The Arabidopsis Class III Peroxidase AtPRX71 Negatively Regulates Growth under Physiological Conditions and in Response to Cell Wall Damage

Abstract

The structure of the cell wall has a major impact on plant growth and development, and alteration of cell wall structural components is often detrimental to biomass production. However, the molecular mechanisms responsible for these negative effects are largely unknown. Arabidopsis (Arabidopsis thaliana) plants with altered pectin composition because of either the expression of the Aspergillus niger polygalacturonase II (AnPGII; 35S:AnPGII plants) or a mutation in the QUASIMODO2 (QUA2) gene that encodes a putative pectin methyltransferase (qua2-1 plants), display severe growth defects. Here, we show that expression of Arabidopsis PEROXIDASE71 (AtPRX71), encoding a class III peroxidase, strongly increases in 35S:AnPGII and qua2-1 plants as well as in response to treatments with the cellulose synthase inhibitor isoxaben, which also impairs cell wall integrity. Analysis of atprx71 loss-of-function mutants and plants overexpressing AtPRX71 indicates that this gene negatively influences Arabidopsis growth at different stages of development, likely limiting cell expansion. The atprx71-1 mutation partially suppresses the dwarf phenotype of qua2-1, suggesting that AtPRX71 contributes to the growth defects observed in plants undergoing cell wall damage. Furthermore, AtPRX71 seems to promote the production of reactive oxygen species in qua2-1 plants as well as plants treated with isoxaben. We propose that AtPRX71 contributes to strengthen cell walls, therefore restricting cell expansion, during normal growth and in response to cell wall damage.

Figure 1.

Elevated AtPRX71 expression in plants with altered cell walls. Expression of AtPRX71 in rosette leaves of 4-week-old wild-type (WT), 35S:AnPGII, and qua2-1 plants (A) or 10-d-old wild-type seedlings treated for 24 h with 0, 30, or 300 nm IXB (B) was determined by qPCR and normalized using the UBQ5 gene. Bars represent average arbitrary units ± sd of three technical replicates. This experiment was repeated three times with similar results.

Figure 2.

Isolation of insertional mutants for AtPRX71. A, Schematic representation of the AtPRX71 locus. Exons and introns are represented in black and white, respectively. Localization of T-DNA insertions (gray) and the primers used for genotyping of atprx71-1 and atprx71-2 are shown. B, Genotyping of atprx71-1 and atprx71-2. Genomic DNA from the wild type (WT) and mutants was subjected to PCR using primer pairs for the wild-type allele (LP + RP) or the T-DNA insertion (Lba1 + RP). LP, 71-1LP or 71-2LP; RP, 71-1RP or 71-2RP. C, Expression of AtPRX71 was analyzed in wild-type, atprx71-1, and atprx71-2 rosette leaves by qPCR using UBQ5 as the reference gene. Bars represent average arbitrary units ± sd of three technical replicates. This experiment was repeated twice with similar results. D, Total peroxidase activity in protein extracts from wild-type, atprx71-1, and atprx71-2 10-d-old seedlings was determined by a guaiacol oxidation-based assay. Bars represent average activity ± se of at least six independent samples. This experiment was repeated twice with similar results. *, Statistically significant differences between the wild type and mutants according to Student’s t test (P < 0.05).

Figure 3.

Loss of AtPRX71 increases growth. A to C, Representative picture (A), fresh weight (B), and dry weight (C) of 4-week-old soil-grown wild-type (WT), atprx71-1, and atprx71-2 rosettes. Bar =1.5 cm. D, Fresh weight of 10-d-old wild-type, atprx71-1, and atprx71-2 seedlings grown in vitro on solid medium in the light. E, Hypocotyl length of 5-d-old wild-type, atprx71-1, and atprx71-2 etiolated seedlings grown in vitro on solid medium. B to E, Bars represent average weight ± se (n > 10 in each experiment); asterisks indicate statistically significant differences between the wild type and mutants according to Student’s t test. *, P < 0.05; ***, P < 0.01.

Figure 4.

Overexpression of AtPRX71 causes reduced growth. A, RNA was extracted from leaves of four-week-old wild-type (WT) plants and four independent lines expressing AtPRX71 under the control of the 35S promoter (35S:AtPRX71). Expression of AtPRX71 was determined by reverse transcription PCR using UBQ5 as reference gene. B, Representative picture of rosettes of the same lines described in A. Bar = 5 cm. C, Average rosette fresh weight ± se (n > 10) of the wild type and 35S:AtPRX71 lines 22 and 24 4-week-old soil-grown plants. D, Peroxidase activity in leaves of wild-type and 35S:AtPRX71 plants grown as in C. Bars represent average enzymatic activity (nanokatal milligram⁻¹ protein) ± sd (n = 3). ***, Significant differences between wild-type and transgenic plants according to Student’s t test (P < 0.01).

Figure 5.

AtPRX71 negatively affects leaf epidermal cell size. Rosette leaves from 3-week-old wild-type (WT; A), atprx71-1 (B), and atprx71-2 (C) plants and two lines overexpressing AtPRX71 (35S:AtPRX71 lines 22 [D] and 24 [E]) were cleared and stained with ruthenium red, and images of the epidermis were taken with a microscope. Bars = 100 µm. F, Bars represent the average area of epidermal cells ± se (n > 20). Asterisks indicate significant differences between the wild type and mutants or transgenic plants according to Student’s t test. 71-1, atprx71-2; 71-2, atprx71-2. *, P < 0.05; ***, P < 0.01.

Figure 6.

Loss of AtPRX71 partially restores qua2-1 growth defects. A, Representative picture of wild-type (WT), atprx71-1, qua2-1, and atprx71-1 qua2-1 3-week-old soil-grown rosettes. Bar = 1 cm. B, Rosette fresh weight of wild-type, atprx71-1, qua2-1, and atprx71-1 qua2-1 4-week-old soil-grown plants. Bars indicate average weight ± se (n > 10). C, Hypocotyl length of 5-d-old wild-type, atprx71-1, and atprx71-2 etiolated seedlings grown in vitro on solid medium (n > 10). Different letters in B and C indicate significant differences according to one-way ANOVA followed by Tukey’s significance test (P < 0.05).

Figure 7.

Peroxidase activity in atprx71 and qua2-1 mutants. Total proteins were extracted from 10-d-old in vitro-grown seedlings (A) or rosette leaves from 4-week-old soil-grown plants (B) and assayed for peroxidase activity. Bars represent average enzyme activity (nanokatal milligram⁻¹ protein) ± sd (n = 3). Letters indicate significant differences according to one-way ANOVA followed by Tukey’s significance test (P < 0.05). 71-1, atprx71-1; 71-2, atprx71-2; WT, wild type.

Figure 8.

Inhibition of root elongation by IXB is partially reduced in atprx71 mutants. Wild-type (WT), atprx71-1, and atprx71-2 seedlings grown for 5 d on solid medium were transferred to solid medium containing the indicated doses of IXB. Primary root elongation was measured after 3 d. Bars indicate average elongation ± se (n > 12). Asterisks indicate significant differences between the wild type and mutants according to Student’s t test. This experiment was repeated twice with similar results. *, P < 0.05; ***, P < 0.01.

Figure 9.

AtPRX71 promotes ROS accumulation in response to cell wall modifications. ROS accumulation in rosette leaves of 4-week-old qua2-1, atprx71-1, and atprx71-1 qua2-1 plants (A) and 35S:AtPRX71 plants belonging to lines 22 and 24 (B) was revealed by DAB staining. A representative picture for each genotype is shown. This experiment was repeated three (A) and two (B) times with similar results. C, Wild-type (WT), atprx71-1, and atrbohD seedlings grown for 3 d in liquid medium were treated with 600 nm IXB, and H₂O₂ production was measured every 10 min for 18 h with a luminol-based assay. Each point represents the average luminescence of 12 seedlings ± sd. This experiment was repeated three times with similar results. RLU, Relative luminescence unit.