Poplar woody taproot under bending stress: the asymmetric response of the convex and concave sides

Abstract

Background and Aims Progress has been made in understanding the physiological and molecular basis of root response to mechanical stress, especially in the model plant Arabidopsis thaliana, in which bending causes the initiation of lateral root primordia toward the convex side of the bent root. In the case of woody roots, it has been reported that mechanical stress induces an asymmetric distribution of lateral roots and reaction wood formation, but the mechanisms underlying these responses are largely unknown. In the present work, the hypothesis was tested that bending could determine an asymmetric response in the two sides of the main root axis as cells are stretched on the convex side and compressed on the concave side. Methods Woody taproots of 20 seedlings were bent to an angle of 90° using a steel net. Changes in the anatomy, lignin and phytohormone content and proteome expression in the two sides of the bent root were analysed; anatomical changes, including dissimilarities and similarities to those found in poplar bent woody stem, were also considered. Key Results Compression forces at the concave side of poplar root induced the formation of reaction wood which presented a high lignin content and was associated with the induction of cambium cell activity. Auxin seemed to be the main hormone triggering lignin deposition and cell wall strengthening in the concave sides. Abscisic acid appeared to function in the water stress response induced by xylem structures and/or osmotic alterations in the compression sides, whereas gibberellins may control cell elongation and gravitropisms. Conclusions Poplar root reaction wood showed characteristics different from those produced in bent stem. Besides providing biomechanical functions, a bent root ensures water uptake and transport in the deforming condition induced by tension and compression forces by two different strategies: an increase in xylem thickness in the compressed side, and lateral root formation in the tension side.

Keywords: Mechanical stress; phytohormones; proteomics; reaction wood; root anatomy.

Fig. 1.

Photographs of the control and of the convex and concave sides of a bent taproot sector. Root sections were stained in Toluidine Blue O. Cross-section of the cambial zone in control (A), bending sector (BS) convex (B) and BS concave (C) roots. Scale bar = 20 μm. Entire cross-section of control (D) and a bent taproot sector (E); in the case of the bent taproot, the BS was analysed. Scale bar = 2 mm.

Fig. 2.

Lignin content in control and convex and concave sides of three bent sectors. Lignin content is expressed as a percentage of the value measured in BBS concave, considered as 100 %. Data represent the mean of three independent extractions ± s.d. Values marked with the same letter are not statistically significant (t-test, P < 0·01). ABS, above bending sector; BS, bending sector; BBS, below bending sector.

Fig. 3.

Content of phytohormones in control and convex and concave sides of three bent sectors. Concentrations represent the amount of each phytohormone in root tissues (μg g⁻¹ of fresh weight) as analysed by HPLC. Data represent the mean of four independent extractions ± s.d. Values marked with the same letter are not statistically significant (t-test, P < 0·05). IAA, indole-3-acetic acid; ABA, abscisic acid; GAs, gibberellins (GA₃ + GA₄); Kin, kinetin. ABS, above bending sector; BS, bending sector; BBS, below bending sector.

Fig. 4.

Relative ACO gene expression levels measured in control and in convex and concave sides of three bent sectors. Data were normalized to cyclophilin gene expression values and represent the mean of three independent extractions ± s.d. Values marked with the same letter are not statistically significant (t-test, P < 0·05). ABS, above bending sector; BS, bending sector; BBS, below bending sector.

Fig. 5.

Master gel. Map showing the 66 proteins differentially represented in unstressed, and in convex and concave sides of three bent sectors (ABS, BS and BBS). Arrows indicate the position of each protein spot and corresponding spot ID reported in Table 2.

Fig. 6.

Heat map. The map reports the expression level of proteins differentially represented in control, and in convex and concave sides of three bent sectors. Proteins are grouped on the basis of assigned functional classification (Table 2). Light green and red block colours indicate, respectively, over- (2-fold) or under- (0·5-fold) representation of each protein in both the convex and concave sides of the three bent sectors, compared with the control. Dark green and dark red block colours indicate, respectively, proteins over- (2-fold) or under- (0·5-fold) represented in a specific side, compared with the opposite side of the same sector. ABS, above bending sector; BS, bending sector; BBS, below bending sector.

Fig. 7.

Two-ways diagram. Proteins are grouped according to their over- or under-representation in the convex and concave sides of the three bending sectors (ABS, BS and BBS). Overlapping regions include proteins over- or under-represented in both sides of a bent region, compared with control; underlined proteins were found to be over- or under-represented in either the convex or the concave side of the same sector.

Fig. 8.

Model summarizing the main anatomical, phytohormonal and proteomical changes observed in the convex and concave sides of three bent taproot sectors (ABS, BS and BBS). Phytohormone (IAA, ABA, GAs, Kin and ethylene) changes are represented by diverse coloured blocks. Proteomic and anatomical changes are indicated by arrows. Zones with the highest lateral root number and lignin content are also reported. ABS, above bending sector; AlaT1, alanine aminotransferase 1 (spot 20); Ara4, ara4-interacting protein (spot 1); BS, bending sector; BBS, below bending sector; CBS1, cystathionine-β-synthase 1 (spot 60); CCN, cambial cell number; CDC48, cell division cycle protein 48 (spot 2); ERP, ethylene-responsive protein; MMSDH, methylmalonate semialdehyde dehydrogenase (spot 17); NDPK, nucleoside diphosphate kinase (spot 64); RPT, relative phloem thickness; RXT, relative xylem thickness; SHMT, serine hydroxymethyltransferase (spot 19); SVA, specific vessel area; SVN; specific vessel number.