Proline-rich protein-like PRPL1 controls elongation of root hairs in Arabidopsis thaliana

Abstract

The synthesis and composition of cell walls is dynamically adapted in response to many developmental and environmental signals. In this respect, cell wall proteins involved in controlling cell elongation are critical for cell development. Transcriptome analysis identified a gene in Arabidopsis thaliana, which was named proline-rich protein-like, AtPRPL1, based on sequence similarities from a phylogenetic analysis. The most resemblance was found to AtPRP1 and AtPRP3 from Arabidopsis, which are known to be involved in root hair growth and development. In A. thaliana four proline-rich cell wall protein genes, playing a role in building up the cross-connections between cell wall components, can be distinguished. AtPRPL1 is a small gene that in promoter::GUS (β-glucuronidase) analysis has high expression in trichoblast cells and in the collet. Chemical or mutational interference with root hair formation inhibited this expression. Altered expression levels in knock-out or overexpression lines interfered with normal root hair growth and etiolated hypocotyl development, but Fourier transform-infrared (FT-IR) analysis did not identify consistent changes in cell wall composition of root hairs and hypocotyl. Co-localization analysis of the AtPRPL1-green fluorescent protein (GFP) fusion protein and different red fluorescent protein (RFP)-labelled markers confirmed the presence of AtPRPL1-GFP in small vesicles moving over the endoplasmic reticulum. Together, these data indicate that the AtPRPL1 protein is involved in the cell's elongation process. How exactly this is achieved remains unclear at present.

Keywords: Arabidopsis thaliana; cell expansion; hypocotyl; pollen Ole allergen; root hairs..

Fig. 1.

In silico analysis of At5g05500. (a) Schematic representation of At5g05500 gene structure, including the start and stop codon, one exon, and the T-DNA insertion position in the mutant. (b) Phylogenetic tree made by Neighbor–Joining analysis using Mega 5.05 software of the 15 most related proteins in Arabidopsis identified by BlastP. The bootstrap values are shown in the tree, and 1000 replicates were used. (c) Alignment of the DNA sequence of At5g05500 and the five most related genes. Prolines are shaded in grey.

Fig. 2.

Expression analysis of At5g05500 using promoter–reporter lines. Bright-field images showing (a) faint GUS activity in a young etiolated hypocotyl, (b) absent GUS activity in a young etiolated hypocotyl where fast cell expansion has started, (c) enrichment in the root of a whole seedling, (d) trichoblast-specific expression in a root and in its close-up, (e) expression in root hairs at the collet, (f) expression in pollen. (g) represents a confocal image of promoter-driven GFP in the trichoblast cell files only. Scale bars are 200 μm in (a, b, and e), 100 μm in (d), and 1mm in (c).

Fig. 3.

Effect of altered ethylene presence and mutated background on AtPRPL1 expression. (a) GUS activity is seen in the trichoblast cell files of a mock-treated root. (b) Treatment with the ethylene precursor ACC induces ectopic root hair formation and concomitant extra AtPRPL1 expression. (c) AVG-inhibited ethylene production interferes with root hair initiation/formation and reduces AtPRPL1 expression. (d, e) Both ethylene effects are mimicked in the eto2 and ein2-1 backgrounds. Scale bars are 100 μm.

Fig. 4.

Root, etiolated hypocotyl, and root hair phenotyping in the wild type, knock-out, and overexpression lines of AtPRPL1. (a) Root length, (b) etiolated hypocotyl length, and (c) root hair length of wild type, knock-out, and overexpression plants. Asterisks refer to significant changes compared with the corresponding wild types (the wild type and lines that are statistically compared with it are presented in the same greyscale). Data shown are the average of three representative biological replicates having at least 15 seedlings; error bars represent the SE. Student’s t-test, P<0.05.

Fig. 5.

Cell wall compositional changes between root hairs of the wild type, knock-out, and overexpression lines of AtPRPL1 revealed by FT-IR. (a) Comparison of the average spectra of Atprpl1-1 (dark blue trace) and its corresponding WT (pink trace) and of 35S::AtPRPL1-10 (green trace) and its corresponding WT (light blue trace). (b) Student’s t-test (P=0.05) of a comparison of FT-IR spectra sampled from root hairs. Comparison between WT and Atprpl1-1 (violet trace) and WT and AtPRPL1-1 overexpression (blue trace). For t-values >2 the absorbance for the corresponding wavenumber is significantly higher in the wild type. Horizontal red lines mark the P=0.01 significance level.

Fig. 6.

Protein–GFP localization. Transient co-localization analysis of AtPRPL1–GFP and RFP-tagged markers for the (a) plasma membrane, (b) Golgi apparatus, (c) peroxisomes, and (d) ER. (e) AtPRPL–GFP, (f) mCherry-fused ER marker ER-rb CD3-960, and (g) overlay of both in 35S::AtPRPL1-GFP lines stably transformed with the ER marker. Scale bar=10 μm.