Plant roots use a patterning mechanism to position lateral root branches toward available water

Abstract

The architecture of the branched root system of plants is a major determinant of vigor. Water availability is known to impact root physiology and growth; however, the spatial scale at which this stimulus influences root architecture is poorly understood. Here we reveal that differences in the availability of water across the circumferential axis of the root create spatial cues that determine the position of lateral root branches. We show that roots of several plant species can distinguish between a wet surface and air environments and that this also impacts the patterning of root hairs, anthocyanins, and aerenchyma in a phenomenon we describe as hydropatterning. This environmental response is distinct from a touch response and requires available water to induce lateral roots along a contacted surface. X-ray microscale computed tomography and 3D reconstruction of soil-grown root systems demonstrate that such responses also occur under physiologically relevant conditions. Using early-stage lateral root markers, we show that hydropatterning acts before the initiation stage and likely determines the circumferential position at which lateral root founder cells are specified. Hydropatterning is independent of endogenous abscisic acid signaling, distinguishing it from a classic water-stress response. Higher water availability induces the biosynthesis and transport of the lateral root-inductive signal auxin through local regulation of tryptophan aminotransferase of Arabidopsis 1 and PIN-formed 3, both of which are necessary for normal hydropatterning. Our work suggests that water availability is sensed and interpreted at the suborgan level and locally patterns a wide variety of developmental processes in the root.

Keywords: adaptive root response; auxin-regulated root patterning; moisture regulation; root development; root system architecture.

Fig. 1.

Hydropatterning of root development in Arabidopsis, maize and rice. (A) Diagram showing asymmetries in the local environment generated when seedlings grow on the surface of an agar-based media or the symmetric environment generated when roots are grown through agar. (B and C) LR primordia emerging from the contact (B) or air side (C) of the primary root. (D) Quantification of LR emergence patterns from the primary root under different conditions (n > 10). Various phenotypic categories are indicated with different colors and are marked in A. (E) Cross-section of a rice primary root grown on agar, stained with calcofluor. Image shows the development of aerenchyma (AE) and root hairs (RH) on the air side and an LR emerging from the contact side. (F) Cross-section of a maize root grown on agar and stained with propidium iodide. (G) Diagram showing the construction of “agar sandwiches” used to test the effects of local differences in media composition on LR development in maize. (H) LR outgrowth is induced on two sides by contact with agar (Mock/Control); this effect is diminished on the “Treatment” side when the water potential of the media is reduced using PEG infusion (PEG/Control). Contact of the root with a glass surface does not induce LR outgrowth (Glass/Control). Growth of roots along a single agar surface results in the suppression of LR development on the air side (Air/Control) (n > 10). (I–K) MicroCT-generated images of maize seedlings grown through a macropore of air (I and K) or a continuous volume of soil (J). The root in K is growing in air, whereas in I the root is contacting the soil surface. Root tissue is false-colored in white, and soil is false-colored in brown. Average number of LRs per seedling (D) and per centimeter of primary root (H) is shown at base of columns in bar charts. Error bars indicate SEM. Significant differences based on Fisher’s exact test (P < 0.05) with similar groups are indicated using the same letter.

Fig. 2.

Hydropatterning acts during FC specification to affect LR patterning. (A) The PromiR390a:GUS-GFP reporter is expressed at stage 1 of LR initiation and later. Confocal imaging of contact and air sides of the primary root showed a strong bias in the number of GFP-positive foci (n ≥ 10). (B) The ProDR5:N7:VENUS reporter marks pericycle cell nuclei at the stage of FC activation (FCA), Stage 1 (S1), Stage 2 (S2), and later stages. Most stages showed greater numbers of primordia on the contact side than on the air side. (C) Seedling expressing the ProDR5:LUC+ reporter. LR emergence patterns were quantified in the region of the primary root containing all emerged LRs (in this example, the region above the yellow arrow). Luciferase activity was then visualized, and foci of reporter activity, included outgrown LRs, were counted (n = 27). Quiescent PBSs (red arrows) are sites of reporter expression that showed no signs of LR emergence. (D) Chart showing the number of emerged LRs and quiescent PBSs (QPBS). (E) Seedlings were grown for 5 d on 1% agar, then a second agar slab was applied to the former air side of the root. Seedlings were grown for five additional days, and the position of emerged LRs was quantified in the region of the primary root that formed before and after treatment. Control seedlings were grown similarly; however, a second agar slab was not applied. The proximal domain is defined as the region of the primary root in contact with the second applied agar slab, whereas the distal domain is toward the original agar slab on which seedlings were germinated. In B, significant differences were analyzed on a per-stage basis using Student’s t test (P < 0.05); statistically similar groups are indicated using the same letter. For E, asterisk indicates a significant difference based on Fisher’s exact test (P < 0.05). Average number of LRs per centimeter of primary root shown at base of columns in bar charts. Error bars indicate SEM. (Scale bar, 50 μm.)

Fig. 3.

Moisture activates auxin biosynthesis and response. (A) IAA levels quantified by liquid chromatography tandem mass spectrometry in whole roots grown on media containing different concentrations of agar (n = 3). (B) Maximum projects of confocal image stacks show DII-VENUS reporter expression is higher on the air side of the root relative to the contact side. Fluorescence intensity is shown using a 16-color look-up table. (C) Quantitation of DII-VENUS average nuclear fluorescence intensity in the epidermis shown for different regions of the root. (D) Cross-sections of rice roots expressing the ProDR5:GUS reporter showing local induction of the reporter on the contact side of the root (on agar) and uniform activation of the reporter when roots are grown in agar. (E and F) The ProTIR2:TIR2:GUS reporter shows stronger expression in the outer tissue layers of seedlings grown on 1% compared with 3% agar, quantified in F. (G) Two mutant alleles of tryptophan aminotransferase of Arabidopsis (TAA1) show a strong suppression of hydropatterning (n ≥ 20). (H) Maximum projections of confocal image stacks show ProTAA1:GFP:TAA1 reporter expression in the LRC and epidermis of the transition and elongation zones. (I) GFP fluorescence quantified for cell types on the air and contact sides (n ≥ 8). (J) The ProWER:TAA1 transgene was able to rescue the wei8-1 hydropatterning defect in multiple independent transgenic lines as was growth of seedlings on media supplemented with IAA (K). Average number of LRs per seedling shown at base of columns in bar charts. Error bars indicate SEM. Significant differences based on Fisher’s exact test (P < 0.05) (G, J, and K) or Student’s t test (P < 0.05) (A, C, F, and I) with similar groups indicated using same letter. (Scale bars, 50 μm.)

Fig. 4.

Auxin efflux transport pathways are necessary for hydropatterning. Effect of IAA (A) or 2,4-D (B) treatment on hydropatterning (n ≥ 20). (C) pin3-4 and pin2/3/7 mutants showed defects in hydropatterning (n ≥ 20). (D) Roots expressing the ProPIN3:PIN3:GFP and ProDR5:N7:VENUS reporters. Optical cross-sections at the cortex cell layer (Upper) or at the pericycle (Lower). Propidium iodide counter stain (magenta), PIN3:GFP (green, plasma membrane localized), and N7:VENUS (cyan, nuclear). (E) A radial cross-section reveals strong localization to the lateral cross-walls between cortex cells (yellow arrow). (F) Frequency with which early stages of LR development are observed on the air and contact sides of the primary root and whether these primordia are associated with PIN3:GFP expression in ground tissue (n = 9). (G) Transactivation of axr3-1 expression in the COR/END had the strongest effect in suppressing hydropatterning (n ≥ 20). Average number of LRs per seedling shown at base of columns in bar charts. Error bars indicate SEM. Significant differences based on Fisher’s exact test (P < 0.05) with similar groups indicated using same letter. (Scale bars, 50 μm.)