodeling players are involved in low pH-induced sensitivity responses such as arrest in root growth or cell mortality in roots [2, 5,9,17,18]. Class III peroxidases (CIII Prxs) and ROS are remarkable CW remodeling players [20,25]. However, information about their involvement with low pH-induced cell death is missing. Altogether, our results show a spatiotemporal correlation in A. thaliana roots, between low pH-induced cell death, CIII Prx activity, AtPrx62 expression, and ROS distribution.

AtPrx62 Expression Is Spatiotemporally Correlated to Low pH-Induced Cell Death in Roots
We mined previously published transcriptomic data [18] to find CIII Prx gene candidates induced during low pH stress that might be possibly involved in low pH-induced cell death. Among the 73 CIII Prx genes predicted in the A. thaliana genome [21], the involvement of AtPrx62 in low pH-induced cell death was examined, because it is the CIII Prx encoding gene with the highest induction of its expression (8.37 fold) upon low pH treatment (Figure 2A, Supplemental Materials Table S1).
Our results indicate that AtPrx62 spatiotemporal expression is positively correlated to the low pH-induced cell death in MZ, TZ, and early EZ as described in our model ( Figure 6). The most compelling indications for this are as follows: (i) the expression of AtPrx62 in low pH-treated Col-0 roots increased in TZ, MZ, and early EZ, and this was correlated with the observed pattern of cell death upon low pH stress; (ii) cell death was greatly suppressed in MZ, TZ, and early EZ of the atprx62-1 KO mutant treated at pH 4.6 compared to Col-0, despite the increase in total CIII Prx activity at pH 4.6 observed in these zones for both Col-0 and atprx62-1.
Class III peroxidases belong to a large family dedicated to CW remodeling with 38 isoforms identified in the A. thaliana root CW proteome [33]. These proteins could play either specific and complementary roles (loosening or stiffening) with possible functional redundancy [20]. It is thus challenging to investigate the biological function of specific isoforms. Thus, it was rather remarkable to find that a KO mutant in a single CIII Prx isoform (AtPrx62) caused an effect on cell mortality due to the fact of low pH. A KO mutation in AtPrx71, the gene with the third most induced expression (3.23 fold) upon 8 h of low pH stress (Figure 2A; Supplementary Materials Table S1) did not result in any significant difference with respect to cell death upon low pH compared to Col-0. Hence, our study illustrates the importance of reverse genetic studies to uncover the functions of CIII Prxs [20]. In situ hybridization has allowed refining the tissue-specific expression pattern of this gene. Indeed, while tissue-specific transcriptomics argued for AtPrx62 expression in early MZ ( Figure 1B), our results clearly showed that the low pH-induced AtPrx62 expression occurred below this zone in MZ, TZ, and early EZ, i.e., in the zones where low pH-induced cell death occurred (Figures 4 and 6), thus suggesting that AtPrx62 was involved in low pH-induced cell death.
Intriguingly, AtPrx62 does not seem to be regulated by SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) [39], a transcription factor involved in low pH and Al 3+ tolerance [8,39,40]. In the same way, AtPrx62 is not a direct target of UPBEAT1, a transcription factor that regulates the expression of other CIII Prx genes necessary to control the balance between H 2 O 2 and O 2 •− in root tips controlling root growth [28]. However, AtPrx62 expression was upregulated in A. thaliana roots after 6 h of aluminum stress [41] which is appreciably toxic for roots at low pH. Unfortunately, cell death was not assessed in the quoted report, neither the gene expression level in a control at a pH higher than 5.0.  Figure 6. Spatio-temporal model proposed for the action of AtPrx62 in roots of A. thaliana upon low pH and the progression of cell death. In Columbia-0 (Col-0) roots, a reliable spatio-temporal correlation was observed in EZs, TZs, and MZs between low pH-induced CIII Prx activity (total activity: without distinction among the isoforms), AtPrx62 mRNA distribution, and cell death. The low-pH-induced cell death and AtPrx62 mRNA accumulation were highly decreased in roots of atprx62-1 KO mutant indicating that AtPrx62 was positively involved in the progression of low pH-induced cell death. However, no decrease could be measured for the CIII activity in atprx62-1.

Col-0
The prominent disruption in H2O2/O2 •− balance in root tips upon low pH stress was not dependent on AtPrx62 gene products. Whether the observed decrease in O2 •− is linked to cell death upon low pH or is caused by arrest of its production or by exacerbated scavenging, remains to be elucidated.
Intriguingly, AtPrx62 does not seem to be regulated by SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) [39], a transcription factor involved in low pH and Al 3+ tolerance [8,39,40]. In the same way, AtPrx62 is not a direct target of UPBEAT1, a transcription factor that regulates the expression of other CIII Prx genes necessary to control the balance between H2O2 and O2 •− in root tips controlling root growth [28]. However, AtPrx62 expression was upregulated in A. thaliana roots after 6 h of aluminum stress [41] which is appreciably toxic for roots at low pH. Unfortunately, cell death was not assessed in the quoted report, neither the gene expression level in a control at a pH higher than 5.0.
Although we have shown the involvement of AtPrx62 in cell death, we do not know yet if upon low pH stress, the presumed AtPrx62 activity in the apoplast contributes to CWDs. The cell wall disturbances seem to be relevant for the sensitive responses due to the low pH in roots [7,17,19]. With excessive loosening, it is a CWD that causes CW yielding in root hairs upon low pH [7]. Hence, if the presumed AtPrx62 activity causes loosening of the root CWs upon low pH, it could accelerate CWDs upon the stress. Recently, it was shown that in seed endosperm, AtPrx62 belongs together Figure 6. Spatio-temporal model proposed for the action of AtPrx62 in roots of A. thaliana upon low pH and the progression of cell death. In Columbia-0 (Col-0) roots, a reliable spatio-temporal correlation was observed in EZs, TZs, and MZs between low pH-induced CIII Prx activity (total activity: without distinction among the isoforms), AtPrx62 mRNA distribution, and cell death. The low-pH-induced cell death and AtPrx62 mRNA accumulation were highly decreased in roots of atprx62-1 KO mutant indicating that AtPrx62 was positively involved in the progression of low pH-induced cell death. However, no decrease could be measured for the CIII activity in atprx62-1. The prominent disruption in H 2 O 2 /O 2 •− balance in root tips upon low pH stress was not dependent on AtPrx62 gene products.
Whether the observed decrease in O 2 •− is linked to cell death upon low pH or is caused by arrest of its production or by exacerbated scavenging, remains to be elucidated.
Although we have shown the involvement of AtPrx62 in cell death, we do not know yet if upon low pH stress, the presumed AtPrx62 activity in the apoplast contributes to CWDs. The cell wall disturbances seem to be relevant for the sensitive responses due to the low pH in roots [7,17,19]. With excessive loosening, it is a CWD that causes CW yielding in root hairs upon low pH [7]. Hence, if the presumed AtPrx62 activity causes loosening of the root CWs upon low pH, it could accelerate CWDs upon the stress. Recently, it was shown that in seed endosperm, AtPrx62 belongs together with AtPrx69, AtPrx16, and AtPrx71 to a CIII Prx co-expression cluster that could contribute to the stiffening of endosperm CW domains to control seed envelop rupture during early germination steps [36]. However, among these four genes, only AtPrx62, and to a lesser extent AtPrx71 were found to be induced by low pH stress [18].
The CIII Prxs can regulate ROS levels by oxidizing aromatic compounds from CW components leading to a stiffened CW structure [20]. Alternatively, they produce ROS which by themselves are able to break covalent bonds between CW polymers causing the loosening of the CW structure [20,42]. This later role is the most plausible explanation for the bursting of root hairs treated with low pH [7,13]. A decrease in CW stiffness was reported in epidermal TZ cells before the onset of cell death in this root zone [19]. Thus, if the enzymatic activity of AtPrx62 is related to CW loosening, it could exacerbate CWDs caused by low pH and also accelerate the progression of cell death ( Figure 6). AtPrx10 and AtPrx71 were also up-regulated after 8 h of low-pH treatment with the ratio of induction in pH 4.5 compared to pH 6.0 of 4.36 and 3.23 fold, respectively, but with lower absolute expression values (Supplementary Table S1). AtPrx62 and AtPrx71 were found as being CW-targeted proteins in seed endosperm of A. thaliana in the region of the envelop rupture [36]. The expression of AtPrx71 was induced after CWDs due to treatment with isoxaben [43]. However, we observed no low pH-induced cell death phenotype in the mutants. Thus, if the enzymatic activity of AtPrx62 exacerbated CWDs upon low pH, it was likely to have consequences on cell survival.
Beyond the above considerations about AtPrx62 and CWDs in low pH-treated roots, the progression of cell death in roots exposed to low pH was reported in a related work of our group as a result of a programmed cell death (PCD) mechanism [19]. Before being targeted to the apoplast, unfolded secreted proteins can accumulate in the endoplasmic reticulum causing stress that disturbs the most vital cellular functions and can activate PCD [44]. The class III peroxidase AtPrx62 was shown to be upregulated upon endoplasmic reticulum stress [45]. The cell wall disturbances can produce fragments of pectin molecules called oligogalacturonides (OGs) [46] which can trigger plant immune response leading to cell death [47]. Root hairs are rich in pectin [48] and very sensitive to low pH [7]. Suspension-cultured cells of A. thaliana treated with OGs showed downregulation of AtPrx62 [49]. This information seems relevant since exogenous low pH stress is assumed to modify pectin structure in roots [2]. Hypoxia stress, which is well known to induce PCD in roots, negatively regulates AtPrx62 expression by the ethylene-responsive factor ERF73/HRE1 [50]. Hence, besides low pH stress, signaling pathways from stress situations that ultimately triggers PCD seem to regulate AtPrx62 expression. Thus, alternatively, we cannot exclude that AtPrx62 might be part of an orchestrated network leading to cell death, rather indirectly acting as a player of ROS signaling pathway.

Low pH Disrupts the O 2 •− /H 2 O 2 Homeostasis in Roots
The CIII Prx activity controls ROS homeostasis by reducing their impaired electrons while oxidizing CW components and, thus, changing, physically, CW properties [22,27,42]. Reactive oxygen species production is linked to several signaling processes such as stomata closure or developmental programs such as pollen tube formation, root hair tip-growth and CW architecture in plants [51]. Reactive oxygen species levels are tightly controlled in intracellular compartments [29] or in the apoplast [22]. High ROS concentration occurs due to exacerbated production or failure in scavenging and can cause oxidative stress damaging proteins, lipids and DNA [29,52]. Altogether, these damages targeted on key cellular macromolecules can trigger cell death [52].
In our study, we observed an interesting pattern of ROS distribution in low pH-treated roots. In Col-0, there was an increased CIII Prx activity in MZ and TZ, but decreased O 2 •− levels in MZs.
No change in H 2 O 2 levels was found in TZs, EZs, and root hairs at pH 5.8 or 4.6, similarly to a previous report [27]. Cell death-induced by low pH coincidently occurred in MZs, TZs, and early EZs, before root hairs fully undergo the tip-growth. Hence, excessive ROS levels could not be associated with low pH-induced cell death in roots, as it could have been expected. The mitochondria-dependent release of ROS was interpreted as a trigger for PCD in response to aluminum stress and low pH treatment in peanut roots [31]. The inhibition of CIII Prx activity with SHAM decreased H 2 O 2 production and cell death due to aluminum stress and low pH in barley roots [32]. Unfortunately, the responses to low pH alone could not be evaluated in the works cited above because of a lack of control at higher pH (>5.5), but perhaps aluminum induced distinct ROS activities in stressed roots compared to low pH alone, as reported here.
A balance between levels of O 2 •− in MZ and H 2 O 2 in TZ and EZ was shown to be accurately adjusted in the root tip through CIII Prx activity [27]. This balance coordinates the rate of cell division in MZ for normal root growth [28]. Low pH caused a striking decrease in O 2 •− levels in MZ and early TZ within 2 h of treatment, and, this apparently preceded both the increase in CIII Prx activity and cell death, as reported here ( Figure 6). It remains to be investigated whether a modification  Figure 6).
Altogether, our results suggest that AtPrx62 could be a positive and spatiotemporal regulator of cell death in root tip cells upon exogenous low pH stress. Our study further confirms that CW remodeling players such as CIII Prxs are crucial for the occurrence of cell death in response to low pH stress. The disruption of the H 2 O 2 /O 2 •− homeostasis in roots upon exogenous low pH may be part of a complex cell death signaling network and must be further elucidated.
Seeds of A. thaliana were sterilized with sodium hypochlorite solution (5%) for 10 min under stirring and then washed with distilled water four times. The seeds were then transferred to Petri dishes containing a modified Hoagland's solution [9] with final pH adjusted to 5. For all low pH treatments, at least 10 five-day-old seedlings were incubated in 250 mL Erlenmeyer's with 20 mL of treatment solution composed of 0.5 mM CaCl 2 and 0.6 mM Homopipes buffer (homopiperazine-1,4-bis(2-ethanesulfonic acid)) upon gentle stirring. The constant growth temperature was 22 • C and the light intensity was approximately 120 µE.m −2 .s −1 .

Evaluation of Total CIII Prx Activity and ROS Distribution in Roots Exposed to Low pH
The total activity of endogenous CIII Prxs in roots was probed using a guaiacol/H 2 O 2 assay [54]. Before the experiments, 0.125 % v/v guaiacol (Fluka, Munich, Germany) diluted in 200 mM phosphate buffer (pH 6.1) and stored at 4 • C. For the reaction, fresh 30% H 2 O 2 was added to the guaiacol solution to reach a final concentration of 1.65 mM and roots were immediately covered with this solution in glass Petri dishes kept in the dark. After 5 min of incubation, the roots were gently washed by adding abundant water to stop the reaction and were instantaneously imaged on bright-field in a Zeiss Axio Zoom.V 16 stereomicroscope (Göttingen, Germany).
A solution of NBT (2 mM) was prepared in 20 mM phosphate buffer (pH 6.1). The roots were covered with this solution in glass Petri dishes kept in the dark for 15 min and the reaction was stopped by adding water. Immediately, the roots were imaged as described above. H 2 O 2 was detected in roots using hydroxyphenyl fluorescein (HPF) [27]. The final concentration was 5 µM HPF in 20 mM phosphate buffer pH 6.1. The staining of roots with this solution was done in the dark for 15 min. The reaction was stopped by washing the roots in 20 mM phosphate buffer (pH 6.1). Immediately, the roots were imaged using a Zeiss Axio Zoom.V 16 stereomicroscope coupled to a GFP long pass filter cube (excitation 485/12 nm and emission >515 nm). The fluorescence background in roots stained with phosphate buffer alone was negligible.
H 2 O 2 was quantified using two methods. (i) measurement of freely diffusing H 2 O 2 : After treatments, 2 cm of the root tips were excised (3 independent experiments each using 10 seedlings) and incubated in plastic tubes containing 1 mL of solution composed of 50 µM Amplex ® Red (10-acetyl-3,7-dihydroxyphenoxazine, ampliflu™ red, Sigma, St. Louis, Missouri, USA) and 2 U/mL horseradish Prx for 10 min in the dark. Following, the reaction was immediately stopped by adding SHAM 3 mM. The fluorescence was read upon 570 nm of excitation and 585 nm of emission with a spectrofluorimeter (Varian Cary Eclipse, Agilent ® ). (ii) measurement of total H 2 O 2 : the same protocol was used except that root tips were first macerated before the reaction.

Transcriptomic Data Mining for CIII Prx Genes Involved in Low pH Response
We analyzed public transcriptomic data to search for CIII Prx encoding genes potentially involved in low pH response. The data set NASC 470 from Lager's work [18] was downloaded at [55] using Expression Console™ 1.4.1.46 [56] to build an edited Microsoft Excel sheet [57]. The mean of the log2(value) for each condition (n = 3) was calculated as well as the ratio of absolute expression at pH 4.5 versus control at pH 6. The 73 CIII Prx genes [37] were searched for within the transcriptomic data using their probeset ID allowing the identification of ambiguous and non-ambiguous CIII Prxs [58]. Absolute heat map was drawn for the expression values (red to yellow to grey) with an arbitrary threshold value set as 5. Absolute heat map was drawn for ratio of absolute expression (blue to yellow).

Evaluation of Cell Death
Cell death was evaluated by probing roots with Evans blue that can penetrate dead cells that lost membrane selectivity [59]. After pH treatments the seedlings were stained with Evans blue aqueous solution (0.25% w/v) for 15 min. Then, they were washed three times for 5 min each with distilled water and bright field images were taken using a Zeiss Axio Zoom V16 stereomicroscope. All procedures were performed in glass Petri dishes taking care of avoiding damages or root dehydration.

Image Analysis
To obtain semi-quantitative data, the images of Evans blue staining or of CIII Prx activity in roots were analyzed using the ImageJ software [60]. These images were used to draw the contour of each root tip, reaching 500 µm and 350 µm from the root tip for Evans blue staining and CIII Prx activity, respectively, constituting the regions of interest (ROIs). In both cases, the mean gray values of these ROIs were obtained. From each of these values, the mean gray value of the background of the corresponding bright-field image was subtracted to compensate variations in the light intensity between each image. The results were expressed as pixel intensity of the mean gray value. Thus, the increase of pixels intensity was straightforward interpreted as an increase in cell death or increase in total CIII Prx activity.

Whole Mount In Situ mRNA Hybridization
The protocol described in Hejatko's work [61] was followed using the solutions described in detail in Francoz et al. [58] using 5 day old seedlings. The samples (10-20 seedlings) were processed in 0.95-1 mL solution/condition in 24 well sterile plate or in 1.5 mL RNase free microtubes for the hybridization step.The following minor modifications were introduced: use of Roti ® Histol (Carl Roth, Karlsruhe, Germany) for sample permeabilization, replacement of heparin with dextran sulfate in the hybridization solution. The digoxigenin-labelled riboprobes for detection of AtPrx62 or AtPrx42 were previously described [52]. The key parameters were as follows: (i) 125 µg mL −1 proteinase K for prehybridization; (ii) hybridization with a digoxigenin-labelled riboprobe at a final concentration of 50 ng/kb/mL for 16 h at 55 • C; (iii) immunodetection of hybridized probes with 1:2000 diluted anti-digoxigenin-alkaline phosphatase (AP) Fab-fragments (Roche, Basel, Switzerland) [52]; (iv) BCIP-NBT reaction for 48 min in the dark; (v) final mounting of samples in 50% (w/v) glycerol; (vi) and microscope analysis using a slide Nanozoomer slide scanner (Hamamatsu, Shizuoka, Japan) to produce whole slide scan at 20× resolution with five 10 µm-z scans to ensure finding the correct focus for all samples. The scans were analyzed using NDP view (Hamamatsu) and the images were directly extracted from the viewer to assemble the Figure.

Statistical Analysis
We conducted randomized experiments. For each parameter analyzed at least three independent experiments were performed. Each biological replicate was composed of at least 10 seedlings. Means were compared by analysis of variance (ANOVA), followed by Duncan's test. Only two means were compared by Student's t-test at the 5% significance level.  Table S1: Transcriptomics data mining for CIII Prx multigenic family expression following low pH treatment of A. thaliana roots reveals AtPrx62 as the most promising candidate gene for involvement in low pH-induced cell death in roots. Supplementary Figure