PIRIN2 suppresses S-type lignin accumulation in a noncell-autonomous manner in Arabidopsis xylem elements

Abstract

PIRIN (PRN) genes encode cupin domain-containing proteins that function as transcriptional co-regulators in humans but that are poorly described in plants. A previous study in xylogenic cell cultures of Zinnia elegans suggested a role for a PRN protein in lignification. This study aimed to identify the function of Arabidopsis (Arabidopsis thaliana) PRN proteins in lignification of xylem tissues. Chemical composition of the secondary cell walls was analysed in Arabidopsis stems and/or hypocotyls by pyrolysis-gas chromatography/mass spectrometry, 2D-nuclear magnetic resonance and phenolic profiling. Secondary cell walls of individual xylem elements were chemotyped by Fourier transform infrared and Raman microspectroscopy. Arabidopsis PRN2 suppressed accumulation of S-type lignin in Arabidopsis stems and hypocotyls. PRN2 promoter activity and PRN2:GFP fusion protein were localised specifically in cells next to the vessel elements, suggesting a role for PRN2 in noncell-autonomous lignification of xylem vessels. Accordingly, PRN2 modulated lignin chemistry in the secondary cell walls of the neighbouring vessel elements. These results indicate that PRN2 suppresses S-type lignin accumulation in the neighbourhood of xylem vessels to bestow G-type enriched lignin composition on the secondary cell walls of the vessel elements. Gene expression analyses suggested that PRN2 function is mediated by regulation of the expression of the lignin-biosynthetic genes.

Keywords: Arabidopsis; PIRIN; lignification; noncell-autonomy; xylem vessel element.

Figure 1

The promoters of the Arabidopsis thaliana PIRIN genes have distinct activities in various tissue types throughout the development of the Arabidopsis plant. Histochemical staining of tissues from plants expressing β‐glucuronidase (GUS) fused to each of the PIRIN (PRN) promoters. Promoter activity of PRN1 (a–f), PRN2 (g–l), PRN3 (m–r), PRN4 (s–x) in the cotyledon (a, g, m, s), the root tip (b, h, n, t), the maturation zone of 5‐d‐old roots (c, i, o, u), the vascular bundle of inflorescence stems in 6‐wk‐old plants (d, j, p, v), and the hypocotyls of 8‐wk‐old plants (e, f, k, l, q, r, w, x). (f, l, r, x) are magnified images from (e, k, q, w), respectively. Bars, 100 μm. Arrowheads and arrows indicate examples of GUS activity in xylem parenchyma cells adjacent to xylem vessels and in xylem vessels, respectively.

Figure 2

PRN2 is localised to cells adjacent to vessel elements in Arabidopsis vasculature. Confocal laser scanning microscopy (CLSM) imaging of proPRN2::PRN2:GFP seedlings. (a) Maximum intensity projection of the main root of a 4‐d‐old seedling stained with propidium iodide (PI). (b) Optically reconstructed transverse section from (a). (c) Maximum intensity projection of CLSM imaging of the main root’s vasculature in a 4‐d‐old seedling similar to (a), with the leftmost vessel elements being the first ones with visible secondary cell walls (SCWs) in the two protoxylem files. White arrowheads indicate protoxylem vessels. Bars, 50 µm.

Figure 3

The effect of the different Arabidopsis PRN proteins on the secondary cell wall composition in hypocotyls. Relative content (percentage of detected cell wall components) of carbohydrates (cellulose and hemicelluloses) (a), total lignin (b), H‐type lignin (c), G‐type lignin (d) and S‐type lignin (e), and the S‐type : G‐type lignin ratio (f) in the cell walls of 8‐wk‐old wild‐type (WT) and single and double mutants for the PRN genes, analysed by pyrolysis–gas chromatography/mass spectrometry. All mutants are in Col‐0 background, except for prn4‐4 which is in Ler‐0 background. Error bars indicate ± SD. For each genotype, five biological replicates were analysed, each composed of a pool of three hypocotyls. The asterisks indicate statistically significant differences from the corresponding WT background, tested by Welch’s t‐test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 4

PRN2 affects lignin composition in the secondary xylem tissues of stems and hypocotyls. (a–c) Pyrolysis–gas chromatography/mass spectrometry analysis of hypocotyls (a, c) and stems (b) from 8‐wk‐old soil‐grown Arabidopsis plants. Relative content (percentage of detected cell wall components) of total lignin, G‐type lignin and S‐type lignin, and the S‐type : G‐type lignin ratio is shown for prn2 mutants, PRN2‐overexpressor lines PRN2OE6 and PRN2OE13, and prn2 complementation lines (prn2‐1 proPRN2::PRN2:myc). For each genotype, five biological replicates were analysed, each composed of a pool of three hypocotyls (a, c) or stems (b). Lines that do not share any letter are significantly different (P < 0.05) from each other according to post ANOVA Fisher’s test. Error bars indicate ± SD. The experiment was repeated at least three times with similar results. (d, e) The aromatic region of the 2D HSQC NMR spectra of wild‐type (WT) (d) and prn2‐1 (e). Two hundred bark‐peeled hypocotyls from each of WT and the prn2‐1 mutant were harvested and pooled to obtain adequate lignin extracts for the analysis. Peaks from the S‐type and G‐type lignin are indicated by ʻSʼ and ‘G’, respectively and the numbers following these letters correspond to the positions in the aromatic rings that give rise to these peaks.

Figure 5

PRN2 acts in a noncell‐autonomous manner on xylem lignification in Arabidopsis. (a) White‐light image of a transverse section showing example positions of the extracted representative FT‐IR spectra; circles indicate cell walls between two vessel elements, and diamonds indicate cell walls between two xylem fibres that are located next to the predicted PRN2‐expressing cells. The PRN2‐expressing cells were predicted on the basis of the results from the proPRN2::GUS plants (see Fig. 1k,l). The image was taken at ×15 magnification (used by the Cassegrain objective of the FT‐IR microscope). Bar, 25 µm. (b–e) FT‐IR microspectroscopic analysis of vessel elements (b, d) and fibres next to the predicted PRN2‐expressing fibres (c, e), in the secondary xylem of the hypocotyls of prn2‐2 mutant and wild‐type (WT). Spectra were collected from three 8‐wk‐old plants per genotype, with at least 10 spectra per plant. Models always use 1 + 2 (predictive + orthogonal) components. (b, c) OPLS‐DA scores plots showing the separation between prn2‐2 (white symbols) and WT (black symbols) in vessel–vessel walls (b) and cell walls between two fibres located next to the predicted PRN2‐expressing cells (c). Each symbol represents one measurement. Model details: vessel elements: N = 60, R2X(cum) = 0.56, R2Y(cum) = 0.39, Q2(cum) = 0.165; fibres next to the predicted PRN2‐expressing fibres: N = 64, R2X(cum) = 0.803, R2Y(cum) = 0.466, Q2(cum) = 0.310. Q2(cum) values stand for the predictive ability of the model, with higher values (closer to the maximum 1) generally meaning better separation for the same dataset. (d, e) The corresponding correlation‐scaled loadings plots for the predictive components, showing factors separating WT from prn2‐2 in vessel elements (d) and fibres next to the predicted PRN2‐expressing fibres (e). Bands on the negative side of the plots have higher relative intensity in the spectra of wild‐type plants, whereas bands on the positive side have higher relative intensity in the spectra of prn2‐2. Loadings higher than 0.4 (more than 40% correlation) can be considered indicative of changes that are specific to their respective genotypes. The bands corresponding to the aromatic –C=C– skeletal vibrations at 1510 and 1595 cm⁻¹, indicating prominent bands that can be correlated to lignin, and a band around 1460 cm⁻¹ are indicated by arrows. The experiments were repeated twice with similar results.

Figure 6

PRN2 suppresses the expression of lignin‐biosynthetic genes and some of the transcription factors regulating secondary cell wall biosynthesis in Arabidopsis. Expression of lignin‐biosynthetic genes (a, c) and transcription factors related to secondary cell wall biosynthesis (b, d) in the hypocotyls (a, b) and the stems (c, d) of prn2‐1, prn2‐2 and the PRN2‐overexpressor lines PRN2OE6 and PRN2OE13. The expression levels were normalised to the expression in the wild‐type (WT). For each genotype, five biological replicates were analysed, each composed of a pool of three hypocotyls (a, b) or stems (c, d). PDF2 and UBQ10 were used as reference genes. Error bars indicate ± SD. The asterisks indicate statistically significant difference from the WT by Welch’s t‐test (*, P < 0.05; **, P < 0.01).