The Arabidopsis glutathione transferases, AtGSTF8 and AtGSTU19 are involved in the maintenance of root redox homeostasis affecting meristem size and salt stress sensitivity
Introduction
Glutathione transferases (GSTs, EC 2.5.1.18) constitute a very ancient protein superfamily that participate in a broad network of catalytic and regulatory functions. Their most known role is the detoxification of exogenous and endogenous harmful compounds, including herbicides, xenobiotics and endogenous stress metabolites. They are also involved in numerous redox, hormone and stress responses. Mediating cross-talks between these signaling pathways, they have important roles in various developmental processes such as apoptosis or growth regulation [[1], [2], [3], [4]]. Plant GSTs are grouped into ten different classes, among them the tau (GSTU), phi (GSTF), lambda (GSTL) and dehydroascorbate reductase (DHAR) are specific to plants. The tau and phi classes are largely responsible for catalyzing conjugation of reduced glutathione (GSH; γ-glu-cys-gly) with wide range of electrophyl substrates [5,6]. That is why their catalytic activity may reduce the GSH pool. These isoenzymes and a significant portion of other GSTs also has glutathione peroxidase (GPX) activity and can convert lipid peroxides and other peroxides to less harmful compounds [7]. DHAR and GSTL enzymes catalyse redox reactions, even deglutathionylation, and participate in the recycling of antioxidants, such as ascorbate (ASC) and flavonols [8,9].
The total amounts of non-enzymatic antioxidants (e. g. GSH, ASC and flavonoids) and their reduced-oxidized status are essential elements of the redox homeostasis of cells [10]. They are linked with the production or enhanced availability of reactive oxygen species (ROS), among them superoxide radical (O2−), hydrogen peroxide (H2O2), and hydroxyl radical (OH). ROS generation accompanies the normal aerobic metabolism, but their level typically increases in plants exposed to different stresses [11]. ROS production induces detrimental oxidation of macromolecules including DNA, proteins, and lipids. On the other hand, endogenous change in oxidant levels can fulfill signaling functions and play a positive role in adaptation to the changed environmental conditions [[12], [13], [14]]. In addition, they regulate many developmental processes [15,16]. The amount and distribution of ROS have been shown to be crucial in maintaining the root meristem size, together with the antagonistic cytokinin/auxin interaction [17].
Plants maintain a high cellular ratio of GSH to its oxidized glutathione disulfide (GSSG) form, but GSH reacts with oxidants during environmental stress and becomes converted into GSSG. Shifts in the cellular glutathione redox status may reversibly modify redox-sensitive thiol groups in target proteins either through glutathionylation or formation of cysteine crossbridges. Many reports indicate that the [GSH]:[GSSG] ratio and the glutathione half-cell reduction potential (EGSSG/2GSH), which depends on the absolute glutathione concentration and the ratio of GSH and GSSG, can be effective markers of the overall redox homeostasis [[18], [19], [20], [21], [22]].
A non-destructive technology to detect changes in redox potential has been developed by the introduction of redox-sensitive green fluorescent protein (roGFP) imaging [[23], [24], [25], [26]]. Engineering of two surface-exposed cysteines into the GFP allows reversible disulfide formation. The thiol-disulfide status of the roGFP can be equilibrated with that of glutathione in the cells. The fluorescence image can be obtained by confocal microscope. Determination of the fluorescent intensity of the fully reduced and fully oxidized form of the probe enables quantitative monitoring of EGSH without destroying the cell [21,25]. Different roGFPs (roGFP1-4, roGFP-iX) have been used as ratiometric redox sensors [[27], [28], [29], [30], [31], [32]]. Jiang et al. [33] analysed the redox potential profile of the primary root tip of 5-day-old Arabidopsis seedlings applying roGFP1 as redox sensor and demonstrated that E values may differ according to root zones. They found that in seedlings grown on agar the most reduced redox status is at, or nearly at, the quiescent center (QC), and moving to proximal direction the redox status becomes more oxidized. The terminal 300 μm including the root cap initials, the QC and the most distal portion of the proximal meristem (PM) exhibited the highest redox difference of 5–10 mV under different growth condition. Treating roots with 50, 100 or 150 mM NaCl resulted in marked changes in root meristem structure and development, and also the redox profile [33].
It was reported that glutathione is specifically required to activate and maintain the cell division cycle in the root apical cells [[34], [35], [36], [37]]. Severe GSH depletion specifically inhibited root meristem development, while low root GSH levels decreased lateral root densities [37]. Low level of GSH significantly increased the redox potentials and caused arrest of the cell cycle in roots but not shoots. Applying the GSH-synthesis-inhibitor buthionine sulphoximine (BSO) resulted in significantly more oxidized redox potential in the root tip cells (the values were ca. 50 mV less negative both in the cytosol and nuclei). Vernoux and his colleagues performed a transcript profiling analysis of an Arabidopsis thaliana rootmeristemless1 (rml1) mutant, which is severly limited in GSH synthesis capacity. Expression of several hundred genes encoding transcription factors and proteins particularly involved in hormone-dependent regulation of plant growth and development had changed [35]. Although low GSH level affects mostly the hormonal homeostasis of plants modulating developmental responses, several results showed that it is also linked to stress responses [22]. Among the genes regulated by low GSH, numerous redox-related proteins were found, such as glutaredoxins (GRXs), h-type thioredoxins (TRXs), glutathione peroxidases (GPXs), DHARs and GSTs [37].
In A. thaliana the tau and phi GSTs are the two most numerous GST classes: they have 28 and 13 members, respectively [38]. Comprehensive expression analysis indicated overlapping and specific roles of GST genes during development and stress responses [39]. Overexpression of several GSTs have been reported to increase the chilling, osmotic stress, salinity and/or herbicide tolerance, and in most cases even the growth of transgenic plants were altered [[40], [41], [42], [43], [44], [45], [46], [47], [48], [49]]. Recently, Bielach et al. [50] suggested that the auxin-inducible AtGSTU5 may represent a general component of the environmental stress response, because it was upregulated by both auxin and cytokinin hormones and all the investigated abiotic stresses.
Analysis of the expression pattern of Arabidopsis GST-coding genes using the Genevestigator online tool revealed that AtGSTF8 and AtGSTU19 express at a very high level especially in roots [51]. The expression of AtGSTF8 was induced by short-term salicylic acid treatment [52,53], ethylene, and H2O2 [54], while that of AtGSTU19 was induced by biotic stress [54], salicylic acid (SA) and H2O2 [55]. Xu et al. [45] demonstrated that the AtGSTU19-overexpressing plants showed not only enhanced tolerance to different abiotic stresses, increased percentage of seed germination and cotyledon emergence, but also the expression levels of several stress-regulated genes were altered. The AtGSTU19 overexpressing plants exhibited increased activities of antioxidant enzymes and had enhanced amount of proline along with decreased malondialdehyde level under stress conditions [45]. Interestingly, although the catalytic activities of GST proteins may reduce the GSH pool by using reduced glutathione as a co-substrate, it was also suggested that GST enzymes may participate in the maintenance of the redox status of cells [8,9]. For instance, in our earlier investigations we found that Atgstf9 mutants accumulated more ASC and GSH than the wild type (WT) plants, and had altered redox homeostasis [56].
Here we report detailed analysis of the redox status across longitudinal zones of roots in one-week-old Col-0 and Atgstf8, Atgstu19 insertional mutant plants expressing the GRX1-roGFP2 fluorescent protein. Our main aim was the comparison of the redox state and ROS levels in different zones of roots under control conditions and after applying salt stress. According to our results, the redox status of un-treated Atgstu19 roots was more oxidized, than that of the Col-0 or Atgstf8, and the size of the mutant’s meristem proved to be shorter compared to the wild type. The redox potential showed the biggest differences in the proximal meristem of the roots. Treatment with 75 or 150 mM NaCl for 3 h resulted in more oxidized redox state generally in all studied zones of the roots of the investigated genotypes, but the highest redox potential values (most oxidized redox status) were detected in the transition zone of the Atgstu19 mutant.
Section snippets
Plant material and growth conditions
Arabidopsis thaliana (L.) ecotype Columbia (Col-0) as a wild-type control and the mutants, Atgstf8 and Atgstu19, related to the At2g47730 and At1g78380 genes, respectively, were used in all experiments. The T-DNA insertional lines, Atgstf8 (N859808) and Atgstu19 (N541942) were obtained from the NASC [57], and tested for homogenisity using gene-specific PCR primers (Fig. S1, Table S1). The seedlings were grown in vitro at a photon flux density of 100 μmol m−2 s-1 (12/12 day/night period), at a
AtGSTF8 and AtGSTU19 differently determines ROS levels and redox status
Our aim was to investigate the involvement of AtGSTF8 and AtGSTU19 in the control of the redox status and ROS homeostasis in the root meristem. For this, the redox-dependent fluorescence of GRX1-roGFP2-expressing roots of the Atgstf8 and Atgstu19 mutants was monitored and compared to that of the Col-0 control. The redox potential values (EGSH) were calculated in the proximal meristem (PM), transition zone (TZ) and elongation zone (EZ). The most reduced, −301.02 mV, redox potential was detected
The AtGSTF8 and AtGSTU19 enzymes are differentially involved in the maintenance of the redox homeostasis of root meristem zones
ROS modulate cell division, differentiation, and expansion via functioning as second messengers and/or affecting the cell wall structure [[67], [68], [69],16]. The responses of cells to cellular oxidation due to abiotic stress or the action of defense phytohormones depend on cell identity [70,71]. Jiang et al. [33] demonstrated using the roGFP1 probe that the redox potentials of various regions of 3–9 day-old Arabidopsis roots differ significantly. In accordance with the above reports, we found
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Author contributions
JC contributed in planning the experiments, wrote and edited the manuscript. EH designed and coordinated the study, analyzed the data, wrote and edited the manuscript. KB, BH, RR, ÁH, ÁBH and EH performed the experiments. AF, KB and ÁG discussed the results and contributed to the writing of the manuscript.
Acknowledgements
This study was supported by the Hungarian National Research, Development and Innovation Office [Grant Numbers: NKFI-6 K 125265 and NKFI-1 PD 121027]. We thank Dr. F. Ayaydin and I. Kelemen for their technical assistance in confocal microscopy observations in the Cellular Imaging Laboratory of BRC, Szeged. We would like to thank Prof. Dr. A. Meyer for the c-GRX1-roGFP2-harbouring plasmids and Dr. G. Rigó for GV3101 Agrobacterium strain. The authors would like to thank Mrs. Erzsébet Porkoláb for
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2021, Developmental CellCitation Excerpt :We prioritized a set of these markers by predicted developmental roles and validated them using in situ hybridization (Figures 2B–2M; Table S1). For example, marker genes for meta-cluster 9, GRMZM2G004528, annotated as ZmMYO-INOSITOL PHOSPHATE SYNTHASE2 (ZmMIPS2), predicted to act in auxin signaling and transport (Chen and Xiong, 2010), and GRMZM2G097989, annotated as ZmGLUTATHIONE TRANSFERASE 41 (ZmGST41), involved in meristem size control (Horváth et al., 2019), showed specific expression in the meristem boundary, similar to BD1 (Figures 2B and 2C; Chuck et al., 2002). ZmGST41 was also a DE marker for meta-cluster 12 and consistently showed vascular trace expression (Figure 2C, arrow).