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. 2020 May 4;39(9):e103894.
doi: 10.15252/embj.2019103894. Epub 2020 Mar 18.

SCHENGEN Receptor Module Drives Localized ROS Production and Lignification in Plant Roots

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Free PMC article

SCHENGEN Receptor Module Drives Localized ROS Production and Lignification in Plant Roots

Satoshi Fujita et al. EMBO J. .
Free PMC article

Abstract

Production of reactive oxygen species (ROS) by NADPH oxidases (NOXs) impacts many processes in animals and plants, and many plant receptor pathways involve rapid, NOX-dependent increases of ROS. Yet, their general reactivity has made it challenging to pinpoint the precise role and immediate molecular action of ROS. A well-understood ROS action in plants is to provide the co-substrate for lignin peroxidases in the cell wall. Lignin can be deposited with exquisite spatial control, but the underlying mechanisms have remained elusive. Here, we establish a kinase signaling relay that exerts direct, spatial control over ROS production and lignification within the cell wall. We show that polar localization of a single kinase component is crucial for pathway function. Our data indicate that an intersection of more broadly localized components allows for micrometer-scale precision of lignification and that this system is triggered through initiation of ROS production as a critical peroxidase co-substrate.

Keywords: Casparian strips; extracellular diffusion barriers; lignin; localized ROS production; polarized signaling.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. Apolar SGN1 leads to ectopic lignin accumulation in endodermal cells

Schematic of Casparian strip development (magenta). Casparian strips start to appear as centrally aligned discontinuous dots in the endodermal cell layer, progressing into a network of fused rings functioning as a root apoplastic barrier.

Lignin accumulation patterns at endodermal surface or median positions with or without the 100 nM CIF2 (Casparian strip integrity factor 2) ligand treatment. Lignin and cellulosic (unmodified) cell walls are stained with Basic Fuchsin and Calcofluor White, shown in magenta and white, respectively. Schematics are indicating the position of optical sections in a 3D illustration. For each condition, at least 10 roots were tested and showed similar results in two independent experiments. White arrows indicate sites of excess lignification on the cortex‐facing (outer) side. Scale Bar = 5 μm.

Localization of SGN1‐Citrine and lignin deposition patterns in pCASP1::SGN1‐Citrine lines in wild‐type (Col) and different mutant backgrounds (sgn1, sgn3, cif1 cif2) (C). myrpalm‐SGN1‐Citrine localization and lignin deposition patterns in pCASP1::myrpalm‐SGN1‐Citrine lines (D). Lignin (Basic Fuchsin) and cell walls (Calcofluor White) are shown in magenta and white, respectively. For this experiment, two or three independent lines were tested. From each transgenic line, 2 positions from 12 roots were observed and representative pictures are shown in the figure. Schematics are indicating the position of optical sections in a 3D illustration. White arrows in (D) highlight excess lignification on the pericycle‐facing (inner) side. Scale bars = 5 μm.

Schematic illustrating how signal activation can be governed by SGN1 localization and peptide ligand diffusion from the stele.

Data information: “inner” designates the stele‐facing endodermal surface, “outer”, the cortex‐facing surface.
Figure EV1
Figure EV1. Apolar SGN1 leads to ectopic lignin accumulation in endodermal cells

PI penetration assay. Scoring of number of cells after the onset of cell elongation until PI signal is excluded from the inner side of the endodermis (10 roots in total were tested in each condition during two independent assays). In the box plot, boxes are showing ranges from the first to third quartiles, and the bold central lines display median. Upper and lower whiskers extend to the maximum or minimum values no further than 1.5 times IQR. Different letters indicate significant statistical differences (P < 0.01, one‐way ANOVA and Tukey test).

Localization of CASP1‐mCherry, driven by CASP1 promoter, in pCASP1::SGN1‐Citrine or pCASP1::myrpalmSGN1‐Citrine transgenic lines. For each crossed line, more than 10 roots were observed and showed similar localization patterns. Scale bar = 10 μm.

Localization patterns of SGN1 (WT, kinase dead (KD)), myrpalm‐SGN1 (WT and KD), and lignin deposition patterns in each indicated transgenic line. Arrowheads indicate excess lignification. For this experiment, two independent lines were tested. From each transgenic line, 2 spots from 12 roots were observed and representative pictures are shown. Scale bars are 10 μm in SGN1‐Cit, 5 μm in lignin and cell wall pictures, 20 μm in overview of lignin deposition.

One‐base pair insertion sites of cif1‐2 and cif2‐2. Red letters indicate inserted bases in each locus.

PI penetration phenotype of the cif1 cif2 double mutant with or without 100 nM peptide treatment. Seedlings were germinated on the medium with or without peptides. At least five roots were observed in each condition. Asterisks indicate the stele. Scale bar = 40 μm.

Whole root views of suberin deposition patterns in polar‐ or apolar‐SGN1 transgenic lines. esb1 (enhanced suberin 1) is shown as a representative oversuberized mutant. Scale bar = 500 μm.

Quantification of the ratio of suberized zones and root lengths in each mutant or transgenic line. esb1 is shown as a representative oversuberized mutant. In the box plot, boxes are showing ranges from the first to third quartiles, and the bold central lines display median. Upper and lower whiskers extend to the maximum or minimum values no further than 1.5 times IQR. Different letters are indicating statistically significant differences (n = 16–36 roots, P < 0.01, ANOVA and Tukey test).

Figure 2
Figure 2. SGN1 acts as a transducer of CIF2 signaling and is phosphorylated by the SGN3 receptor

Quantification of defects in CSD formation as number of holes per 100 μm in the CASP1‐GFP domain at around 10 cells after onset of CASP1‐GFP expression in 5‐day‐old seedlings. In the box plot, boxes indicate ranges from first to third quartiles, and the bold central lines display median. Upper and lower whiskers extend to maximum or minimum values no further than 1.5 times IQR (interquartile range, the distance between the first and third quartiles). One‐way ANOVA was performed followed by Tukey's test. Different letters show significant statistical differences (P < 0.05, one‐way ANOVA and Tukey's test, 12 roots in total were observed for each condition in two independent assays).

Propidium iodide (PI) penetration assay in the presence or the absence of CIF2. CS barrier function was scored as exclusion of PI signal from the inner side of endodermal cells. In the box plot, boxes indicate ranges from first to third quartiles, and the bold central lines display median. Upper and lower whiskers extend to the maximum or minimum values no further than 1.5 times IQR. Different letters show significant statistical differences (P < 0.05, one‐way ANOVA and Tukey's test. During two independent experiments, 10 roots in total were tested for each condition).

[γ‐32P]ATP radioactive in vitro kinase assay of SGN3 kinase domain against SGN1. Autoradiograph is shown on top. Coomassie‐stained gel below illustrates presence and equal loading of recombinant proteins. Note that a kinase‐dead SGN1 variant was used to avoid autophosphorylation activity of SGN1. Also note that trigger factor represents a very big tag protein, accounting for the high migration of TF‐SGN1. Representative result of three independent experiments is shown.

Figure 3
Figure 3. Both RBOHD and F are required for CIF2‐induced excess lignin accumulation

Localization of Citrine‐RBOHD (left) or Citrine‐RBOHF (right) in endodermal (en) cells. Both proteins were expressed under the control of pCASP1, an endodermis‐specific promoter. Representative pictures are shown; 2 positions from 10 roots for each transgenic line were inspected. Scale bar = 5 μm.

Lignin accumulation in WT and rbohD and rbohF single mutants and a double mutant with or without 2‐h 100 nM CIF2 peptide treatment. Arrowheads indicate excess lignification. Pictures are shown as overviews (maximum projection) or median sections. Lignin and cell walls are shown with magenta (stained with Basic Fuchsin) and gray (stained with Calcofluor White), respectively. Representative pictures are shown; 12 roots (overview) and 2 positions in 12 roots (median section) were inspected. Scale bars = 20 μm (lignin overviews), 5 μm (median sections). “inner” designates the stele‐facing endodermal surface, “outer”, the cortex‐facing surface.

Figure EV2
Figure EV2. Both RBOHD and F are required for CIF2‐induced excess lignin accumulation

Co‐treatment experiments with the NADPH oxidase inhibitor DPI (diphenyleneiodonium chloride) and CIF2. Pretreatment was done on medium with or without DPI for 30 min, and seedlings were then transferred to each medium (100 nM for CIF2 and 10 μM for DPI, respectively). The seedlings were incubated for 2 h in each condition. Arrowheads indicate excess lignification on the cortex‐facing side. Representative pictures are shown from two independent experiments (2 spots from 5 roots in each condition for one experiment) with similar results. Scale bar = 5 μm.

Whole root views of suberin deposition patterns in the rbohD and rbohF mutants with or without CIF2 treatment. Five‐day‐old seedlings were treated for 24 h with or without CIF2 and stained. Scale bar = 1 mm.

Quantification of the ratio of suberized zones to root lengths in each mutant from (B) (n = 13–24 roots). In the box plot, boxes are showing ranges from the first to third quartiles, and the bold central lines display median. Upper and lower whiskers extend to the maximum or minimum values no further than 1.5 times IQR. Different letters indicate statistical significance (one‐way ANOVA, Tukey's test).

Figure 4
Figure 4. ROS production is enhanced by SGN3/CIFs and requires RBOHD and F

Co‐visualization of pCASP1::SGN1‐mCherry and pCASP1::SGN3‐GFP. Note that their respective localization at the PM has only restricted overlap at the cortical side of the CS domain (Arrow). Representative picture is shown; 3–6 roots in four different transgenic lines were inspected. Scale bar = 10 μm. “inner” designates the stele‐facing endodermal surface, “outer”, the cortex‐facing surface.

Overview of endodermal cells after with or without 1 μM CIF2 treatment for 24 h. Red arrowheads are indicating ROS production sites, and boxes in dotted lines are corresponding to the regions in (sky blue and creme boxes in C and black boxes in D). Similar patterns were obtained in 39 or 41 cells from 5 roots with or without the peptide treatment. Scale bars = 500 nm (B) and 100 nm (C).

In situ H2O2 detection at Casparian strips in WT, sgn3, sgn1, rbohF, rbohD, and rbohDF with or without 24‐h treatment of 1 μM CIF2. Brackets and arrowheads indicate Casparian strips (seen as uniformly whitish cell wall areas) and H2O2 production sites (black area), respectively. Scale bar = 1000 nm.

Quantification of ROS production as a number of dark pixels area (n = 23–44 sites from 5 roots of each condition in one experiment.). For quantification methods, see Fig EV3. In the box plot, boxes indicate ranges from first to third quartiles, and the bold central lines display median. Upper and lower whiskers extend to the maximum or minimum values no further than 1.5 times IQR. Different letters mean significant statistical differences (P < 0.01, one‐way ANOVA and Tukey's test.)

Figure EV3
Figure EV3. ROS production is enhanced by SGN3/CIFs and requires RBOHD and F

A schematic illustrating the protocol for pixel area quantification of ROS as measured by the cerium chloride method. Pictures were normalized to a picture of non‐treated WT. Following the normalization, the area was chosen manually from the cortex side corner to the end of the CS at the pericycle side. Pixels below the threshold were marked as pink dots and counted. For more details, see the materials methods part. Note that the picture of WT (+CIF2) was reused from Fig 4D. Scale bar = 1000 nm.

Figure 5
Figure 5. SGN1 directly activates NADPH oxidases in a cellular context

[γ‐32P]ATP radioactive in vitro kinase assay of TF‐SGN1 against GST‐N‐terminal cytoplasmic domains of RBOHD or F. Autoradiograph is shown on top. Coomassie‐stained gel below illustrates presence and equal loading of recombinant proteins. Experiments were done independently three times with similar results.

HEK293T cell‐based NOX activation assay. Cells were transfected with the indicated plasmid combinations. The phosphatase inhibitor Calyculin A was added directly before the start of the measurements. Each data point represents the mean of six wells analyzed in parallel; bars indicate SD. Experiments were repeated three times (another set of results is shown in Fig EV4B).

Figure EV4
Figure EV4. SGN1 directly activates NADPH oxidases in a cellular context

Localization patterns of SGN1‐mCherry of myrpalm‐SGN1 in HEK293T cells. Myrpalm‐SGN1 is efficiently recruited to the plasma membrane, while wild‐type SGN‐mCherry fusions remain in the cytoplasm. Scale bar = 5 μm.

Independent HEK cell ROS production assay. The phosphatase inhibitor CalyculinA was added directly before the start of the measurements. Each data point represents the mean of six wells analyzed in parallel. Bars indicate SD.

Figure 6
Figure 6. CIF2 induces large‐scale transcriptional changes for cell wall remodeling

CASP1‐GFP and lignin deposition in WT, sgn3, rbohD, rbohF, and rbohDF. CASP1‐GFP and lignin (fuchsin) are presented in green and magenta, respectively. Pictures were obtained from more than 10 roots from each background with similar results. Scale bar = 10 μm.

Time lapse imaging of single or co‐treatment of 10 nM CIF2 with 25 μM cycloheximide (CHX) on CASP1‐GFP in cif1 cif2. Seedlings were pretreated with or without CHX for 30 min and transferred onto each medium. Scale bar = 10 μm (see also Movie EV1).

Quantification of (B). Relative numbers of holes in CASP1‐GFP domain after single or co‐treatment with CIF2 or CHX from the pictures in (B). Bars are SD. * indicates statistical significance from all other conditions (P < 0.01) after one‐way ANOVA and Tukey test. Six roots in total for each condition were observed during two independent tests.

Fold change of 930 genes (P < 0.05 and log2(fold change) ≥ 1 or ≤ −1 at least one time point in one genotype) after CIF2 treatment at indicated time points in WT, cif1,2, and sgn3. Degree of the fold changes is shown in color code as indicated.

Relative expression levels of PER15 to CLATHRIN control in each genotype with or without 2‐h CIF2 treatment. Bars are SD (n = 3). Different characters indicate statistically significant differences (P < 0.01, ANOVA and Tukey test).

Fold changes of PER15 in each genotype with or without 2‐h CIF2 treatment. Bars are SD (n = 3). Different characters indicate statistical significance differences (P < 0.01, ANOVA and Tukey test).

Relative expression levels of PER49 to CLATHRIN control in each genotype with or without 2‐h CIF2 treatment. Bars are SD (n = 3). Different characters indicate statistically significant differences (P < 0.01, ANOVA and Tukey test).

Fold changes of PER49 in each genotype with or without 2‐h CIF2 treatment. Bars are SD (n = 3). Different characters indicate statistically significant differences (P < 0.01, ANOVA and Tukey test).

Relative fold changes of PER15 in pCASP1::SGN1‐Citrine and pCASP1::myrpalm‐SGN1‐Citrine compared to the expression level in WT. Bars are SD (n = 3). Different characters indicate statistically significant differences (P < 0.01, ANOVA and Tukey test).

Relative fold changes of PER49 in pCASP1::SGN1‐Citrine and pCASP1::myrpalm‐SGN1‐Citrine compared to the expression level in WT. Bars are SD (n = 3). Different characters indicate statistically significant differences (P < 0.01, ANOVA and Tukey test).

Figure EV5
Figure EV5. CIF2 induces large‐scale transcriptional changes to remodel cell walls

PCA of the most differentially expressed genes. The 1,000 most differentially expressed genes are clustered by condition at each time point for all replicates.

Immunoblot with phospho‐specific antibody against p42,44. Seedlings were treated with or without 1 μM CIF2 peptide for 15 min. Ponceau S‐stained membranes and IB with anti‐MPK6 antibody were shown as loading controls. This experiment was repeated three times with independent biological samples with the same result.

Heatmaps of MYB36 and CASPs expression fold changes with or without peptide treatment at the indicated time points. Asterisks indicate significant differentially regulated transcripts (P ≤ 0.05) at each condition.

Heatmaps of MYB41 and suberin biosynthesis‐related gene expression fold changes with or without the peptide treatment at the indicated time points. Asterisks indicate significant differentially regulated transcripts (P ≤ 0.05) at each condition.

Heatmaps of MYB15 and PEROXIDASES expression fold changes with or without peptide treatment at the indicated time points. Asterisks indicate significant differentially regulated transcripts (P ≤ 0.05) at each condition.

Heatmaps of MYB15 and LACCASES expression fold changes with or without the peptide treatment at the indicated time points. Asterisks indicate significant differentially regulated transcripts (P ≤ 0.05) at each condition.

Figure 7
Figure 7. Overview of the plasma membrane‐based and nuclear branches of the SGN3 pathway

(A) Schematic of the spatially restricted activation of ROS production at the plasma membrane after CIF stimulation (B) Comparison of the components of the cytoplasmic/nuclear signaling cascades induced by the flg22 bacterial pattern peptide (left) and the CIF peptides (right). Note that all of the signaling components in the two pathways belong to the same gene families, with the exception of the transcription factors. Components for which there is currently no direct experimental evidence are marked in gray, as are arrows indicating activation events that have not yet been experimentally established.

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