Reduced RG-II pectin dimerization disrupts differential growth by attenuating hormonal regulation

Abstract

Defects in cell wall integrity (CWI) profoundly affect plant growth, although, underlying mechanisms are not well understood. We show that in Arabidopsis mur1 mutant, CWI defects from compromising dimerization of RG-II pectin, a key component of cell wall, attenuate the expression of auxin response factors ARF7-ARF19. As a result, polar auxin transport components are misexpressed, disrupting auxin response asymmetry, leading to defective apical hook development. Accordingly, mur1 hook defects are suppressed by enhancing ARF7 expression. In addition, expression of brassinosteroid biosynthesis genes is down-regulated in mur1 mutant, and supplementing brassinosteroid or enhancing brassinosteroid signaling suppresses mur1 hook defects. Intriguingly, brassinosteroid enhances RG-II dimerization, showing hormonal feedback to the cell wall. Our results thus reveal a previously unrecognized link between cell wall defects from reduced RG-II dimerization and growth regulation mediated via modulation of auxin-brassinosteroid pathways in early seedling development.

Fig. 1.. RG-II dimerization plays a key role in apical hook development.

(A) Kinematics analyses of apical hook development in dark-grown wild-type (WT), mur1-1, and mur1-2 seedlings. For each genotype and condition, n ≥ 15. Error bars represent the SE of the mean. (B) Kinematics analyses of apical hook development in dark-grown WT and mur1-2 seedlings without or after supplementation with 250 μM boric acid (boron). Error bars represent the SE of the mean (n ≥ 15). (C) Cell elongation rates of the outer and inner epidermis of dark-grown WT and mur1-2 mutant plants without or with supplementation with 250 μM boric acid (boron). The lengths of epidermal cells in the region of 0 to 400 μM from the shoot apical meristem were measured every 2 hours and the plot shows the average cell elongation rate over 8 hours. For each genotype, n = 6. Error bars represent the SE of the mean. Significant differences according to Tukey’s post hoc test and Duncan’s test [P < 0.05, one-way analysis of variance (ANOVA)] are indicated by different lowercase letters above the columns.

Fig. 2.. Reduced RG-II dimerization perturbs auxin response maxima.

(A to D) Auxin response maxima on the inner and outer sides of the apical hook were visualized using the synthetic auxin response reporter DR5-Venus in the WT and the mur1-2 mutant without or with supplementation with 250 μM boric acid (boron). Scale bars, 20 μm. (E) The fluorescence intensity of the Venus protein in the apical hook with and without boron in the WT and mur1-2 was then analyzed. The plotted values are means ± SE (n ≥ 60 to 70 cells from 10 seedlings for each genotype). Significant differences according to Tukey’s post hoc test and Duncan’s test (P < 0.05) are indicated by different lowercase letters.

Fig. 3.. Plasma membrane levels of auxin transport carriers are altered in mur1-2 mutant.

Representative confocal images of the plasma membrane of (A) PIN3-GFP, (B) PIN4-GFP, and (C) AUX1-YFP in the apical hook of WT and mur1-2 mutant in the presence and absence of boric acid (boron). Scale bars, 20 μm. Quantification of plasma membrane fluorescence intensity of (D) PIN3-GFP, (E) PIN4-GFP, and (F) AUX1-YFP in WT and mur1-2 mutant in the presence and absence of 250 μM boric acid. Seedlings were imaged 48 hours after germination. For each genotype and treatment, five cells from each of the 10 seedlings were analyzed. Significant differences according to Tukey’s post hoc test and Duncan’s test (P < 0.05) are indicated by different lowercase letters. (G to I) Transcript levels of (G) PIN3, (H) PIN4, and (I) AUX1 in WT, mur1-2 mutant in the presence and absence of 250 μM boric acid (boron). Graphs represent averages of three biological replicates. Ubiquitin was used as an internal control. Plotted values are averages for three independent biological replicates. Significant differences according to Tukey’s post hoc test and Duncan’s test (P < 0.05) are indicated by different lowercase letters.

Fig. 4.. The mur1 hook defect is mediated by the ARF7 and ARF19 pathway.

(A and B) qRT-PCR analysis of ARF7 (A) and ARF19 (B) expression in the WT and the mur1 mutant in the presence and absence of 250 μM boric acid (boron). Expression of ARF7 and ARF19 relative to ubiquitin is plotted on the y axis. Plotted values are averages for three independent biological replicates. Significant differences according to Tukey’s post hoc test and Duncan’s test (P < 0.05) are indicated by different lowercase letters.

Fig. 5.. Hook defects resulting from reduced RG-II dimerization are associated with attenuation of the BRs pathway.

(A) The transcript level of the BR biosynthetic genes DWF4 and (B) ROT3 expression in the WT and mur1-2 mutant in the presence or absence of exogenous boron (250 μM boric acid). The values on the y axis indicate expression relative to ubiquitin and are averages of three independent biological replicates. Significant differences according to Tukey’s post hoc test and Duncan’s test (P < 0.05) are indicated by different lowercase letters. (C) Kinematics analyses of apical hook development in dark-grown WT and mur1-2 seedlings with and without 24-epibrassinolide (EBR) (100 nM). (D) Kinematics analyses of apical hook development in dark-grown WT, mur1-2, and bes1-D/mur1-2 seedlings (the gain-of-function mutant bes1-D suppress mur1 hook defect). For each genotype and condition, n ≥ 15. Error bars represent the SE of the mean.