COI1-dependent jasmonate signalling affects growth, metabolite production and cell wall protein composition in arabidopsis

Abstract

Background and aims: Cultured cell suspensions have been the preferred model to study the apoplast as well as to monitor metabolic and cell cycle-related changes. Previous work showed that methyl jasmonate (MeJA) inhibits leaf growth in a CORONATINE INSENSITIVE 1 (COI1)-dependent manner, with COI1 being the jasmonate (JA) receptor. Here, the effect of COI1 overexpression on the growth of stably transformed arabidopsis cell cultures is described.

Methods: Time-course experiments were carried out to analyse gene expression, and protein and metabolite levels.

Key results: Both MeJA treatment and the overexpression of COI1 modify growth, by altering cell proliferation and expansion. DNA content as well as transcript patterns of cell cycle and cell wall remodelling markers were altered. COI1 overexpression also increases the protein levels of OLIGOGALACTURONIDE OXIDASE 1, BETA-GLUCOSIDASE/ENDOGLUCANASES and POLYGALACTURONASE INHIBITING PROTEIN2, reinforcing the role of COI1 in mediating defence responses and highlighting a link between cell wall loosening and growth regulation. Moreover, changes in the levels of the primary metabolites alanine, serine and succinic acid of MeJA-treated Arabidopsis cell cultures were observed. In addition, COI1 overexpression positively affects the availability of metabolites such as β-alanine, threonic acid, putrescine, glucose and myo-inositol, thereby providing a connection between JA-inhibited growth and stress responses.

Conclusions: This study contributes to the understanding of the regulation of growth and the production of metabolic resources by JAs and COI1. This will have important implications in dissecting the complex relationships between hormonal and cell wall signalling in plants. The work also provides tools to uncover novel mechanisms co-ordinating cell division and post-mitotic cell expansion in the absence of organ developmental control.

Fig. 1.

Detection of COI1::HA in Ler and COI1-overexpressing cell suspension cultures. Total protein was extracted from the wild type (Ler) and the COI1-overexpressing cell suspensions (COV1 and COV2) at 4 DASU, and run on a 12 % SDS–polyacrylamide gel. COI1 and COI::HA mass was approx. 67–68 kDa. A 10 μg aliquot of protein extract were loaded for Ler and COV1, and 2 μg for COV2.

Fig. 2.

Cell number (A) and volume (B) of Ler and COV cell suspensions. Cells were treated with 50 μm MeJA at 1 DASU. MeJA-treated and untreated cell suspensions were collected at 2 d intervals and subjected to enzymatic digestion to release protoplasts, from which the cell number (cell mL^–1) and cell volume (μm³) was determined using the Image J software (Schneider et al., 2012). (A and B) Data represent the average of five independent biological replicates ± s.e. per line with n = 417–3145.

Fig. 3.

MeJA alters cell cycle progression in Ler and COV cell suspensions. Quantitative analysis of nuclear DNA content in Ler and COV cell suspensions performed by flow cytometry analysis of cell suspensions at 4 and 6 DASU treated with 50 μm (+) and 200 μm (++) MeJA at 1 DASU. (A and B) Average frequencies of the observed ploidy levels of a minimum of three independent biological replicates ± s.e. (C and D) Cell cycle analysis of flow cytometry data at 4 (C) and 6 (D) DASU. The analyses were performed on at least 20 000 nuclei isolated for each ploidy measurement.

Fig. 4.

qRT-PCR analysis of cell cycle (A–G) and cell wall (H–L) remodelling markers over time. Transcript levels in Ler and COV cell suspensions by extracting RNA from 0, 2, 4 and 6 DASU cell suspensions. AT5G55480 was used as a reference gene as per Noir et al. (2013), and the ΔΔCt (Schmittgen and Livak, 2008) method was applied for the calculations. MeJA (50 μm) was applied to Ler, COV1 and COV2 cell suspensions at 1 DASU. The allene oxide synthase (AOS) gene was analysed to test the effectiveness of the MeJA treatment (M). Data are the averages ± s.e. of three independent biological replicates, and reactions were performed in triplicate. Results are expressed as log2 fold changes normalized to the 0 h time point for each genotype.

Fig. 5.

SDS–PAGE analysis of apoplastic proteins in Ler and COV cell suspensions. Ler and COV cell cultures grown for 7 d were gently vacuum filtered and then incubated for 30 min in 200 mm CaCl₂ as described in the Materials and Methods. Proteins were precipitated with chloroform/methanol, resuspended in sample buffer and separated by SDS–PAGE. The gels were stained with Coomassie blue R250 for protein visualization. Arrows indicate bands selected for in-gel trypsin digestion and sequencing. Protein IDs are listed in Table 1 and peptides are listed in Supplementary data Table SIII. A representative SDS–polyacrylamide gel is shown of three independent experiments.

Fig. 6.

Differentially regulated metabolites in arabidopsis cell culture associated with the overexpression of the JA receptor COI1. Significant metabolite changes in COV1 cell culture compared with Ler (both untreated) using GC-MS. (A) Volcano plot of metabolomics data. The x-axis is the mean ratio fold change (plotted on a log2 scale) of the relative abundance of each metabolite between untreated Ler and COV1. The y-axis represents the statistical significance P-value (plotted on a –log10 scale) of the ratio of relative abundances for each metabolite. Metabolites highlighted in red (and also represented in B) hyperaccumulate in COV1 cell culture and have significant P-values (orange threshold bar represents P < 0.05) and high fold change values (>2). (B) The vertical scale bars (log10) represent the relative metabolite abundance normalized to the total peak areas. Metabolites shown are significantly different (P < 0.05) according to pair-wise comparison using Tukey HSD test. Data represent the means of four independent biological replicates and error bars represent the s.e.

Fig. 7.

MeJA contributes to the regulation of the trade-off between defence mode and plant growth. Schematic representation of the cellular processes regulated by MeJA through the JA receptor COI1. MeJA inhibits cell proliferation via regulation of key components of the plant cell cycle and promotes changes in cell wall composition. Such modifications halt cell expansion while enhancing defence responses. MeJA induces metabolic reprogramming in plant cells to adjust to stress conditions, compromising growth. Shaded red and green shapes indicate accumulation or reduction in transcript, protein or metabolite levels, respectively.