The Mechanistic Underpinnings of an ago1-Mediated, Environmentally Dependent, and Stochastic Phenotype

Abstract

The crucial role of microRNAs in plant development is exceedingly well supported; their importance in environmental robustness is studied in less detail. Here, we describe a novel, environmentally dependent phenotype in hypomorphic argonaute1 (ago1) mutants and uncover its mechanistic underpinnings in Arabidopsis (Arabidopsis thaliana). AGO1 is a key player in microRNA-mediated gene regulation. We observed transparent lesions on embryonic leaves of ago1 mutant seedlings. These lesions increased in frequency in full-spectrum light. Notably, the lesion phenotype was most environmentally responsive in ago1-27 mutants. This allele is thought to primarily affect translational repression, which has been linked with the response to environmental perturbation. Using several lines of evidence, we found that these lesions represent dead and dying tissues due to an aberrant hypersensitive response. Although all three canonical defense hormone pathways (salicylic acid, jasmonate, and jasmonate/ethylene pathways) were up-regulated in ago1 mutants, we demonstrate that jasmonate perception drives the lesion phenotype. Double mutants of ago1 and coronatine insensitive1, the jasmonate receptor, showed greatly decreased frequency of affected seedlings. The chaperone HEAT SHOCK PROTEIN 90 (HSP90), which maintains phenotypic robustness in the face of environmental perturbations, is known to facilitate AGO1 function. HSP90 perturbation has been shown previously to up-regulate jasmonate signaling and to increase plant resistance to herbivory. Although single HSP90 mutants showed subtly elevated levels of lesions, double mutant analysis disagreed with a simple epistatic model for HSP90 and AGO1 interaction; rather, both appeared to act nonadditively in producing lesions. In summary, our study identifies AGO1 as a major, largely HSP90-independent, factor in providing environmental robustness to plants.

Figure 1.

Hypomorphic ago1 mutants showed increased frequency of seemingly stochastic lesions. A, Experimental setup. When grown in yellow, filtered light conditions for 10 d, few ago1 seedlings showed lesions on cotyledons. When grown in full-spectrum light, many more ago1 seedlings developed lesions. B, In full-spectrum light, ago1-46, ago1-27, and ago1-25 mutants showed a significantly increased frequency of affected seedlings compared with the wild type. ago1-27 and ago1-46 mutants showed significantly more affected individuals in unfiltered versus filtered light conditions; a similar, albeit nonsignificant, tendency was observed for seedlings carrying the ago1-25 allele. Error bars indicate se across at least three replicates with n = 25 to 72 seedlings per replicate. Statistical significance was calculated using the χ² test of significance (**, P < 1 × 10⁻⁴ and *, P < 0.05; for all possible P values, see Supplemental Table S1). n.s., Not significant. C to E, Lesions in wild-type and ago1-27 mutant seedlings show distinct morphology. C, Full-spectrum light-grown, 10-d-old, unaffected ago1-27 seedling. D, Full-spectrum light-grown, 10-d-old, affected ago1-27 seedling with lesions denoted by arrows. E, Full-spectrum light-grown, 10-d-old, affected wild-type seedling with lesion denoted by the arrow. Bars = 1 mm. F to K, Scanning electron micrographs of 10-d-old cotyledons grown in full-spectrum light. F, Unaffected wild-type cotyledon. G, Wild-type cotyledon with lesion denoted by the arrow. H, Magnified view of the lesion from G. I, Unaffected ago1-27 cotyledon. J, ago1-27 cotyledon with lesion denoted by the arrow. K, Magnified view of the lesion from I. Bars = 1 mm (F, G, I, and J) and 500 μm (H and K).

Figure 2.

Vital staining of unaffected and affected tissue from wild-type and ago1-27 cotyledons. A and G, Unaffected wild-type cotyledons prior to staining. B, Unaffected wild-type cotyledon after Trypan Blue staining showed little dye accumulation. H, Unaffected wild-type cotyledon after DAB staining showed little dye polymerization. C and I, Unaffected ago1-27 cotyledons prior to staining. D, Unaffected ago1-27 cotyledon after Trypan Blue staining showed some dye accumulation. J, Unaffected ago1-27 cotyledon after DAB staining showed no dye polymerization. E and K, Affected ago1-27 cotyledons prior to staining. F, Affected ago1-27 cotyledon after Trypan Blue staining showed strong accumulation of dye in lesions (arrows). L, Affected ago1-27 cotyledon after DAB staining showed strong dye polymerization in lesions (arrows). Bars = 1 mm.

Figure 3.

Marker genes of all three canonical defense hormone pathways were up-regulated in ago1 cotyledons. PR1 (SA responsive; A), VSP1 (JA responsive; B), and PDF1.2 (JA/ET responsive; C) expression were dramatically up-regulated in 10-d-old ago1-46, ago1-27, and ago1-25 cotyledons in full-spectrum light. Expression was measured by quantitative reverse transcription (RT)-PCR; all assayed genes were normalized to UBC21 (UBIQUITIN-CONJUGATING ENZYME 21) expression. Error bars indicate se across at least two replicates of cotyledons harvested from 18 pooled seedlings.

Figure 4.

Genes in all three defense hormone pathways, including miRNA target genes, were up-regulated in ago1-27 cotyledons. A, Top, pathway diagram indicates the pathway connection of three tested SA genes, none of which are miRNA targets. Bottom, NPR1, EDS1, and SNC1 were up-regulated in ago1-27 seedlings relative to the wild type. B, Top, pathway diagram indicates the pathway connection of four tested JA genes, the most upstream of which, TCP4, is an miRNA target gene. Bottom, the canonical jasmonate signaling and biosynthesis genes TCP4, LOX2, MYC2, VSP2, and JAR1 were strongly up-regulated. C, Top, pathway diagram indicates the pathway connection of four tested JA/ET genes. The most upstream gene, EIN2, acts in an incoherent feed-forward loop with mir164ab and its target gene ORE1. ET and JA pathways show cross-regulation via ORA59 and MYC2. Bottom, the JA/ET markers ORE1, ORA59, and ERF1 were significantly up-regulated in ago1-27 cotyledons relative to the wild type; in contrast, EIN2 was significantly down-regulated in ago1-27 cotyledons. All assayed genes are normalized to UBC21 (UBIQUITIN-CONJUGATING ENZYME 21) expression. Error bars indicate se across three to four replicates of cotyledons harvested from 18 pooled seedlings. Black asterisks represent significant up-regulation in ago1-27 cotyledons; the gray asterisk (EIN2) represents significant down-regulation in ago1-27 cotyledons.

Figure 5.

Methyl jasmonate (MeJA) treatment was sufficient to induce lesions. A, Under high-SA conditions, fewer seedlings were affected for both the wild type and ago1-27 in full-spectrum light. B, At 10 μm MeJA, ago1-27 was significantly more responsive compared with the wild type (GLMM, P < 1 × 10⁻⁴), whereas at the 50 μm MeJA treatment, the wild type was more responsive (GLMM, P < 1 × 10⁻⁴). C, At 1 μm ACC (ET precursor molecule), ago1-27 was more responsive than the wild type (GLMM, P < 1 × 10⁻⁴), reaching a frequency of affected seedlings similar to ago1-27 seedlings at 10 μm MeJA. At higher doses, ACC repressed the lesion phenotype in ago1-27 (GLMM, P < 1 × 10⁻⁴) and had no further effect on the wild type. D, Combining 10 μm MeJA with varying ACC concentrations yielded no significant change, albeit the frequencies of affected seedlings declined. n = 72 to 144 individuals per replicate, with two to five replicates each. The difference in responsiveness between genotypes was modeled using GLMM, testing for interaction between genotype and treatment, with replicate and number of seedlings set as random variables (*, P < 1 × 10⁻⁴; for all possible P values, see Supplemental Table S4).

Figure 6.

Disruption of JA perception suppresses lesion phenotype. coi1-1 mutant seedlings showed significantly fewer affected seedlings than the wild type (P < 0.05). ago1-27;coi1-1 double mutant seedlings showed dramatically fewer affected seedlings than ago1-27 single mutant seedlings (P < 1 × 10⁻³); however, the frequency of affected seedlings was significantly higher than in the wild type (P < 0.05). Statistical significance was calculated using the χ² test of significance (**, P < 1 × 10⁻³ and *, P < 0.05; for all reported P values, see Supplemental Table S4). n = 72 to 144 individuals per replicate, with four replicates each. Error bars represent se across biological replicates.

Figure 7.

AGO1 and HSP90 interact genetically. A, HSP90 RNAi-A1 seedlings showed significantly more affected seedlings than the wild type (P < 0.05). ago1-27;HSP90 RNAi-A1 double mutant seedlings showed significantly more affected seedlings than ago1-27 single mutant seedlings (P < 1 × 10⁻⁴). Error bars represent se across two replicates of 108 seedlings. B, The ago1-27 mutation showed higher penetrance in the presence of HSP90 RNAi-A1 (ago1-27; A1) in full-spectrum light (GLMM, P < 1 × 10⁻⁴). WT, Wild type. Statistical significance was calculated using the χ² test of significance (***, P < 1 × 10⁻⁴; **, P < 1 × 10⁻³; and *, P < 0.05; for all possible P values, see Supplemental Table S4). n = 72 to 144 individuals per replicate, with two replicates each. Error bars represent se across biological replicates.