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. 2020 Nov 13;10(11):1550.
doi: 10.3390/biom10111550.

Glutathione Enhances Auxin Sensitivity in Arabidopsis Roots

Affiliations

Glutathione Enhances Auxin Sensitivity in Arabidopsis Roots

Taras Pasternak et al. Biomolecules. .

Abstract

Root development is regulated by the tripeptide glutathione (GSH), a strong non-enzymatic antioxidant found in plants but with a poorly understood function in roots. Here, Arabidopsis mutants deficient in GSH biosynthesis (cad2, rax1, and rml1) and plants treated with the GSH biosynthesis inhibitor buthionine sulfoximine (BSO) showed root growth inhibition, significant alterations in the root apical meristem (RAM) structure (length and cell division), and defects in lateral root formation. Investigation of the molecular mechanisms of GSH action showed that GSH deficiency modulated total ubiquitination of proteins and inhibited the auxin-related, ubiquitination-dependent degradation of Aux/IAA proteins and the transcriptional activation of early auxin-responsive genes. However, the DR5 auxin transcriptional response differed in root apical meristem (RAM) and pericycle cells. The RAM DR5 signal was increased due to the up-regulation of the auxin biosynthesis TAA1 protein and down-regulation of PIN4 and PIN2, which can act as auxin sinks in the root tip. The transcription auxin response (the DR5 signal and expression of auxin responsive genes) in isolated roots, induced by a low (0.1 µM) auxin concentration, was blocked following GSH depletion of the roots by BSO treatment. A higher auxin concentration (0.5 µM) offset this GSH deficiency effect on DR5 expression, indicating that GSH deficiency does not completely block the transcriptional auxin response, but decreases its sensitivity. The ROS regulation of GSH, the active GSH role in cell proliferation, and GSH cross-talk with auxin assume a potential role for GSH in the modulation of root architecture under stress conditions.

Keywords: auxin; auxin biosynthesis; auxin response; auxin transport; glutathne (GSH); lateral root formation; root apical meristem.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Root traits of wild type, cad2, rax1, and rml1 mutants. (A) Semi-quantitative analysis of low molecular weight thiols using fluorescent in situ labeling with bromobimane. Scale bars represent 20 µm; (B) Root length after seven days of growth. Error bars represent the means ± SDs (n = 8); (C) Length of the root apical meristem at seven days after germination. Error bars represent the means ± SDs (n = 8). Different letters indicate a significant difference at p < 0.05. Fisher’s protected LSD test was used to compare the individual means; (D) root apical meristem (RAM) structure after propidium iodide (PI) staining. White arrow indicates the end of meristematic zone borders; Scale bars represent 100 µm.
Figure 2
Figure 2
Pharmacological manipulation affecting tripeptide glutathione (GSH) contents and root growth. (A) Root length after five days of exposure to different concentration of L-buthionine-(R,S)-sulfoximine (BSO). Error bars represent the means ± SDs (n = 8–10); (B) GSH concentrations in roots of Arabidopsis seedlings after treatments with L-buthionine-(R,S)-sulfoximine (BSO) and BSO plus IAA. Error bars represent the means ± SEs (n = 3); (C) Root length after 5 days of exposure to different concentrations of GSH with and without adjustment of the pH of the growth medium. Error bars represent the means ± SDs (n = 10–16). Different letters indicate a significant difference at p < 0.05. Fisher’s protected LSD test was used to compare the individual means; (D) RAM structure after propidium iodide (PI) staining. White arrows show the end of meristematic zone borders. Scale bar −50 µm.
Figure 2
Figure 2
Pharmacological manipulation affecting tripeptide glutathione (GSH) contents and root growth. (A) Root length after five days of exposure to different concentration of L-buthionine-(R,S)-sulfoximine (BSO). Error bars represent the means ± SDs (n = 8–10); (B) GSH concentrations in roots of Arabidopsis seedlings after treatments with L-buthionine-(R,S)-sulfoximine (BSO) and BSO plus IAA. Error bars represent the means ± SEs (n = 3); (C) Root length after 5 days of exposure to different concentrations of GSH with and without adjustment of the pH of the growth medium. Error bars represent the means ± SDs (n = 10–16). Different letters indicate a significant difference at p < 0.05. Fisher’s protected LSD test was used to compare the individual means; (D) RAM structure after propidium iodide (PI) staining. White arrows show the end of meristematic zone borders. Scale bar −50 µm.
Figure 3
Figure 3
Effect of GSH level on auxin response markers and auxin biosynthesis gene expression. (A) BSO dose-dependently increases AXR3NT-GUS stability in response to auxin. Seven-day-old seedlings were treated for 12 h with different L-buthionine-(R,S)-sulfoximine (BSO) concentrations in the liquid medium and/or pre-treated in the control medium. Seedlings were subjected to heat shock by cultivation at 37 °C for 1.5 h, allowed to recover for the next 10 min, and then subjected to GUS assays for the next 1–1.5 h; (B) BSO increases DII::VENUS stability without and with NAA application; (C) DR5::GUS signal in wildtype seedlings, rml1 mutant seedlings, and seedlings treated with 1 mM BSO; (D) TAA1::GFP signal in RAM upon BSO treatment. Scale bar −50 µm. (E) DII::VENUS quantification in response to BSO concentrations. Error bars represent the means ± SDs (n = 6). Different letters indicate a significant difference at p < 0.05; (F) DII::VENUS quantification following BSO and NAA treatments (represented image in B). Error bars represent the means ± SEs (n = 6); (G) TAA1::GFP quantification in RAM (represented image from D). Error bars represent the means ± SDs (n = 8). Means of treatments with * and ** are significantly different from means of the control at p < 0.05 and p < 0.01, respectively. Fisher’s protected LSD test was used to compare the individual means.
Figure 4
Figure 4
Effect of BSO, NAA, and proteasome inhibitors on the stability of AXR3NT-GUS. Four-day-old HS::AXR3NT-GUS seedlings were adapted in liquid medium and then treated for 12 h with BSO and 20 min with proteasome inhibitors. The seedlings were then treated for 1.5 h with 100 nM NAA and subjected to heat-shock at 37 °C for 1.5 h. GUS assays were performed for next 1.5 h. Scale bar −50 µm.
Figure 5
Figure 5
GSH level affects protein ubiquitination status. Protein was isolated from 4-day-old WT and homozygotic rml1 plants and subjected to immunoblotting with Ubi11 AS08 307 (Agrisera) antibody. Each lane contains 10 µg protein. Arrows show the accumulations of high MW ubiquitinated proteins in rml1 mutant.
Figure 6
Figure 6
GSH level altered the PIN signal localization in the RAM. PIN1, PIN2, and PIN4 expression and localization in the RAM of Arabidopsis seedlings grown in a medium with different L-buthionine-(R,S)-sulfoximine (BSO) concentrations. Scale bar −20 µm.
Figure 7
Figure 7
GSH level altered the PIN signal localization in the RAM. PIN1, PIN2, and PIN4 localization in rml1 mutant at 3 and 5 days after germination and rml1 mutants treated with 0.5 mM GSH. Scale bar −20 µm.
Figure 8
Figure 8
GSH affected the induction of lateral root primordia and the DR5 signal in isolated roots. (A) The effect of 1 mM L-buthionine-(R,S)-sulfoximine (BSO) and different NAA concentrations on GSH concentrations in isolated roots. Error bars represent the means ± SEs (n = 3); (B) BSO inhibited de novo formation of the lateral root primordia. Error bars represent the means ± SEs (n = 10). Different letters indicate a significant difference at p < 0.05. Fisher’s protected LSD test was used to compare the individual means; (C) BSO inhibited the auxin response, determined as DR5 activity, after NAA treatments. Scale bar −100 µm.
Figure 9
Figure 9
GSH is important for the auxin-induced response. GSH regulated the transcription of classical auxin-responsive genes according to real-time qPCR assays. Isolated roots from 7-day-old plants were pre-treated with 0 and 2 mM L-buthionine-(R,S)-sulfoximine (BSO) for 12 h and then treated with 100 nM NAA for the indicated times. Error bars represent the means ± SEs (n = 3). Means of treatments marked with * and ** are significantly different from means of the control at p < 0.05 and p < 0.01, respectively. Student’s t-test was used.

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