Spatial differences in stoichiometry of EGR phosphatase and Microtubule-associated Stress Protein 1 control root meristem activity during drought stress

Abstract

During moderate severity drought and low water potential (ψw) stress, poorly understood signaling mechanisms restrict both meristem cell division and subsequent cell expansion. We found that the Arabidopsis thaliana Clade E Growth-Regulating 2 (EGR2) protein phosphatase and Microtubule-Associated Stress Protein 1 (MASP1) differed in their stoichiometry of protein accumulation across the root meristem and had opposing effects on root meristem activity at low ψw. Ectopic MASP1 or EGR expression increased or decreased, respectively, root meristem size and root elongation during low ψw stress. This, along with the ability of phosphomimic MASP1 to overcome the EGR-mediated suppression of root meristem size and the observation that ectopic EGR expression had no effect on unstressed plants, indicated that during low ψw EGR activation and attenuation of MASP1 phosphorylation in their overlapping zone of expression determines root meristem size and activity. Ectopic EGR expression also decreased root cell size at low ψw. Conversely, both the egr1-1 egr2-1 and egr1-1 egr2-1 masp1-1 mutants had similarly increased root cell size but only egr1-1egr2-1 had increased cell division. These observations demonstrated that EGRs affect meristem activity via MASP1 but affect cell expansion via other mechanisms. Interestingly, EGR2 was highly expressed in the root cortex, a cell type important for growth regulation and environmental response.

Figure 1

EGRs and MASP1 affect growth during low ψ_w stress. A, Representative seedlings of EGR and MASP1 mutants and overexpression lines. Five-day-old seedlings were transferred to either fresh control plates (−0.25 MPa) or PEG-infused agar plates for moderate severity low ψ_w (−0.7 MPa) treatment. Plants were photographed at the end of the experiment (6 days after transfer). Scale bars indicate 1 cm. B, Primary root elongation rates over 6 days after transfer of seedlings to control or stress (−0.7 MPa) treatments. Blue or red symbols show elongation rates for individual seedlings. Black bars and error bars indicate the mean and standard deviation for each genotype (n = 22–67). Lowercase letters indicate statistical differences (analysis of variance [ANOVA] with Tukey’s post hoc test, corrected P ≤ 0.05). Genotypes sharing the same letter did not significantly differ from one another. Data are combined from three independent experiments and three independent transgenic lines per construct. Both 35S:EGR1 and 35S:YFP-EGR1 as well as 35S:MASP1 and 35S:YFP-MASP1 lines were used. Data from additional experiments with each individual transgenic line are shown in Supplemental Figure S2 and data from egr single mutants is shown in Supplemental Figure S1). The lines expressing untagged or YFP-tagged proteins had consistent results and the combined data are labeled as 35S:MASP1 and 35S:EGR1 for clarity of presentation. Details of the statistical analysis are shown in Supplemental Data Set S3.

Figure 2

EGRs and MASP1 affect root cell division and cell expansion during low ψ_w stress. A, Representative images of CYCB1::GUS staining in primary root tips of the indicated mutant and ectopic expression genotypes 4 days after transfer of 5-day-old seedlings to either control or low ψ_w (−0.7 MPa). Scale bars indicate 100 µm. B, Quantification of CYCB1::GUS foci in the primary root meristem. Symbols indicate counts of GUS foci from individual roots, while black bars and error bars indicate the mean and standard deviation for each genotype. Within the control or stress treatments, genotypes sharing the same letter do not significantly differ from one another (ANOVA with Tukey’s post hoc test, corrected P ≤ 0.05). In the WT, the number of CYCB1::GUS foci was significantly different between control and stress treatment (t test, P ≤ 0.001). Data are combined from three or four independent experiments (n = 21–66) for each genotype and at least two independent transgenic lines for 35S:MASP1 and 35S:EGR1. Data plotted separately for each transgenic line are shown in Supplemental Figure S4. Cell length of egr single mutants is shown in Supplemental Figure S1. Details of the statistical analysis are shown in Supplemental Data Set S3. C, Cell length at 2 mm from the root tip measured 4 days after transfer of mutant or ectopic expression seedlings to control or low ψ_w (−0.7 MPa). Format of data and statistical analysis are the same as in (B) (n = 133–224). Both 35S:EGR1 and 35S:YFP-EGR1 as well as 35S:MASP1 and 35S:YFP-MASP1 lines were used. The lines expressing untagged or YFP-tagged proteins all had consistent results and the combined data are labeled as 35S:MASP1 and 35S:EGR1 for clarity of presentation. Data plotted separately for each transgenic line are shown in Supplemental Figure S4. Details of the statistical analysis are shown in Supplemental Data Set S3.

Figure 3

Ectopic EGR or MASP1 expression alters root meristem size during low ψ_w stress. A, Representative images of PI-stained root tips to show the cell size and organization of cell files in the proximal meristem region. Scale bars indicate 20 µm. The epidermal (Ep.), cortical (Cor.), and endodermal (En.) cell layers are labeled on the WT control image. B, Meristem cell number and meristem length 5 days after transfer of 5-day-old mutant and ectopic expression seedlings to unstressed control or to low ψ_w (−0.7 MPa). Data are combined from two to three independent experiments for each genotype (n = 19–31 in the control, n = 33–65 in the stress treatment). Symbols indicate data from individual roots, while black bars and error bars indicate the mean and standard deviation for each genotype. Within the stress treatments, genotypes sharing the same letter do not significantly differ from one another (ANOVA with Tukey’s post hoc test, corrected P ≤ 0.05). There were no significant differences among genotypes for meristem cell number in the unstressed control. In the WT, meristem size was significantly different between control and stress treatment (t test, P ≤ 0.001). In the stress treatment, the increase of meristem length in 35S:MASP1 was marginally nonsignificant compared to the WT (adjusted P = 0.08) and egr1-1 egr2-1 may have a slightly longer meristem than egr1-1 egr2-1 masp1-1 (adjusted P = 0.06). Representative images showing differences in root meristem length are shown in Supplemental Figure S6). For 35S:EGR1 and 35S:MASP1, three independent transgenic lines were used for each gene. Data for each individual transgenic line plotted separately are shown in Supplemental Figure S7. Details of the statistical analysis are shown in Supplemental Data Set S3.

Figure 4

EGR and MASP1 have differing spatial patterns of expression in the root tip. A, Representative images of EGR2_pro:GUS and MASP1_pro:GUS staining in primary root tips 4 days after transfer to control or low ψ_w (−0.7 MPa). Red scale bars indicate 100 µm. Orange bars indicate 1 mm. Images are representative of the expression patterns seen in three independent transgenic lines for each construct. B, Quantitation of EGR2_pro:EGR2-YFP and MASP1_pro:MASP1-YFP fluorescence intensity along the root tip. Roots were imaged with a fluorescence imager and ImageJ was used to perform line scans of root tips for 6–10 roots (from two independent transgenic lines for each construct assayed in two independent experiments) at 4 days after transfer of seedlings to control or low ψ_w (−0.7 MPa) treatments. Representative root tip images used for the scans are shown in Supplemental Figure S12C. Dashed line boxes indicate the end of the root meristem for control and stress treatments (based on data in Figure  3B). Gray error bars (gray shading) indicate the standard error. Note the log scale of the y-axis. C, Root tip expression pattern of EGR2_pro:EGR2-YFP and MASP1_pro:MASP1-YFP in unstressed plants (Control) and low ψ_w-treated plants (−0.7 MPa, 4 days). The gray dashed lines in the whole root tip images indicate the positions of the radial scans (50, 150, 350, and 450 µm from the QC). The whole root tip images were made by merging individual images using Leica LAS X software. Images are representative of the expression pattern seen in three independent transgenic lines for each construct. In the whole root tip images, scale bars indicate 100 µm. In the radial scan images, scale bars indicate 20 µm.

Figure 5

EGR attenuation of MASP1 S670 phosphorylation in their zone of overlapping expression controls root meristem size at low ψ_w. A, Root meristem cell number at low ψ_w for the WT and two independent transgenic lines expressing phosphonull MASP1 (35S:MASP1^S670A) or phosphomimic MASP1 (35S:MASP1^S670D). The MASP1 phosphonull and phosphomimic lines were previously described in Bhaskara et al. (2017). Data are combined from two to three independent experiments (n = 35 for the WT and 10–18 for each transgenic line). The transgenic lines used here are in the GFP-TUA6 background (Supplemental Data Set S1). It was previously shown that the presence of GFP-TUA6 did not affect the growth phenotypes of any EGR-MASP1 mutant or transgenic lines (Bhaskara et al., 2017). Symbols indicate data from individual roots, while black bars and error bars indicate the mean and standard deviation for each genotype. Different letters indicate significant differences between genotypes (ANOVA with Tukey’s post hoc test, corrected P ≤ 0.05). Details of the statistical analysis are shown in Supplemental Data Set S3. B, Representative root meristem images of each genotype in (A). Scale bars indicate 100 µm. C, Quantitation of meristem size in F₁ seedlings of 35S:EGR1 crossed to lines ectopically expressing nonmutated MASP1, phosphonull MASP1 (S670A) or phosphomimic MASP1 (S670D). All the MASP1 lines express untagged MASP1 in the GFP-TUA6 background. Root meristem size was measured 5 days after transfer to low ψ_w (−0.7 MPa). Bars indicate the mean, error bars indicate the standard deviation from two independent experiments for each genotype (n = 56 for the WT, n = 21–29 for F₁ genotypes). Asterisks directly above the bars indicate a significant difference compared to the WT, brackets show the results of comparison between the indicated genotypes (ANOVA with Tukey’s post hoc test, Asterisk indicates corrected *P ≤ 0.05; **P ≤ 0.005; ****P ≤ 0.0001). Note that the difference in meristem cell number between the WT and WT × 35S:EGR1 was marginally nonsignificant (adjusted P = 0.07). Details of the statistical analysis are shown in Supplemental Data Set S3. D, Model of how EGR-MASP1 signaling affects meristem function and root growth at low ψ_w. MASP1 is highly expressed in all cell types of the proximal root meristem where it promotes cell division. MASP1 decreases in expression farther from the QC, especially in the inner cell layers and cortex. Conversely, EGR2 is low in the proximal meristem but high in the distal meristem and elongation zone where it influences cell expansion. EGR2 is highly expressed in cortical cells, while MASP1 does not have cell-type-specific expression. During low ψ_w stress, EGR2 expression encroaches closer to the proximal meristem region. In their overlapping zone of expression, EGRs attenuate phosphorylation of MASP1, particularly in cortex cells, to suppress MASP1 activity and restrict meristem size. The high expression of EGR2 in cortex is consistent with recent data indicating that this cell layer is important for growth responses to low ψ_w. Low ψ_w activates (or de-represses) EGRs such that EGR-MASP1 signaling is a dominating factor controlling root elongation and meristem function during low ψ_w stress but has less or no effect on unstressed plants.