Arabidopsis sfd mutants affect plastidic lipid composition and suppress dwarfing, cell death, and the enhanced disease resistance phenotypes resulting from the deficiency of a fatty acid desaturase

Abstract

A loss-of-function mutation in the Arabidopsis SSI2/FAB2 gene, which encodes a plastidic stearoyl-acyl-carrier protein desaturase, has pleiotropic effects. The ssi2 mutant plant is dwarf, spontaneously develops lesions containing dead cells, accumulates increased salicylic acid (SA) levels, and constitutively expresses SA-mediated, NPR1-dependent and -independent defense responses. In parallel, jasmonic acid-regulated signaling is compromised in the ssi2 mutant. In an effort to discern the involvement of lipids in the ssi2-conferred developmental and defense phenotypes, we identified suppressors of fatty acid (stearoyl) desaturase deficiency (sfd) mutants. The sfd1, sfd2, and sfd4 mutant alleles suppress the ssi2-conferred dwarfing and lesion development, the NPR1-independent expression of the PATHOGENESIS-RELATED1 (PR1) gene, and resistance to Pseudomonas syringae pv maculicola. The sfd1 and sfd4 mutant alleles also depress ssi2-conferred PR1 expression in NPR1-containing sfd1 ssi2 and sfd4 ssi2 plants. By contrast, the sfd2 ssi2 plant retains the ssi2-conferred high-level expression of PR1. In parallel with the loss of ssi2-conferred constitutive SA signaling, the ability of jasmonic acid to activate PDF1.2 expression is reinstated in the sfd1 ssi2 npr1 plant. sfd4 is a mutation in the FAD6 gene that encodes a plastidic omega6-desaturase that is involved in the synthesis of polyunsaturated fatty acid-containing lipids. Because the levels of plastid complex lipid species containing hexadecatrienoic acid are depressed in all of the sfd ssi2 npr1 plants, we propose that these lipids are involved in the manifestation of the ssi2-conferred phenotypes.

Figure 1.

Impact of the ssi2 Mutant on Defense Signaling in Arabidopsis. SA and JA are important signaling molecules in plant defense. JA signaling is required for the pathogen-activated expression of PDF1.2 and resistance to the necrotrophic pathogen B. cinerea. SA signaling is required for the pathogen-activated expression of the PR genes and resistance to Psm. The EDS5 and SID2 genes are required for SA synthesis; the loss-of-function eds5 and sid2 mutations block SA synthesis. SA signaling is activated via both NPR1-dependent and -independent mechanisms. In addition to SA, an unknown pathogen-activated factor is required for signaling through the NPR1-independent pathway. The loss of the SSI2-encoded stearoyl-ACP desaturase activity in the ssi2 mutant has pleiotropic effects on plant defense responses. The ssi2 mutant allele promotes (+) the spontaneous development of lesions containing dead cells, the accumulation of increased SA levels, and the constitutive expression of NPR1-dependent and -independent defense mechanisms, which confer high-level expression of PR genes and enhanced resistance to Psm. By contrast, the ssi2 mutant interferes with (−) the ability of JA/MeJA to activate PDF1.2 expression and exhibits enhanced susceptibility to B. cinerea. 18:1 application restores the JA-inducible expression of PDF1.2 in the ssi2 mutant plant (Kachroo et al., 2001), suggesting a role for an 18:1-derived factor, which is limiting in the ssi2 mutant plant, in promoting JA signaling.

Figure 2.

Comparison of Morphological and Cell Death Phenotypes of the sfd ssi2 npr1 Mutants. (A) Comparison of the morphology of 4-week-old npr1, ssi2 npr1, sfd1-1 ssi2 npr1, sfd1-2 ssi2 npr1, sfd2-1 ssi2 npr1, sfd2-1/+ ssi2 npr1, sfd2-2 ssi2 npr1, and sfd4 ssi2 npr1 plants. The sfd2-1/+ ssi2 npr1 plant is heterozygous for the sfd2-1 mutant allele. All plants were photographed from the same distance. (B) Light microscopy of trypan blue–stained leaves from npr1, ssi2 npr1, sfd1-1 ssi2 npr1, sfd2-1 ssi2 npr1, and sfd4 ssi2 npr1 plants. The arrows mark areas containing intensely stained dead cells in ssi2 npr1. All photographs were taken at the same magnification.

Figure 3.

Defense Gene Expression in the sfd Mutants. (A) Comparison of PR1 and BGL2 expression in leaves of 4-week-old soil-grown wild-type (WT), npr1, ssi2, ssi2 npr1, sfd1-1 ssi2 npr1, sfd1-2 ssi2 npr1, sfd4 ssi2 npr1, sfd2-3 ssi2 npr1, sfd2-2 ssi2 npr1, sfd2-1 ssi2 npr1, and sfd2-1 ssi2 plants. (B) Comparison of PR1 expression in leaves of 4-week-old wild-type, npr1, ssi2, ssi2 npr1, sfd1-1 ssi2 npr1, and sfd2-1 ssi2 npr1 plants 48 h after treatment with 500 μM SA. (C) Comparison of PR1 expression in leaves of 4-week-old soil-grown ssi2, ssi2 npr1, wild-type, npr1, and sfd1-1 ssi2 plants. (D) Comparison of PR1 expression in sfd2-1 ssi2 npr1, sfd2-1/+ ssi2 npr1, and ssi2 npr1 plants. (E) Comparison of PDF1.2 expression in leaves of 4-week-old wild-type, npr1, ssi2, ssi2 npr1, ssi2 npr1 nahG, sfd1-1 ssi2 npr1, sfd4 ssi2 npr1, and sfd2-1 ssi2 npr1 plants 48 h after treatment with 5 μM MeJA dissolved in 0.1% ethanol. All RNAs were resolved on denaturing gels, transferred to Nytran Plus membranes (Schleicher & Schuell), and probed for the indicated genes. Gel loading was monitored by photographing the ethidium bromide–stained gel (EtBr) before transferring the RNA to a Nytran Plus membrane.

Figure 4.

Growth of Pathogen in the sfd Mutants. (A) Bacterial numbers in the sfd mutants. Psm (OD₆₀₀ = 0.0002) was infiltrated into the abaxial surfaces of leaves of wild-type (WT), npr1, ssi2, ssi2 npr1, sfd1-1 ssi2 npr1, sfd1-2 ssi2 npr1, sfd2-1 ssi2 npr1, sfd2-2 ssi2 npr1, sfd2-3 ssi2 npr1, and sfd4 ssi2 npr1 plants with a needleless syringe. Leaf discs were harvested from the inoculated leaves at 3 days after inoculation, weighed, and ground in 10 mM MgCl₂, and the bacterial numbers were titered. Each sample contained five leaf discs. The bacterial numbers, presented as colony-forming units (CFU) per mg of leaf tissue, represent the average of five samples ± sd. (B) B. cinerea disease ratings in the sfd mutants. Leaves of 4-week-old soil-grown wild-type, npr1, ssi2, ssi2 npr1, sfd1-1 ssi2 npr1, sfd1-2 ssi2 npr1, sfd2-1 ssi2 npr1, sfd2-2 ssi2 npr1, and sfd4 ssi2 npr1 plants were inoculated with spores of B. cinerea. Four days later, plants were scored for the extent of spreading necrosis. Leaves from each line were grouped based on the extent of necrosis. A four-step grading system was used. Leaves with <25%, 25 to 50%, 50 to 75%, and 75 to 100% of leaf area exhibiting necrosis were given scores of 0.25, 0.5, 0.75, and 1, respectively. The leaves in each category were counted, multiplied by the score, and divided by the total number of leaves inoculated (given next to each bar) to give an infection rating for each line. The “relative infection rating” for each line was calculated as the ratio of the infection rating for the line to the infection rating of the wild type.

Figure 5.

Lipid Profiles of the sfd Mutants. (A) Electrospray ionization–tandem mass spectrometry-generated profiles of the plastidic glycerolipids PG, DGDG, and MGDG in wild-type (WT), npr1, ssi2, ssi2 npr1, sfd1-1 ssi2 npr1, sfd2-1 ssi2 npr1, and sfd4 ssi2 npr1 plants. (B) Electrospray ionization–tandem mass spectrometry-generated profiles of the extraplastidic glycerolipids PC, PE, and PI in wild-type (WT), npr1, ssi2, ssi2 npr1, sfd1-1 ssi2 npr1, sfd2-1 ssi2 npr1, and sfd4 ssi2 npr1 plants.

Figure 5.

Lipid Profiles of the sfd Mutants. (A) Electrospray ionization–tandem mass spectrometry-generated profiles of the plastidic glycerolipids PG, DGDG, and MGDG in wild-type (WT), npr1, ssi2, ssi2 npr1, sfd1-1 ssi2 npr1, sfd2-1 ssi2 npr1, and sfd4 ssi2 npr1 plants. (B) Electrospray ionization–tandem mass spectrometry-generated profiles of the extraplastidic glycerolipids PC, PE, and PI in wild-type (WT), npr1, ssi2, ssi2 npr1, sfd1-1 ssi2 npr1, sfd2-1 ssi2 npr1, and sfd4 ssi2 npr1 plants.

Figure 6.

sfd4 Contains a Mutation in the FAD6 Gene. (A) Alignment of amino acid sequences in a conserved region of plastidic ω6 desaturases from Arabidopsis thaliana (A.t.), rape (Brassica napus; B.n.), soybean (Glycine max; G.m.), spinach (Spinacia oleracea; S.o.), and the green alga Chlamydomonas sp W80 (C.w.). The Ser-133 that is mutated to Phe-133 in sfd4 is shown in boldface. Numbers at left and right indicate amino acid positions. (B) EcoRV restriction polymorphism generated by a C→T mutation at the FAD6 locus in the sfd4 ssi2 npr1 plant. PCR-amplified products from the wild type (WT) and sfd4 ssi2 npr1 were digested with EcoRV and resolved on an agarose gel. The size of each band in base pairs is listed at left. (C) Comparison of PR1 expression in the leaves of 4-week-old soil-grown ssi2 plants that are homozygous for the wild-type FAD6 allele (ssi2), heterozygous for the fad6-1 mutant allele (ssi2 fad6/+), and homozygous for the fad6-1 mutant allele (ssi2 fad6). All RNAs were resolved on denaturing gels, transferred to Nytran Plus membranes, and probed for the indicated genes. Gel loading was monitored by photographing the ethidium bromide–stained gel (EtBr) before transferring the RNA to a Nytran Plus membrane. (D) Morphological phenotype of 4-week-old soil-grown ssi2 plants that are homozygous for the wild-type FAD6 allele (ssi2), heterozygous for the fad6-1 mutant allele (ssi2 fad6/+), and homozygous for the fad6-1 mutant allele (ssi2 fad6). All plants were photographed from the same distance. (E) Light microscopy of trypan blue–stained leaves of 4-week-old soil-grown ssi2 plants that are homozygous for the wild-type FAD6 allele (ssi2), heterozygous for the fad6-1 mutant allele (ssi2 fad6/+), and homozygous for the fad6-1 mutant allele (ssi2 fad6). All photographs were taken at the same magnification.

Figure 7.

Working Model of the Interplay of ssi2, sfd1, sfd2, and sfd4 in Defense Signaling in Arabidopsis. This model is a refinement of Figure 1. The sfd1, sfd2, and sfd4 mutant alleles suppress (−) the ssi2-conferred dwarfing, spontaneous development of lesions containing dead cells, NPR1-independent expression of PR1, and enhanced resistance to Psm. In addition, sfd1 and sfd4 also suppress the ssi2-conferred accumulation of high SA levels. However, SA application is ineffective in restoring PR1 expression in sfd1 ssi2 npr1, sfd2 ssi2 npr1, and sfd4 ssi2 npr1 plants, implicating the involvement of another ssi2-contributed factor in the activation of the NPR1-independent pathway leading to the expression of PR1 and enhanced resistance to Psm. The absence of SA accumulation in the ssi2 nahG and ssi2 eds5 plants does not ameliorate the ssi2-conferred cell death phenotype, suggesting that high levels of SA do not have a causal role in the cell death phenotype. The sfd2 ssi2 npr1 plants accumulate increased SA levels despite the lack of spontaneous cell death, suggesting that cell death is not the primary factor that promotes SA accumulation in the ssi2 mutant. Hence, ssi2-conferred cell death and SA accumulation are shown to be independent of each other. The sfd1 mutant alleles restore JA-inducible PDF1.2 expression in sfd1 ssi2 npr1 plants. sfd1 is shown to impinge on a step that is common to the activation of cell death, SA accumulation, the activation of the NPR1-independent defense pathway, and the repression of JA signaling in the ssi2 mutant. However, because sfd1 does not restore resistance to B. cinerea, despite restoring PDF1.2 expression in the sfd1 ssi2 npr1 plants, an additional ssi2-modulated mechanism is shown to suppress (−) the defense against B. cinerea. sfd4 does not restore JA-inducible PDF1.2 expression in sfd4 ssi2 npr1. Hence, sfd4 is shown to suppress (−) a step that is common to the activation of cell death and dwarfing, SA accumulation, and the activation of the NPR1-independent defense pathway. sfd2 is shown to interfere with (−) a step common to the activation of ssi2-conferred cell death and the activation of NPR1-independent signaling.