A humidity-sensitive Arabidopsis copine mutant exhibits precocious cell death and increased disease resistance

Abstract

The copines are a newly identified class of calcium-dependent, phospholipid binding proteins that are present in a wide range of organisms, including Paramecium, plants, Caenorhabditis elegans, mouse, and human. However, the biological functions of the copines are unknown. Here, we describe a humidity-sensitive copine mutant in Arabidopsis. Under nonpermissive, low-humidity conditions, the cpn1-1 mutant displayed aberrant regulation of cell death that included a lesion mimic phenotype and an accelerated hypersensitive response (HR). However, the HR in cpn1-1 showed no increase in sensitivity to low pathogen titers. Low-humidity-grown cpn1-1 mutants also exhibited morphological abnormalities, increased resistance to virulent strains of Pseudomonas syringae and Peronospora parasitica, and constitutive expression of pathogenesis-related (PR) genes. Growth of cpn1-1 under permissive, high-humidity conditions abolished the increased disease resistance, lesion mimic, and morphological mutant phenotypes but only partially alleviated the accelerated HR and constitutive PR gene expression phenotypes. The disease resistance phenotype of cpn1-1 suggests that the CPN1 gene regulates defense responses. Alternatively, the primary function of CPN1 may be the regulation of plant responses to low humidity, and the effect of the cpn1-1 mutation on disease resistance may be indirect.

Figure 1.

Humidity-Dependent Developmental and Lesion Mimic Phenotypes of cpn1-1. The four columns of images, from left to right, represent Col-0 wild type grown in LH, cpn1-1 grown in LH, Col-0 wild type grown in HH, and cpn1-1 grown in HH, respectively. All plants were grown in SD light conditions. (A) to (D) Four-week-old whole plants. Bar in (A) = 1 cm for (A) to (D). (E) to (H) Close-up images of leaves. Arrowhead in (F) denotes a lesion. Bar in (E) = 0.5 cm for (E) to (H). (I) to (L) Sectors of leaves stained with the vital stain trypan blue. Bar in (I) = 200 μm for (I) to (L). (M) to (P) UV autofluorescence images of leaf sectors. Bar in (M) = 100 μm for (M) to (P). (Q) to (T) Leaf sectors stained with aniline blue to detect callose accumulation. The scale is the same as in (M) to (P). (U) to (X) Scanning electron micrographs of the abaxial surfaces of leaves. Bar in (U) = 50 μm for (U) to (X).

Figure 2.

Accelerated HR in cpn1-1. The macroscopic HR was first observed at ∼12 hr after inoculation. Fifty to 100 leaves of each type were inoculated for each experiment. For details, see Methods. (A) Proportions of inoculated Col-0 wild-type and cpn1-1 leaves exhibiting partial and complete collapse over time after inoculation. Asterisks indicate percentages of leaves with partial or complete HR collapse in cpn1-1 plants that are significantly higher than Col-0 wild-type plants grown under the same humidity conditions, at the same time during the experiment, using Student's t test. A leaf was scored as showing complete HR when the leaf had collapsed totally; a leaf was scored as showing partial HR when from 10 to 90% of the infiltrated leaf had collapsed. Leaves showing <10% collapse were not scored as having an HR. (B) Electrolyte leakage from inoculated LH-grown Col-0 wild-type and cpn1-1 leaves over time after infiltration with P.s.m. (avrRpt2) or 10 mM MgCl₂ (control). (C) Electrolyte leakage from inoculated HH-grown Col-0 wild-type and cpn1-1 leaves over time after infiltration with P.s.m. (avrRpt2) or 10 mM MgCl₂ (control). WT, wild type. Bars in (B) and (C) indicate standard errors.

Figure 3.

Stomatal Conductance in Col-0 Wild-Type and cpn1-1 Leaves. Data represent the average stomatal conductance for LH-grown cpn1-1 and Col-0 wild-type (WT) plants. Stomatal conductance represents the rate of passage of water vapor and carbon dioxide through the stomata of the plant. Bars indicate standard errors.

Figure 4.

Humidity-Dependent Resistance of cpn1-1 to Virulent P.s.t. (A) LH-grown Col-0 wild-type plants 4 days after inoculation with virulent P.s.t. (B) LH-grown cpn1-1 plants 4 days after inoculation with virulent P.s.t. (C) HH-grown Col-0 wild-type plants 4 days after inoculation with virulent P.s.t. (D) HH-grown cpn1-1 plants 4 days after inoculation with virulent P.s.t.

Figure 5.

Humidity-Dependent Inhibition of Bacterial Growth in cpn1-1. (A) Growth of virulent P.s.t. (DC3000) and avirulent P.s.t. (avrRpt2) bacteria in LH-grown Col-0 wild-type (WT) and cpn1-1 plants. Plants were inoculated with the bacterial strains (10⁵ cfu/mL) by syringe infiltration of the leaf mesophyll. For details, see Methods. (B) Growth of virulent P.s.t. (DC3000) and avirulent P.s.t. (avrRpt2) bacteria in HH-grown Col-0 wild-type (WT) and cpn1-1 plants. Plants were inoculated with the bacterial strains (2 × 10⁷ cfu/mL) by dipping, as described in Methods. This accounts for the higher initial bacterial count in (B) compared with that in (A). Bars in (A) and (B) indicate standard errors.

Figure 6.

PR Gene Transcript Accumulation in cpn1-1 and Col-0 Wild Type. RNA gel blot analyses of the transcript levels of PR1, PR2, and PR5 in LH- and HH-grown Col-0 wild-type (WT) and cpn1-1 leaf tissues. Twenty micrograms of leaf total RNA were loaded in each lane. rRNA, 28S rRNA stained with methylene blue to show equal RNA loading in each lane.

Figure 7.

Resistance of cpn1-1 to P. parasitica. (A) LH-grown Col-0 wild-type leaf 7 days after inoculation with P. parasitica. Bar = 2 mm. Inset, close-up of sporangiophore at double magnification. (B) LH-grown cpn1-1 leaf 7 days after inoculation with P. parasitica. The scale is the same as in (A). (C) Sporangiophore counts on LH-grown Col-0 wild-type (WT) and cpn1-1 leaves 7 days after inoculation. Bars represent standard errors.

Figure 8.

Molecular Identification of the CPN1 Gene. (A) Scheme of the CPN1 gene with the 16 exons depicted as boxes. In cpn1-1, two copies of the T-DNA are inserted in a head-to-head configuration into exon 7 of CPN1. The numbers indicate the base pair positions in Transformation-competent artificial chromosome (TAC) clone K22G18 for the start and stop codons of CPN1 and the exact site of the T-DNA insertion in cpn1-1. RB, right T-DNA border; LB, left T-DNA border. (B) 5′ and 3′ RACE PCR products resolved on a 1% agarose gel and visualized with ethidium bromide. The 5′ RACE reaction produced a single 1.5-kb band corresponding to the 5′ end of the CPN1 gene transcript. The 3′ RACE reaction produced two bands. The 750-bp band, denoted by the arrow, represents the 3′ end of the CPN1 gene transcript. The 1.8-kb band, denoted by the asterisk, was determined by DNA sequencing to represent a portion of one of the other copine homologs in the Arabidopsis genome (accession number AL163912). (C) Complete CPN1 cDNA sequence and predicted amino acid translation. The C2 domains are solid underlined. The A domain is dashed underlined. (D) Reverse transcriptase–mediated PCR analysis of CPN1 transcript accumulation in LH- and HH-grown Col-0 wild-type (WT) and cpn1-1 leaf tissue. CPN1, a 467-bp portion of the CPN1 transcript; Actin, a 900-bp portion of an actin gene.