ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE 1 (ADPG1) releases latent defense signals in stems with reduced lignin content

Abstract

There is considerable interest in engineering plant cell wall components, particularly lignin, to improve forage quality and biomass properties for processing to fuels and bioproducts. However, modifying lignin content and/or composition in transgenic plants through down-regulation of lignin biosynthetic enzymes can induce expression of defense response genes in the absence of biotic or abiotic stress. Arabidopsis thaliana lines with altered lignin through down-regulation of hydroxycinnamoyl CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) or loss of function of cinnamoyl CoA reductase 1 (CCR1) express a suite of pathogenesis-related (PR) protein genes. The plants also exhibit extensive cell wall remodeling associated with induction of multiple cell wall-degrading enzymes, a process which renders the corresponding biomass a substrate for growth of the cellulolytic thermophile Caldicellulosiruptor bescii lacking a functional pectinase gene cluster. The cell wall remodeling also results in the release of size- and charge-heterogeneous pectic oligosaccharide elicitors of PR gene expression. Genetic analysis shows that both in planta PR gene expression and release of elicitors are the result of ectopic expression in xylem of the gene ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE 1 (ADPG1), which is normally expressed during anther and silique dehiscence. These data highlight the importance of pectin in cell wall integrity and the value of lignin modification as a tool to interrogate the informational content of plant cell walls.

Keywords: cell wall remodeling; defense response; elicitor; lignin modification; polygalacturonase.

Fig. 1.

Cell wall remodeling in reduced lignin Arabidopsis lines. (A) (Lower) Glycome analysis of cell wall polysaccharides from water extracts of cell walls of control, HCT-RNAi, and ccr1 mutant Arabidopsis stems. The nondialyzed water-soluble cell wall extracts were screened against a panel of 155 monoclonal antibodies directed against diverse epitopes in noncellulosic plant glycans. The resulting heat map depicts antibody binding strength based on optical density (OD) depicted as a color gradient ranging from black (no binding) to yellow (strongest binding), as indicated by the key at the lower right. Antibodies are grouped into clades according to the glycans that they predominantly recognize, as indicated by the panel on the right side of the glycome profiles. Upper shows carbohydrate recovery from water-extracted AIRs. Details of monoclonal antibodies are given in ref. . (B–D) Lignin modification alleviates the need for pectinase action to enable a cellulolytic bacterium to access Arabidopsis biomass. (B) Growth of wild-type (WT) C. bescii on ground biomass from WT and HCT-down-regulated Arabidopsis plants. (C) Growth of C. bescii JWCB005 (ΔpyrFA). (D) Growth of C. bescii JWCB010 (ΔpyrFA ΔpecABCR). Cells were collected and stained with acridine orange at the times shown and counted using an epifluorescence microscope with a counting chamber lens. Two biological replicates were taken. Asterisks indicate significant differences from WT (P < 0.05) by pairwise multiple comparison Tukey test.

Fig. 2.

Defense gene expression in Arabidopsis cell cultures in response to water-soluble elicitors from cell walls of wild-type (WT), HCT-RNAi, ccr1, and f5h (fah-1) mutant and F5H overexpressor (OE) Arabidopsis plants. (A) PR1 and PR2 induction in response to crude elicitors from the plant lines shown. (B) PR10 induction in response to crude elicitors from the plant lines shown. (C) PR10 and PR1 induction by ion exchange fractionated elicitors from cell walls of HCT-RNAi plants. (D) PR10 and PR1 induction by ion exchange fractionated elicitors from cell walls of ccr1 plants. The elicitor activity of selected fractions was determined by measuring their ability to induce defense gene transcripts (PR2, PR10, PR1) in cell suspension cultures. Analysis of transcript levels in cell cultures was by qRT-PCR performed with total messenger RNA (mRNA) from suspension cells harvested 12 h postelicitation, and incubated in the dark at 25 °C. Transcript levels are expressed relative to AtPP2A. Results are means ± SD of three biological replicates. Asterisks in A and B indicate significant differences from WT (P < 0.05) by pairwise multiple comparison Tukey test. Elicitor extracts were prepared from the AIR fraction of cell walls. Extracts were added directly to cell cultures (−), or pretreated with PGase (+). PGA was also tested as elicitor for a positive control. Elicitor fractions are as shown in SI Appendix, Fig. S7 A–D.

Fig. 3.

ADPG1 is required for elicitor release and PR gene induction in HCT-RNAi Arabidopsis. (A) Overall growth phenotypes of wild-type (WT), HCT-RNAi, adpg1, and HCT-RNAi adpg1 lines at 8 wk old. (Scale bar, 2 cm.) (B) Lignin content and composition of the above lines as determined by thioacidolysis. (C) PR1 and PR 10 transcript levels in the above lines. (D) Induction of PR1 and PR10 transcripts in Arabidopsis cell cultures by elicitors derived from WT, HCT-RNAi, and HCT-RNAi adpg1 Arabidopsis. Cell wall extracts from WT and HCT-RNAi plants were also pretreated with RC-ADPG1 prior to testing of elicitor activity. Genotypes are represented as uppercase (WT) and lowercase (mutant) alleles. HCT-RNAi x WT is heterozygous for the RNAi construct. Error bars represent SD of three technical replicates (individual assays). Separate bars are shown for biological replicates. Asterisks indicate significant differences from WT (P < 0.05) by pairwise multiple comparison Tukey test.

Fig. 4.

ADPG1 is required for PR1 induction in the ccr1 mutant of Arabidopsis. (A) Phenotype of F2 progeny of the cross ccr1 x adpg1. Wild-type (WT), ccr1, and F2 plants are 7 wk old. (Scale bar, 2 cm.) (B) Lignin content and composition of F2 progeny of the cross ccr1 x adpg1 as determined by thioacidolysis. Results are means ± SD of five technical replicates. Individual biological replicates are indicated by R1, R2, etc. (C) Levels of CCR1 transcripts in WT and ccr1/adpg1 mutant lines. (D) Levels of ADPG1 transcripts in WT and ccr1/adpg1 mutant lines. (E) Levels of PR1 transcripts in WT and ccr1/adpg1 mutant lines. Genotypes are represented as uppercase (WT) and lowercase (mutant) alleles. Transcript analyses were performed on 8-wk-old plants, and expressed relative to ACT2. In C–E, each replicate is a pool of four plants of the same genotype. (F) Restoring CCR expression to xylem prevents ADPG1-mediated induction of PR proteins in the Arabidopsis ccr1 mutant. PR1 transcript levels were determined in inflorescence stems of ccr1 mutant plants (two independent lines, ccr1-3 and ccr1-6), and the ccr1 mutants in which a WT copy of CCR1 was expressed under a xylem-specific promoter (ProSNBE:CCR1) (see ref. for details of the lines). Results are mean ± SD of three biological replicates; WT PR1 transcript value is normalized to 1.0. Asterisks in B, E, and F indicate significant differences from WT (P < 0.05) by pairwise multiple comparison Tukey test.

Fig. 5.

Model for the activation of PR genes in HCT-RNAi and ccr1 Arabidopsis plants. In the proposed model, changes in lignin content in xylem cells of HCT-RNAi or ccr1 Arabidopsis are perceived initially by the cell through activation of plasma membrane-localized cell wall integrity receptors. This results in initiation of a signaling cascade that induces the expression of cell wall remodeling genes, including PECTATE LYASES, XYLOGLUCAN ENDO-TRANSGLYCOSYLASES (XTHs), and ADGP1. ADPG1 activity may contribute to solubilization of pectin, but is necessary for release of elicitor fragments, most likely from RG-II. The soluble elicitors activate expression of PR defense response genes through a signaling pathway involving SA (66). Many of the other transcriptomic changes occurring in the lignin-modified plants, such as the activation of seed-specific genes in stems of ccr1, may result from secondary effects. The modification of pectin is also, at least in part, responsible for the reduced recalcitrance of the biomass.