Root growth is modulated by differential hormonal sensitivity in neighboring cells

Abstract

Coherent plant growth requires spatial integration of hormonal pathways and cell wall remodeling activities. However, the mechanisms governing sensitivity to hormones and how cell wall structure integrates with hormonal effects are poorly understood. We found that coordination between two types of epidermal root cells, hair and nonhair cells, establishes root sensitivity to the plant hormones brassinosteroids (BRs). While expression of the BR receptor BRASSINOSTEROID-INSENSITIVE1 (BRI1) in hair cells promotes cell elongation in all tissues, its high relative expression in nonhair cells is inhibitory. Elevated ethylene and deposition of crystalline cellulose underlie the inhibitory effect of BRI1. We propose that the relative spatial distribution of BRI1, and not its absolute level, fine-tunes growth.

Keywords: brassinosteroids; cell size determination; cell wall; hormone signaling; intercellular communication; root development.

Figure 1.

The impact of BRs on root cell elongation is determined by the relative expression of BRI1 in neighboring epidermal cells. (A) Cross-section of the Arabidopsis primary root showing radial organization of its constituent tissues. (N) Nonhair cells; (H) hair cells; (c) cortex; (st) stele. Asterisks mark the endodermis. pGL2 and pCOBL9 promoter fragments mark nonhair and hair cells, respectively. Bar, 10 µm. (B–D) Expression patterns of BRI1-GFP in the different transgenic lines, all in the bri1 mutant background. Note the GFP signal (green, with intensified contrast in the bottom panels) in nonhair cells in pGL2-BRI1 (B), hair cells in pCOBL9-BRI1 (C), and throughout the epidermis of a cross between pCOBL9-BRI1 and pGL2-BRI1 (D). Arrowheads mark hair cells. Cells were stained with propidium iodide (gray). Bar, 20 µm. (E) Confocal microscopy image of these same lines and wild type (Col-0), untreated or treated with BL, with the cortical cell highlighted in red. Bar, 50 µm. (F) pGL2-BRI1 root length is shorter when exposed to low BL concentrations. In contrast, the root length of lines with BRI1 expression and overexpression throughout the epidermal tissue (as in wild type [Col-0] and pGL2-BRI1;pCOBL9-BRI1, respectively) remained similar (mean ± SE; 17 < n < 30). (G,H) Average mature cortical cell length (G) and width (H) in roots of wild-type and transgenic lines untreated or treated with BL (mean ± SE; 26 < n < 95 [G]; 32 < n < 45 [H]). Note the opposing effect of BRI1 on cell elongation upon its high relative expression in hair (pCOBL9-BRI1) versus nonhair (pGL2-BRI1) cells. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001 with two tailed t-test.

Figure 2.

Ethylene mediates the BRI1-triggered inhibitory effect on root cell elongation in nonhair cells. (A,B) Analysis of relative expression of ACS genes using immunopurified polysomal RNA from hair and nonhair cells of wild-type plants in the absence and presence of BL (mean ± SE; n = 2). (C) Analysis of relative expression of ACS9 from whole root tips of various transgenic lines. Note the high relative ACS expression levels in roots of nonhair cell targeted BRI1 and BES1-D (pGL2-BES1-D), while only a minimal response is detected in pUBQ10-BRI1 and in lines expressing pGL2-BRI1 in a background with endogenous BRI1 [pGL2-BRI1(WT)] (mean ± SE; n > 2). (D) Roots expressing BRI1 (pGL2-BRI1) and BES1-D (pGL2-BES1-D) in nonhair cells are hypersensitive to the ethylene precursor ACC as compared with wild type (Col-0) (mean ± SE; 27 < n < 42). (E) Expression of a dominant active version of ACS5 in nonhair cells (pGL2-eto2; independent transgenic lines are shown) is sufficient to inhibit whole-root growth, similar to the endogenous eto2 mutant. pGL2-BRI1 and wild-type plants served as controls (mean ± SE; 23 < n < 28). (F) The ethylene signaling component EIN2 is necessary for BRI1-driven inhibition of cell elongation. Mature cortical cell length is marked. Bar, 50 µm. (G) Average mature cortical cell length of roots untreated or treated with BL (mean ± SE; 29 < n < 49). (***) P < 0.001 with two tailed t-test.

Figure 3.

High relative BRI1 expression in nonhair cells triggers local accumulation of crystalline cellulose. (A–C) Polarized light microscopy images of longitudinal (A) and transverse (B,C) sections of the root elongation zone showing microfibril angle and quantity, respectively. (A) The inset highlights the signal at the cell wall surface. Note the similar microfibril angle in pGL2-BRI1 and wild-type (Col-0) roots, as inferred by the color key. Bar, 50 µm. (B) pGL2-BRI1 and wild type have similar levels of cellulose crystallinity in meristematic cells. Bar, 50 μM. (C) High accumulation of crystalline cellulose in the elongation zone of nonhair cells of pGL2-BRI1 as compared with wild type (note the light blue–yellow signal in the cell wall). (D) Quantification of retardance in the outer cell wall of hair and of nonhair cells. Values are expressed as the ratio of retardance between the outer epidermal cell wall and the inner cortical cell wall. Note that the high deposition of crystalline cellulose in nonhair cells was unique to pGL2-BRI1 roots in the bri1 background and required the ethylene signaling component EIN2 (mean ± SE; 40 < n < 600). (*) P < 0.05; (**) P < 0.01; (***) P < 0.001 with two tailed t-test.

Figure 4.

Moderate inhibition of cellulose production facilitates unidirectional growth in pGL2-BRI1 lines. (A) The effect of increasing concentrations of isoxaben on the root length of wild-type (Col-0) and pGL2-BRI1 roots (mean ± SE; 32 < n < 36). Note the enhanced effect of low isoxaben concentrations on pGL2-BRI1 root growth. (B) Confocal microscopy image of wild-type (Col-0) and pGL2-BRI1 roots untreated and treated with 1 nM isoxaben in the presence of BL, with the cortical cell highlighted in red. Bar, 50 µm. (C,D) Quantification of root length and mature cortical cell length of roots untreated and treated with 1 nM isoxaben in the absence and presence of BL (mean ± SE; 22 < n < 32 and 16 < n < 88, respectively). (E) Quantification of retardance in the inner cortical and outer cell wall of hair and of nonhair cells. Absolute values are shown. Note the small reduction in crystalline cellulose levels in nonhair cells of pGL2-BRI1 as compared with wild type in response to a mild isoxaben treatment (mean ± SE; 22 < n < 60). (*) P < 0.05; (**) P < 0.01; (***) P < 0.001 with two tailed t-test.

Figure 5.

Schematic representation of BRI1-regulated root cell elongation. A model illustrating that the relative expression level of BRI1 in neighboring epidermal cells determines the intensity of its downstream signaling and subsequent whole-root growth via positive (A) and negative (B) effects (see the text).