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Abstract
In plants, root hairs undergo a highly polarized form of cell expansion called tip-growth, in which cell wall deposition is restricted to the root hair apex. In order to identify essential cellular components that might have been missed in earlier genetic screens, we identified conditional temperature-sensitive (ts) root hair mutants by ethyl methanesulfonate mutagenesis in Arabidopsis thaliana. Here, we describe one of these mutants, feronia-temperature sensitive (fer-ts). Mutant fer-ts seedlings were unaffected at normal temperatures (20°C), but failed to form root hairs at elevated temperatures (30°C). Map based-cloning and whole-genome sequencing revealed that fer-ts resulted from a G41S substitution in the extracellular domain of FERONIA (FER). A functional fluorescent fusion of FER containing the fer-ts mutation localized to plasma membranes, but was subject to enhanced protein turnover at elevated temperatures. While tip-growth was rapidly inhibited by addition of rapid alkalinization factor 1 (RALF1) peptides in both wild-type and fer-ts mutants at normal temperatures, root elongation of fer-ts seedlings was resistant to added RALF1 peptide at elevated temperatures. Additionally, at elevated temperatures fer-ts seedlings displayed altered reactive oxygen species (ROS) accumulation upon auxin treatment and phenocopied constitutive fer mutant responses to a variety of plant hormone treatments. Molecular modeling and sequence comparison with other Catharanthus roseus receptor-like kinase 1L (CrRLK1L) receptor family members revealed that the mutated glycine in fer-ts is highly conserved, but is not located within the recently characterized RALF23 and LORELI-LIKE-GLYCOPROTEIN 2 binding domains, perhaps suggesting that fer-ts phenotypes may not be directly due to loss of binding to RALF1 peptides.
Isolation of a temperature-sensitive root hair growth defect mutant. ( A ), Seven…
Figure 1 Isolation of a temperature-sensitive root hair growth defect mutant. (A), Seven day-old seedlings were grown vertically in 1/4 MS media under 20°C and transferred to 30°C for 6 h, followed by 24 h recovery at 20°C. Bright field images were collected with a Nikon Eclipse E600 wide-field microscope with a 10× Plan Apo DIC (0.75 NA) lens. Dashed lines indicate root tip positions when seedling plants were transferred to 30°C for 6 h, and again when they were transferred back to 20°C. Scale bars = 200 µm. B, Localization of EYFP-RabA4b protein in growing root hair cells of fer-ts mutants at 20°C and 30°C. Medial root hair sections were collected using spinning-disk confocal microscopy from growing root hair cells of 7-d-old seedlings stably expressing EYFP-RabA4b in the fer-ts mutant in 20°C (left) or 30°C (right) using a Zeiss 40× Plan-Apochromat (1.3 NA) lens and appropriate EYFP fluorescence filter sets. Scale bars = 10 µm. Insets, magnified images to show details of EYFP-RabA4b subcellular localization in root hair tips. Scale bars = 2 µm. C, Quantification of root hair length in WT and fer-ts mutants under 20°C [WT {n = 392}, fer-ts {n = 454}] and 30°C [WT {n = 185}, fer-ts {n = 23}] conditions. D, Calculation of root hair densities in WT and fer-ts mutants at 20°C [WT {n = 392}, fer-ts {n = 454}] and 30°C [WT {n = 185}, fer-ts {n = 23}] in fully expanded primary roots of 7-d old plants. In each case, root hair lengths and densities were measured from n = 20 individual seedlings. Error bars represent sd. **P < 0.001 by Student’s t test.
Figure 2
Root hair growth dynamics in…
Figure 2
Root hair growth dynamics in WT and fer-ts seedlings. ( A ) Root…
Figure 2 Root hair growth dynamics in WT and fer-ts seedlings. (A) Root hair tip-growth in WT and fer-ts mutant plants under normal (20°C) and elevated (30°C) temperatures by time-lapse microscopy. Bright field images of growing root hairs of WT and fer-ts mutant plants were collected every minute by time-lapse microscopy using a Zeiss 40× Plan-Apochromat (1.3 NA) lens. Representative images of WT and fer-ts mutant root hair elongation are presented at 10-min intervals. Scale bars = 10 µm. (B) Quantitative analysis of WT (n = 4) and fer-ts mutant (n = 4) root hair lengths upon transition to 30°C. WT (black squares) and fer-ts (black triangles) mutant root hair elongation were measured every 5 min and root hair lengths were determined using the measure function in Image J. The dashed line indicates the transition from 20°C to 30°C. Error bars represent sd. *P < 0.05, **P < 0.01 by Student’s t test.
Figure 3
Temperature-sensitive subcellular dynamics of EYFP-RabA4b…
Figure 3
Temperature-sensitive subcellular dynamics of EYFP-RabA4b labeled compartments in growing root hairs in fer-ts …
Figure 3 Temperature-sensitive subcellular dynamics of EYFP-RabA4b labeled compartments in growing root hairs in fer-ts mutants. (A) Localization of EYFP-RabA4b protein in growing root hair cells of WT and fer-ts mutant plants at normal (20°C) or elevated (30°C) temperature conditions. Growing WT and fer-ts root hairs were imaged at 1-min intervals for 30 min at 20°C at which the growth chamber temperature was raised to 30°C. For each time point, both bright field (Bright) and fluorescence (YFP) images were collected sequentially, and tip-localized EYFP-RabA4b compartments were monitored by spinning-disk fluorescence confocal microscopy using a Zeiss 40× Plan-Apochromat (1.3 NA) lens with appropriate EYFP fluorescence filter sets. The vertical line indicates when the temperature was raised to 30°C. Scale bars = 10 µm. Quantification of root hair elongation and EYFP-RabA4b root hair tip localization in WT (B) and fer-ts mutant (C) plants. Root hair length and EYFP-RabA4b fluorescence were quantified by Image J every 1 min. The gray box indicates when the temperature was raised to 30°C.
Figure 4
Map-based cloning of fer-ts .…
Figure 4
Map-based cloning of fer-ts . ( A ) A linear diagram of the…
Figure 4 Map-based cloning of fer-ts. (A) A linear diagram of the Arabidopsis third chromosome is shown, with a magnified F24M12 marker region displayed below. The centromere is indicated with a filled-rectangle. Low-resolution map-based cloning resulted in identification of the fer-ts locus within ∼2 Mb region of chromosome III bounded by markers NIT1.2 and CIW19. (B) SNPs specific to the ts-mutant within this region were identified using whole genomic resequencing, followed by targeted resequencing of genomic DNA of fer-ts and WT parental lines. A single G→A substitution was found in FERONIA (At3g51550). The arrowhead indicates the G121A substituted mutation in the FERONIA gene locus. (C) Schematic diagram of FERONIA protein domains and mutation regions, composed of an N-terminal extracellular domain (tandem repeat malectin-like domains; ML1 and ML2), TM (transmembrane) domain in the middle region and a C-terminal kinase domain (serine/threonine kinase), the end of the N-terminus has a signal peptide (SP) sequence for plasma-membrane trafficking. The fer-4 and fer-5 mutants displayed that T-DNA was inserted in malectin-like domain 1 and kinase domain, respectively. (D) RT-PCR analysis of the T-DNA inserted mutants and EMS mutants. The FER(ExDo) and FER(K) domains were amplified using P1 and P2 primers and P3 and P4 primers, respectively, Actin was used as a loading control.
Figure 5
Localization of the highly conserved…
Figure 5
Localization of the highly conserved glycine mutation in fer-ts in Arabidopsis FERONIA:RALF23:LLG2 and …
Figure 5 Localization of the highly conserved glycine mutation in fer-ts in Arabidopsis FERONIA:RALF23:LLG2 and X. laevis malectin structures. (A) Several residues important for binding carbohydrate ligands are conserved in plant CrRLK1L receptor kinase family members. Sequences analyzed include animal Malectin (X. laevis and Homo sapiens) FERONIA and other well-characterized CrRLK1L homologs in Arabidopsis (ANXUR1; ANX1, ANXUR1; ANX2, HERCULES1; HERK1 and THESEUS1; THE1). Putative N-terminal SPs are indicated as black dashed lines and malectin and CrRLK1L ML1 domains by solid lines, respectively. The highly conserved G41 of FER is marked by an arrowhead. Black boxes indicate fully conserved residues; shaded boxes indicate similar and partially conserved residues. Conserved residues that have been shown to participate in binding nigerose in the X. laevis malectin structure are marked by asterisks. Sequence alignment analysis was performed by CLUSTAL Omega program (http://www.ebi.ac.uk/Tools/msa/clustalo/) and displayed by using BOXSHADE software (www.ch.embnet.org/software/BOX_form.html). (B) Crystal structure of the X. laevis malectin protein (PDB ID: 2K46) with binding pocket aromatic residues and the highly conserved glycine residue based on sequence similarity to FER shown in red. C, Crystal structure of FER protein (green) in complex with RALF23 ligand (magenta) and glycosylphosphatidylinositol-anchored protein LLG2 (blue) (PDB ID: 6A5E). No analogous binding pocket is observed on the ML1 domain, as all conserved aromatic residues (red) are buried within the protein. Both (B and C) were generated using PyMol (DeLano Scientific).
Figure 6
The fer-ts mutant confers ts-root…
Figure 6
The fer-ts mutant confers ts-root hair growth defects when crossed with fer-4 and …
Figure 6 The fer-ts mutant confers ts-root hair growth defects when crossed with fer-4 and fer-5 mutants. (A) WT, fer-4, fer-5, and F1 progeny from crosses (paternal = fer-ts, maternal = fer-4 or fer-5) of fer-ts/fer-4 and fer-ts/fer-5 were grown vertically for 7 d at 20°C, transferred to 30°C for 6 h, and then grown for an additional 24 h at 20°C. Both fer-ts/fer-4 and fer-ts/fer-5 progeny clearly demonstrated a ts-dependent root hair phenotype. Scale bars = 200 µm. (B) Schematic diagram of the FERONIA gene structure. Open and filled boxes indicate untranslated regions and exon regions, respectively. The locations of T-DNA insertion mutants (fer-4 and fer-5) and fer-ts are indicated by triangles and arrows, respectively. (C) Genotyping of crossed F1 plants. Genomic DNA was extracted from F1 generation plants and subjected to PCR to confirm the presence of the fer-4 and fer-5 genotypes. (D) Both fer-4 and fer-5 display temperature-dependent root hair phenotypes when transformed with a fluorescently tagged FER construct containing the fer-ts mutation (pFER-FER(G41S)-EYFP). Seven-day old seedlings stably transformed and homozygous for (pFER-EYFP(G41S)-EYFP) were grown vertically at 20°C, transferred to 30°C for 6 h, and then grown for an additional 24 h at 20°C. The presence of the transgenic pFER-FER(G41S)-EYFP construct clearly demonstrated a ts-dependent root hair phenotype. Scale bars = 200 µm. (E) Temperature-sensitive root hair growth defects of fer-ts are fully rescued when transformed with a fluorescently tagged FER construct (pFER-FER(WT)-EYFP). Seven-day old seedlings stably transformed and homozygous for (pFER-EYFP(WT)-EYFP) were grown vertically at 20°C, transferred to 30°C for 6 h, and then grown for an additional 24 h at 20°C. Bright field images were collected with a Nikon Eclipse E600 wide-field microscope with a 10× Plan Apo DIC (0.75 NA) lens. Scale bars = 200 µm. (F) Subcellular localization of FER(WT)-EYFP protein in roots and root hairs in a rescued fer-ts mutant plant. Fluorescent confocal images displaying the subcellular distribution of FER(WT)-EYFP protein was detected from growing root, and mature root hair cells of 7-d-old fer-ts seedlings stably transformed and homozygous for pFER-FER(WT)-EYFP. Cells were counter-stained by incubating for 5 min in FM4-64 to visualize cell membranes. Images were collected by spinning-disk fluorescence confocal microscopy using a Zeiss 40× Plan-Apochromat (1.3 NA) lens with appropriate EYFP and FM4-64 fluorescence filter sets. Scale bars = 10 µm.
Figure 7
Subcellular localization of FER(WT)-EYFP and…
Figure 7
Subcellular localization of FER(WT)-EYFP and FER(G41S)-EYFP fluorescent fusion proteins in stably transformed Arabidopsis.…
Figure 7 Subcellular localization of FER(WT)-EYFP and FER(G41S)-EYFP fluorescent fusion proteins in stably transformed Arabidopsis. (A) Subcellular localization of FER(WT)-EYFP protein in various tissues. Fluorescent confocal images displaying the subcellular distribution of FER(WT)-EYFP protein were detected from growing root, leaf and root hair cells of 7-d-old seedlings in pFER-FER(WT)-EYFP/WT transgenic plants. Cell walls were counter-stained by incubating for 5 min in a PI solution (10 µg/ml). Images were collected by spinning-disk fluorescence confocal microscopy using a Zeiss 40× Plan-Apochromat (1.3 NA) lens with appropriate EYFP and PI fluorescence filter sets. Scale bars = 20 µm. (B) Magnified images of FER(WT)-EYFP fluorescence. FER(WT)-EYFP in WT of growing root cells of 7-d-old Arabidopsis seedlings was detected by spinning-disc confocal microscopy using a Zeiss 100× Plan-Apochromat (1.46 NA) oil immersion objective with appropriate EYFP and PI filter sets. Scale bars = 10 µm. (C and D) Subcellular localization of FER(WT)-EYFP (C) and FER(G41S)-EYFP (D) fluorescent fusions in root and root hair cells at normal (20°C) and elevated (30°C) temperatures. Cells from fer-4 plants stably transformed and homozygous for FER(WT)-EYFP or FER(G41S)-EYFP were counterstained with FM4-64 (5 µM) for 5 min to visualize cell membranes. Images were collected by spinning-disk fluorescence confocal microscopy using a Zeiss 40× Plan-Apochromat (1.3 NA) lens with appropriate EYFP and FM4-64 fluorescence filter sets. Scale bars = 20 µm; root, 10 µm; root hair.
Figure 8
Subcellular localization dynamics of FER(WT)-EYFP…
Figure 8
Subcellular localization dynamics of FER(WT)-EYFP and FER(G41S)-EYFP proteins in growing root hair cells…
Figure 8 Subcellular localization dynamics of FER(WT)-EYFP and FER(G41S)-EYFP proteins in growing root hair cells under both normal (20°C) and elevated (30°C) temperatures. (A) Root hair tip-growth in stably transformed lines expressing FER(WT)-EYFP and FER(G41S)-EYFP fluorescent fusion proteins under (20°C) and (30°C) temperature by time-lapse microscopy. Confocal images were acquired using a Leica confocal laser-scanning microscope SP8 with a 63× oil lens (Numerical Aperture = 1.4) with 5 s intervals. Representative images are presented at 50 s intervals (10 frames). Scale bar: 10 µm. (B) Schematic diagram showing areas and lines used for fluorescence intensity quantification. Apical (red line) and peripheral (blue line) plasma membrane domains were measured, and the area between the apical plasma membrane and green line represents the vesicle-rich zone. (C) Quantitative analysis of root hair growth in stably transformed lines expressing FER(WT)-EYFP (n = 3) and FER(G41S)-EYFP (n = 3). Root hair elongation was measured every 50 s using the measurement function in ImageJ. (D) Quantitative analysis of YFP intensity of the vesicle-rich zone. (E) Quantitative analysis of YFP intensity of the apical/peripheral ratio. Values are normalized using 0 s as 100%. The dashed line indicates the time point of transition from permissive (20°C) to (30°C). Error bars in (C–E) represent sd. (F) Protein turnover rates of FER(WT)-EYFP and FER(G41S)-EYFP at elevated temperature (30°C). Five-day-old seedlings were grown at 20°C and then treated with 200 µM cycloheximide and transferred to 30°C. Total proteins were extracted at each time point and the relative levels were determined using immunoblotting with anti-GFP and anti-actin antibodies. FER(G41S)-EYFP levels rapidly decreased during the time course, while levels of FER(WT)-EYFP were not significantly reduced. Actin was used as a loading control.
Figure 9
RALF peptide treatment terminates root…
Figure 9
RALF peptide treatment terminates root hair growth in the FER(WT)-EYFP expressing line. ( …
Figure 9 RALF peptide treatment terminates root hair growth in the FER(WT)-EYFP expressing line. (A) Root hair tip-growth in stably transformed lines expressing the FER(WT)-EYFP fluorescent fusion protein under permissive (20°C) temperature by time-lapse microscopy. Bright field images of growing root hair cells (n = 3) were collected using a Nikon Eclipse E600 wide-field microscope with a ×20 Plan Apo (0.45 NA) lens with 5 s intervals. The dashed line represents when media containing RALF1 (1 µM) was perfused into the growth chamber. Representative images are presented at 200 s intervals. Scale bar: 50 µm. (B) Quantification of root hair growth. Root hair elongation was measured every 50 s using the measurement function in ImageJ. Error bars represent sd.
Figure 10
fer-ts mutants are partially insensitive…
Figure 10
fer-ts mutants are partially insensitive to RALF1 peptide-mediated root growth inhibition at elevated… Figure 10 fer-ts mutants are partially insensitive to RALF1 peptide-mediated root growth inhibition at elevated temperatures. WT, fer-ts, or fer-5 plants were germinated and grown for 3 d in 1/2 MS liquid media at 20°C, and then transferred to 1/2 MS liquid media containing 1 µM RALF1 peptide (RALF+) or a mock buffer control (RALF−) and grown an additional 3 d at 20°C (A) or 30°C (B). Images of representative seedlings were collected using an Olympus SZX12 stereoscopic microscope. Quantification of primary root lengths (n = 10 seedlings) in the presence or absence of RALF1 peptide treatment in normal, 20°C (C) and elevated, 30°C (D) conditions. Primary root lengths were determined using Image J. Error bars represent sd. *P < 0.05, **P < 0.01 by Student’s t test.
Figure 11
Detection of ROS in WT, …
Figure 11
Detection of ROS in WT, fer-ts and fer-5 primary roots and root hairs.…
Figure 11 Detection of ROS in WT, fer-ts and fer-5 primary roots and root hairs. (A) ROS accumulation in normal and elevated temperature conditions with or without auxin treatments. WT, fer-ts, fer-4, or fer-5 seedlings were grown vertically on 1/4 MS media plates for 7 d at normal (20°C) or elevated (30°C) temperatures in the presence or absence of 10 nM NAA. Plates were bathed with 5 mL of 50 µM in H2DCF-DA suspended in 1/4 MS liquid media for 5 min, followed by two gentle washes with 10 mL of 1/4 MS. Fluorescence images were collected with a Zeiss Axio Imager Z1 fluorescence microscope with 2.5× objective and green (GFP) filter set. The WT ROS image was acquired by auto-exposure and all other images were acquired using the WT exposure conditions. Scale bars = 500 µm. (B and C) The rectangle in (A) indicates a representative ROI where average ROS intensity was quantified for the samples at 20°C (B) and 30°C (C). Intensities of ROS were quantified by image J program. Error bars represent sd. ** P < 0.01 by Student’s t test. All figures (11) See this image and copyright information in PMC
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