Shoring up the base: the development and regulation of cortical sclerenchyma in grass nodal roots

Abstract

Plants depend on the combined action of a shoot-root-soil system to maintain their anchorage to the soil. Mechanical failure of any component of this system results in lodging, a permanent and irreversible inability to maintain vertical orientation. Models of anchorage in grass crops identify the compressive strength of roots near the soil surface as key determinant of resistance to lodging. Indeed, studies of disparate grasses report a ring of thickened, sclerenchyma cells surrounding the root cortex, present only at the base of nodal roots. Here, in the investigation of the development and regulation of this agronomically important trait, we show that development of these cells is uncoupled from the maturation of other secondary cell wall-fortified cells, and that cortical sclerenchyma wall thickening is stimulated by mechanical forces transduced from the shoot to the root. We also show that exogenous application of gibberellic acid stimulates thickening of lignified cell types in the root, including cortical sclerenchyma, but is not sufficient to establish sclerenchyma identity in cortex cells. Leveraging the ability to manipulate cortex development via mechanical stimulus, we show that cortical sclerenchyma development alters root mechanical properties and improves resistance to lodging. We describe transcriptome changes associated with cortical sclerenchyma development under both ambient and mechanically stimulated conditions and identify SECONDARY WALL NAC7 as a putative regulator of mechanically responsive cortex cell wall development at the root base.

Figure 1.. Histological characterization of Brachypodium distachyon nodal root development.

Representative plant (A). Scale bar 5 cm. Phloroglucinol-HCI stained sections from the same root at 10x (B) and 20x (C) magnification. Scale bars 100 μm. Brackets and arrows indicate the root region corresponding to each section. Quantification of cell wall thickness (D) and phloroglucinol-HCI stain intensity (8-bit grayscale saturation value) (E). N = 4 plants. Plant means calculated from the mean of 15 measurements of cortex and exodermis, and casparian strip and 9 measurements for pith and metaxylem.

Figure 2.. Identification of basal cortical sclerenchyma in crop species.

Overlay of fluorescence (red) and brightfield (grayscale) micrographs of the proximal (A) and distal (B) basal regions of a Basic Fuchsin stained maize nodal root. Scale bars 500 μm. Characterization of secondary cell wall traits in maize (C-F) and wheat (G-J). Fluorescence micrograph of peripheral cortex cell in proximal (C) and distal (D) regions. Images shown in grayscale and brightened by the same factor for visualization. Scale bars 50 μm. Quantification of local cell wall thickness (E,l) and fluorescence signal (F,J). Student’s t-test, **p ≤ 0.01, ***p ≤ 0.001

Figure 3.. Time course of nodal root development.

Phloroglucinol-HCI stained root sections, harvested at 22 (A), 31 (B) and 37 (C) days after sowing. Scale bars 50 μm. Quantification of nodal root secondary wall development. Cell wall thickness (D) and phloroglucinol-HCI stain intensity (8-bit grayscale saturation value)(E). Each point represents the cell type mean of one plant. Three roots of each plant were sectioned and 15 cells per root were measured for cortex and exodermis, 9 per root were measured cells for pith and metaxylem.

Figure 4.. Mechanical stimulus induced wall thickening.

Overlay of brightfield (greyscale) and Basic Fuchsin fluorescence (red) images of the proximal basal region of control (A) and mechanically stimulated (B) roots. Scale bar 100 μm. Quantification of cell wall characteristics. Wall thickness of proximal basal cortex (C) and pith (E) cells. Fluorescence intensity of proximal basal cortex (D) pith (F) cells. Tukey HSD pairwise comparisons, *p ≤ 0.05, **p ≤ 0.01, ns - not significant.

Figure 5.. Testing resistance to lodging. Schematic of lodging resistance assay.

(A). Peak force measured during 30° bending test (B) Tukey HSD pairwise comparisons, *p ≤ 0.05, ns - not significant. N = 12–13 plants.

Figure 6.. Testing root mechanical properties.

Raw force-displacement curves (A). Root diameter inferred from testing data (B). Force measured at different percentages of root penetration (C). Each point represents mean force value for one plant, three roots per plant were assayed. N = 6 plants per treatment. Groups that do not share a letter are significantly different using Tukey’s HSD test, p ≤ 0.05.

Figure 7.. Phytohormone regulation of root secondary cell wall development.

Transverse section of the nodal root base of plants grown under control (A), 10 μM GA3 (B), 0.1 μM paclobutrazol (C) treatment. Scale bars indicate 50 μm. Quantification cell wall thickness (D,F,H) and phloroglucinol stain intensity (E,G,I). Groups that do not share a letter are significantly different using Tukey’s HSD test, p ≤ 0.05.

Figure 8.. DNA affinity purification sequencing to determine SECONDARY WALL NAC7 binding sites.

Most statistically enriched sequence motif in protein binding sites (A). Distribution of binding sites across genome annotation features, relative to primary transcripts of the Brachypodium distachyon annotation v 3.2 (B). Relative distribution of binding sites centered on the transcriptional start site (TSS, blue dashed line), transcriptional termination site (TTS, red dashed line) represents the average length of all annotated transcripts, approximately 4.5 kb away from the TSS (C).

Figure 9.. Secondary cell wall genes and the transcriptional regulators with expression associated with wall thickening in nodal roots were often DAP-seq binding targets of SECONDARY WALL NAC7 protein.

Box plots are the average transcript abundance of 4–5 biological replicates for each tissue/treatment. Binding site determined as peaks of sequence alignment and denoted by green ovals. Scale bar unit is bases. Direction of transcription is shown with arrows on the gene model, 5’ and 3’ UTRs are depicted by narrowed rectangles on the gene model. * adj-p ≤ 0.05, ** adj-p ≤ 0.01, *** adj-p ≤ 0.001, **** adj-p ≤ 0.0001.

Figure 10.

Model of the effect of mechanical stimulation on transcriptional regulation of nodal root basal cortex secondary wall thickening. NAC, GRAS, and MYB family DNA binding transcription factors are orange, pink, and blue ovals, respectively. Cellulose associated CELLULOSE SYNTHASE As and COBRA-like (COBL), green rectangles, hemicellulose associated GT43D3, violet rectangle, and lignin, yellow rectangles, genes are binding targets of the transcription factor proteins. Arrows indicate activation and bars repression. Black lines are supported by RNA- and DAP-seq results in this study. Connections depicted by gray lines are previously described in the literature for various species (reviewed by Rao and Dixon, 2018; Zhang et al., 2018). Mechanical forces acting on the stem induce bending at the base of the root and cells at the root periphery begin to thicken. Under ambient conditions, thickening of outer cell files stiffen the root against bending, protecting interior cortex cells. Under strong mechanical stimulus inner cell files develop thick walls.