A Role for the F-Box Protein HAWAIIAN SKIRT in Plant microRNA Function

Abstract

As regulators of gene expression in multicellular organisms, microRNAs (miRNAs) are crucial for growth and development. Although a plethora of factors involved in their biogenesis and action in Arabidopsis (Arabidopsis thaliana) has been described, these processes and their fine-tuning are not fully understood. Here, we used plants expressing an artificial miRNA target mimic (MIM) to screen for negative regulators of miR156. We identified a new mutant allele of the F-box gene HAWAIIAN SKIRT (HWS; At3G61590), hws-5, as a suppressor of the MIM156-induced developmental and molecular phenotypes. In hws plants, levels of some endogenous miRNAs are increased and their mRNA targets decreased. Plants constitutively expressing full-length HWS-but not a truncated version lacking the F-box domain-display morphological and molecular phenotypes resembling those of mutants defective in miRNA biogenesis and activity. In combination with such mutants, hws loses its delayed floral organ abscission ("skirt") phenotype, suggesting epistasis. Also, the hws transcriptome profile partially resembles those of well-known miRNA mutants hyl1-2, se-3, and ago1-27, pointing to a role in a common pathway. We thus propose HWS as a novel, F-box dependent factor involved in miRNA function.

Figure 1.

Characterization of the hws-5 mutant. A, Phenotype of 21-d-old Col-0, MIM156, hws-5; MIM156 and hws-5; and MIM156 + gHWS plants. B, Chromosome 3 SHOREmap results for hws-5. C, Location and effects of the mutations on the HWS protein. Annotated or predicted domains are marked in blue. D, Sepal-fusion skirt phenotype and phyllotactic distortion in hws-5 compared to Col-0.

Figure 2.

Effects of hws mutation on MIM transgene-dependent and -independent phenotypes. A, Fraction of differentially expressed genes in 35S::HWS, hws-1, hws-5, hws-5; MIM156 (compared to Col-0 and to MIM156), MIM156 (n = 2), miRNA-mutants cpl1-7, hyl1-2, and se-3 (n = 3; data from Manavella et al., 2012) and ago1-27 (n = 3, data from SRA PRJNA309714) with 95% confidence intervals. For P values, see Supplemental Table S4. B, Suppression of MIM159, MIM319, and MIM164 phenotypes in hws-1 background. C, miR156-resistant rSPL9 and rSPL9; hws-5 plants. D, Relative expression of IPS and PHO2 in Col-0, hws-5, and hws-1 plants harboring 35S::HWS. E, Relative expression of MIM156, miR156, SPL9 in Col-0, MIM156, and hws-5; MIM156 and of MIM164, miR164 and CUC2 in Col-0, MIM164, and hws-1; MIM164. Dots represent biological replicates as means of technical replicates (n = 3); bars indicate means of biological replicates.

Figure 3.

Effects of hws and HWS overexpression on miRNA and miRNA target steady-state levels. A, Phenotype of 28-d-old T1 35S::HWS plant in the hws-1 background. Note hyponastic, serrated leaves. B and C, Levels of mature miRNAs and miRNA targets in Col-0, hws-1, and 35S::HWS as measured by RT-qPCR. Dots represent biological replicates (n = 3 to 4); bars indicate mean of biological replicates as means of technical replicates. D, Mature miRNA levels in Col-0, hws-1, hws-5, MIM156, hws-5; MIM156 and 35S::HWS as determined by RNA blotting.

Figure 4.

Epistasis analysis of hws-1 and mutations in major miRNA biogenesis factors. A, Rosette phenotype of single and double homozygous F₃ plants between hws-1, hyl1-2, se-3, ago1-25, ago1-27, hst-3, and abh1-753 at approximately 21 DAS. B, Abscission phenotype of double mutants of hws-1, hws-5, hyl1-2, hst-3, se-3, and ago1-27 as well as T1 of 35S::HWS and 35S::mHWS in hws-1 background. C, Odds ratio of enrichment of differentially expressed genes also found in the miRNA-mutants hyl1-2, se-3, cpl1-7, and ago1-27 as calculated by two-tailed Fisher’s exact test in 35S::HWS, hws-1, hws-5, hws-5; MIM156 (compared to Col-0 and to MIM156; n = 2), MIM156 and miRNA-mutants cpl1-7, hyl1-2, and se-3 (n = 3; data from Manavella et al., 2012) and ago1-27 (n = 3, data from SRA; PRJNA309714), with 95% confidence intervals. For P values, see Supplemental Table S2.

Figure 5.

Transcriptome effects of hws. A, Odds ratio of enrichment of differentially expressed genes in the validated miRNA target gene subset as calculated by two-tailed Fisher’s exact test in 35S::HWS, hws-1, hws-5, hws-5; MIM156 (compared to Col-0 and to MIM156; n = 2), MIM156 and miRNA-mutants cpl1-7, hyl1-2, and se-3 (n = 3; data from Manavella et al., 2012) and ago1-27 (n = 3, data from SRA PRJNA309714), with 95% confidence intervals. For P values, see Supplemental Table S2; for gene list, see Supplemental Table S6. B, Venn diagram of differentially expressed genes in the hws mutants combined compared to MIM156 and hws-5; MIM156.