Acyl CoA Binding Proteins are Required for Cuticle Formation and Plant Responses to Microbes

Abstract

Fatty acids (FA) and lipids are well known regulators of plant defense. Our previous studies have shown that components of prokaryotic (plastidal) FA biosynthesis pathway regulate various aspects of plant defense. Here, we investigated the defense related roles of the soluble acyl CoA binding proteins (ACBPs), which are thought to facilitate the intracellular transport of FA/lipids. We show that ACBP3 and 4 are required for maintaining normal lipid levels and that ACBP3 contributes to the lipid flux between the prokaryotic and eukaryotic pathways. We also show that loss of ACBP3, 4, or 6 impair normal development of the cuticle and affect both basal and resistance protein-mediated defense against bacterial and fungal pathogens. Loss of ACBP3, 4, or 6 also inhibits the induction of systemic acquired resistance (SAR) due to the plants inability to generate SAR inducing signal(s). Together, these data show that ACBP3, ACBP4, and ACBP6 are required for cuticle development as well as defense against microbial pathogens.

Keywords: acyl CoA binding proteins; cuticle; fatty acids; plant defense; systemic acquired resistance.

Figure 1

FA and lipid levels in ACBP KO plants. (A) Levels of total FAs in 4-week-old Col-0 and acbp mutant plants. The values are presented as mean of six to eight replicates. FW indicates fresh weight. The error bars represent SD. The experiment was repeated five times with similar results. (B) Total lipid levels in Col-0 and acbp mutant plants. The values are presented as a mean of five replicates. The error bars represent SD. Asterisks denote a significant difference with Col-0 (t-test, P < 0.05). DW indicates dry weight. (C) Profile of total lipids extracted from Col-0 and acbp mutant plants. The values are presented as a mean of five replicates. The error bars represent SD. Asterisks denote a significant difference with Col-0 (t-test, P < 0.05). Symbols for various components are: DGD, digalactosyldiacylglycerol; MGD, monogalactosyldiacylglycerol; PG, phosphatidylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PA, phosphatidic acid.

Figure 2

Evaluation of cuticle associated phenotypes in acbp mutant plants. (A) Toluidine blue stained leaves from 4-week-old plants. The stain was spotted on the adaxial or abaxial surface of the leaves and the leaves were washed with water after 20 (left) or 40 min (right) incubations. The experiment was repeated three times with similar results. (B) Measurement of water lost from the leaves subjected to drought conditions for 4 days. The error bars represent SD (n = 25). Asterisks denote a significant difference with Col-0 (t-test, P < 0.05). The experiment was repeated three times with similar results. (C) Transmission electron micrographs showing cuticle layer on adaxial surface of leaves from indicated genotypes. At least two independent leaves were sectioned and analyzed. Arrows indicate cuticle. CW indicated cell wall (scale bars, 50 nm). (D) Scanning electron micrographs showing adaxial (left panel) and abaxial (right panel) surface of leaves from indicated genotypes (scale bars, 200 μM). Two-three independent leaves were analyzed per genotype.

Figure 2

Evaluation of cuticle associated phenotypes in acbp mutant plants. (A) Toluidine blue stained leaves from 4-week-old plants. The stain was spotted on the adaxial or abaxial surface of the leaves and the leaves were washed with water after 20 (left) or 40 min (right) incubations. The experiment was repeated three times with similar results. (B) Measurement of water lost from the leaves subjected to drought conditions for 4 days. The error bars represent SD (n = 25). Asterisks denote a significant difference with Col-0 (t-test, P < 0.05). The experiment was repeated three times with similar results. (C) Transmission electron micrographs showing cuticle layer on adaxial surface of leaves from indicated genotypes. At least two independent leaves were sectioned and analyzed. Arrows indicate cuticle. CW indicated cell wall (scale bars, 50 nm). (D) Scanning electron micrographs showing adaxial (left panel) and abaxial (right panel) surface of leaves from indicated genotypes (scale bars, 200 μM). Two-three independent leaves were analyzed per genotype.

Figure 3

Biochemical profiles of cuticular wax and cutin monomers in acbp mutant plants. (A) Analysis of wax components from leaves of 4-week-old Col-0 and acbp plants. C16:0-C30:0 are FAs, C25-C33 are alkanes, C26-OH-C32-OH are primary alcohols. The values are presented as a mean of five replicates. The error bars represent SD. Asterisks denote a significant difference with Col-0 (t-test, P < 0.05). DW indicates dry weight. (B) Analysis of lipid polyester monomer content of 4-week-old Col-0 and acbp plants. Error bars in (A,B) represent SD. Statistical significance was calculated using Student’s t-test (t-test, P < 0.05). Symbols for various components are: 16:0-DCA, 1,16-hexadecane dioic acid; 16-OH-16:0, 16-hydroxyhexadecanoic acid; 10,16-OH-16:0, 10,16-dihydroxyhexadecanoic acid; 18:0-DCA, 1,18-octadecane dioic acid; 18:1-DCA, 1,18-octadecene dioic acid; 18-OH-18:1, 18-hydroxyoctadecenoic acid, 18:2-DCA, 1,18-octadecadiene dioic acid; 18-OH-18:2, 18-hydroxyoctadecadienoic acid; 18-OH-18:3, 18-hydroxyoctadecadienoic acid.

Figure 4

The acbp mutant plants show compromised response to fungal and bacterial pathogens. (A) Disease symptoms on indicated genotypes spot-inoculated with water or 10⁶ spores/ml of C. higginsianum or B. cinerea. The experiment was carried out five times and three of these showed enhanced susceptibility in acbp plants. (B) Lesion size in spot-inoculated genotypes. The plants were spot-inoculated with 10⁶ spores/ml of C. higginsianum and the lesion size was measured from 20 to 30 independent leaves at 6 dpi. Statistical significance was determined using Student’s t-test. Asterisks indicate data statistically significant from that of control (Col-0; P < 0.05). Error bars indicate SD. (C) Growth of virulent P. syringae on leaves from Col-0 or acbp mutant plants. Error bars indicate SD. Asterisks indicate data statistically significant from that of control (Col-0; P < 0.05, n = 4). (D) Growth of avirulent (avrRpt2) P. syringae strains on Col-0 or acbp mutant plants. Error bars indicate SD. Asterisks indicate data statistically significant from that of control (Col-0; P < 0.05, n = 4). Bacterial growth presented as the LOG of colony forming units (CFU) per leaf disk, was monitored at 0 and 3 dpi. Experiments in (C,D) were repeated six times each with similar results.

Figure 5

The acbp mutants show compromised SAR. (A) SAR response in Col-0 and acbp6 (a6), acbp3 (a3), acbp4 (a4), and lacs2 plants. Primary leaves were inoculated with MgCl₂ (gray bars) or P. syringae containing avrRpt2 (black bars). The distal leaves were inoculated with the virulent P. syringae and growth of the virulent bacteria was monitored at 3 dpi. The SAR impaired lacs2 plants were used as a positive control (Xia et al., 2009). This experiment was repeated six times with similar results. Asterisk denotes significant difference from plants of the same genotype pre-infiltrated with MgCl₂ (t-test, n = 4, P < 0.0001). (B–D) SAR response in Col-0 and acbp plants infiltrated with exudates (Ex) collected from wt or acbp plants that were treated either with MgCl₂ (blue and pink bars) or P. syringae expressing avrRpt2 (red and yellow bars). Error bars indicate SD (n = 4). Statistical significance was calculated using Student’s t-test (P < 0.0001). Experiments shown in (B–D) were repeated twice with similar results. Bacterial growth presented as the LOG of colony forming units (CFU) per leaf disk, was monitored at 0 and 3 dpi. (E) Glucose levels in petiole exudates collected from indicated genotypes. Error bars indicate SD. The experiment was repeated twice and the second repeat showed wild-type-like glucose levels in acbp3 plants. Error bars indicate SD (n = 3). No statistical significance was observed in the levels from different genotypes per Student’s t-test.

Figure 6

The acbp mutants accumulate reduced levels of SA. (A) SA and SAG levels in local (inoculated) and distal (uninoculated) leaves of Col-0 and acbp plants inoculated with MgCl₂ or P. syringae expressing avrRpt2. Leaves were harvested at 3 dpi. Error bars indicate SD. Asterisks indicate data statistically significant from that of control (Col-0; P < 0.05, n = 4). The experiment was repeated twice with similar results. (B) RNA gel blot showing transcript levels of PR-1 gene in plants treated with water or BTH for 48 h. Ethidium bromide staining of total RNA was used as the loading control. The experiment was repeated twice with similar results. (C) SAR response in Col-0 and acbp plants pretreated with water (purple and pink bars) or the SA analog BTH (orange and black bars) for 48 h prior to mock (purple and orange bars) or avr (pink and black bars) inoculation. The error bars represent SD (n = 4). Asterisks denote statistical differences from water and mock treated plants of corresponding genotype (t-test P < 0.001). Statistical difference from BTH and mock treated plants is indicated by “a” (P < 0.001). The experiment was repeated three times with similar results. (D–F) SAR response in Col-0 and acbp plants infiltrated with exudates (Ex) collected from wt or acbp plants that were treated either with MgCl₂ (mock, blue, and red bars) or P. syringae expressing avrRpt2 (pink and yellow bars). Exudates were mixed with water (blue and pink bars) or 100 μM BTH (red and yellow bars) prior to infiltration into a fresh set of plants. Error bars indicate SD (n = 4). Statistical significance was calculated using Student’s t-test. Asterisks denote statistical differences from mock + water-treated plants (blue bars) of corresponding genotype (t-test P < 0.005). Statistical difference from avrRpt2 + water-treated plants (pink bars) is indicated by “a” (P < 0.01). Bacterial growth presented as the LOG of colony forming units (CFU) per leaf disk, was monitored at 0 and 3 dpi. Experiments in (B–D) were repeated twice with similar results. a3, a4, a6 indicate acbp3, acbp4, and acbp6, respectively.

Figure 7

The acbp mutants are responsive to MeSA and accumulate normal levels of AA. (A) AA levels in mock (MgCl₂, gray bars) and avrRpt2 (black bars) inoculated wild-type (Col-0) and acbp mutants. Error bars indicate SD (n = 3). Statistical significance was calculated using Student’s t-test. Asterisks denote significant differences from mock-inoculated plants of corresponding genotype. Numbers above black bars indicate P values. The experiment was repeated twice with similar results. (B) RNA gel blot showing transcript levels of PR-1 gene in plants treated with water or MeSA for 48 h. Ethidium bromide staining of total RNA was used as the loading control. The experiment was repeated twice with similar results. (C) SAR response in Col-0 and acbp6 (a6), acbp3 (a3), acbp4 (a4) plants, pretreated with water (blue and pink bars) or 100 μM MeSA (orange and black bars) prior to infiltration with MgCl₂ (mock, blue, and orange bars) or P. syringae expressing avrRpt2 (pink and black bars). Bacterial growth presented as the LOG of colony forming units (CFU) per leaf disk, was monitored at 0 and 3 dpi. Error bars indicate SD (n = 4). Statistical significance was calculated using Student’s t-test (P < 0.005).

Figure A1

Reverse transcription-PCR analysis showing ACBP transcript levels in wild-type (Col-0) and acbp3 (A), acbp4 (B), and acbp6 (C) mutant plants. The level of β-tubulin was used as an internal control to normalize the amount of cDNA template. The RT-PCR analysis was repeated with two independent cDNA templates per genotype.

Figure A2

Levels of MGDG and DGDG containing 34:6 or 36:6 FAs in Col-0 and acbp mutant plants. The values are presented as a mean of five replicates. The error bars represent SD. Asterisks denote significant differences with Col-0 (t-test, P < 0.05).

Figure A3

Cuticular phenotypes of acbp plants. (A) Whole leaf toluidine blue staining. Leaves were incubated in the stain for 10 min and the experiment was repeated four times with similar results. (B) A time-course measurement of chlorophyll leaching in various genotypes at indicated times. The values are presented as a mean of four replicates. The error bars represent SD (P < 0.05). The experiment was repeated four times.

Figure A4

Disease symptoms on indicated genotypes spray-inoculated with 10⁶ spores/ml of C. higginsianum. The experiment was carried out five times, three of which showed enhanced susceptibility in acbp plants, while the remaining two showed wild-like infection phenotypes on acbp plants. The upper and lower panels show two independent experiments that showed enhanced susceptibility in acbp plants.

Figure A5

Pathogen response of Col-0 and acbp plants after exogenous application of the SA analog BTH. The Col-0 and acbp plants were pretreated with water (mock, solid bars) or 100 μM BTH (shaded bars) prior to infiltration with P. syringae expressing avrRpt2. Bacterial growth presented as the LOG of colony forming units (CFU) per leaf disk, was monitored at 0 and 3 dpi. Error bars indicate SD (n = 4). Statistical significance was calculated using Student’s t-test (P < 0.001). The experiment was repeated twice with similar results.

Figure A6

Systemic acquired resistance response in Col-0 and acbp6 (a6), acbp3 (a3), acbp4 (a4) plants, pretreated with water (blue and pink bars) or 50 μM SA (orange and black bars) prior to infiltration with MgCl₂ (mock, blue, and orange bars) or P. syringae expressing avrRpt2 (pink and black bars). Bacterial growth presented as the LOG of colony forming units (CFU) per leaf disk, was monitored at 0 and 3 dpi. Error bars indicate SD (n = 4). Asterisks denote statistical differences from water + mock-inoculated plants (blue bars) of corresponding genotype, calculated using Student’s t-test (P < 0.001).