Involvement of Arabidopsis RACK1 in protein translation and its regulation by abscisic acid

Abstract

Earlier studies have shown that RACK1 functions as a negative regulator of abscisic acid (ABA) responses in Arabidopsis (Arabidopsis thaliana), but the molecular mechanism of the action of RACK1 in these processes remains elusive. Global gene expression profiling revealed that approximately 40% of the genes affected by ABA treatment were affected in a similar manner by the rack1 mutation, supporting the view that RACK1 is an important regulator of ABA responses. On the other hand, coexpression analysis revealed that more than 80% of the genes coexpressed with RACK1 encode ribosome proteins, implying a close relationship between RACK1's function and the ribosome complex. These results implied that the regulatory role for RACK1 in ABA responses may be partially due to its putative function in protein translation, which is one of the major cellular processes that mammalian and Saccharomyces cerevisiae RACK1 is involved in. Consistently, all three Arabidopsis RACK1 homologous genes, namely RACK1A, RACK1B, and RACK1C, complemented the growth defects of the S. cerevisiae cross pathway control2/rack1 mutant. In addition, RACK1 physically interacts with Arabidopsis Eukaryotic Initiation Factor6 (eIF6), whose mammalian homolog is a key regulator of 80S ribosome assembly. Moreover, rack1 mutants displayed hypersensitivity to anisomycin, an inhibitor of protein translation, and displayed characteristics of impaired 80S functional ribosome assembly and 60S ribosomal subunit biogenesis in a ribosome profiling assay. Gene expression analysis revealed that ABA inhibits the expression of both RACK1 and eIF6. Taken together, these results suggest that RACK1 may be required for normal production of 60S and 80S ribosomes and that its action in these processes may be regulated by ABA.

Figure 1.

Analysis of DNA microarray data. A, A Venn diagram shows the number of genes that are co-up-regulated 2.0-fold or more by 50 μm ABA treatment and by rack1 mutation. The number of genes that were coregulated by ABA treatment and rack1 mutation appears in the overlapped portion of the circles, and the number of genes that were not coregulated appears in the nonoverlapping portions for the 2-fold up-regulated genes. B, A Venn diagram shows the number of genes that are co-down-regulated 2.0-fold or more by 50 μm ABA treatment and by rack1 mutation. C, Scatterplot shows the correlation of the genes that were regulated by ABA treatment and of genes that were regulated by rack1a rack1b mutation. The calculated Pearson correlation coefficient was 0.494, indicating a moderate correlation level. [See online article for color version of this figure.]

Figure 2.

Functional categorization of genes that were up-regulated 2-fold or more in the rack1a rack1b mutant background treated with ABA compared with Col treated with ABA. Functional categorization of genes was obtained through The Arabidopsis Information Resource Gene Ontology (GO) Annotations tool (http://www.arabidopsis.org/tools/bulk/go/index.jsp).

Figure 3.

Complementation assay for failed pseudohyphal growth in the diploid S. cerevisiae cpc2 mutant using three Arabidopsis RACK1 genes. Transformants were patched on nitrogen starvation plates and grown for 5 d before photographs were taken. A, RH2656 (wild type [WT]) + p424MET25 (empty vector). B, RH3246 (cpc2) + p424MET25 (empty vector). C, RH3246 (cpc2) + p424MET25-CPC2. D, RH3246 (cpc2) + p424MET25-RACK1A. E, RH3246 (cpc2) + p424MET25-RACK1B. F, RH3246 (cpc2) + p424MET25-RACK1C.

Figure 4.

Physical interaction between RACK1 and eIF6 detected in yeast two-hybrid assays and in the BiFC system. A, Interactions between RACK1s and eIF6s in the yeast two-hybrid assay. eIF6 genes were cloned into pDEST32 and RACK1 genes were cloned into pDEST22. The interaction between eIF6 and the empty prey vector (EV) was used as a negative control. The ability of yeast cells to grow on synthetic medium lacking Leu, Trp, and His and containing 10 mm 3-amino-1,2,4-triazole is scored as a positive interaction. B, Interactions between RACK1 and eIF6 in BiFC. RACK1 proteins were fused with the N-terminal half of YFP and eIF6 proteins were fused with C-terminal half of YFP. CHE/DIC, Overlay of mCherry images and differential interference contrast images of the same field. The interaction between AtOFP1 (Wang et al., 2007) and RACK1/eIF6 proteins was used as a negative control. The HY5-mCherry is included in each transfection to serve as a control for successful transfection as well as for nuclear localization. Image shown are the same transformants photographed under YFP fluorescence and differential interference contrast microscopic setups. Images were pseudocolored with ImageJ for easy visualization.

Figure 5.

Arabidopsis eIF6 homologs. A, RT-PCR assay for the expression of eIF6 genes in different Arabidopsis tissues and organs. PCR was performed with 30 cycles. B, In silico analysis of the relative transcript levels of eIF6A (At3g55620) and eIF6B (At2g39820) in various tissue and organs in Arabidopsis. Data were imported from the Genevestigator Arabidopsis microarray database (https://www.genevestigator.com/gv/index.jsp; Zimmermann et al., 2004).

Figure 6.

eif6 mutant alleles. A, T-DNA insertional mutant alleles of eIF6A and eIF6B in Arabidopsis. The exons are depicted by boxes, and the introns and intergenic regions are depicted by lines. The T-DNA insertion sites are drawn as triangles (not to scale). LB, T-DNA left border. B, RT-PCR analysis of eif6b-1 and eif6b-2 alleles. C, The eif6a mutants are embryo lethal. Each pair of images is representative of the green seeds (top) and white seeds (bottom) from the same silique. WT, Wild type. D, Three-week-old eif6b-1 and eif6b-2 mutant plants grown under a 14-h/10-h photoperiod.

Figure 7.

The synergistic effect of anisomycin treatment and rack1 mutation on Arabidopsis seedling root growth. A, Root growth of rack1 single mutants in the presence of 10 μm anisomycin. B, Root growth of rack1 double mutants in the presence of 5 μm anisomycin. C, The primary root length of Col and the rack1 mutants in the absence of anisomycin treatment. The experiments were repeated three times, and the same data trends were obtained. Data from one experiment are presented here with the se (n = 20) indicated on the top of each column. Asterisks indicate significant differences from Col using Student’s t test (P < 0.05).

Figure 8.

Ribosome profiling of the rack1a rack1b mutant and ABA-treated Arabidopsis seedlings. A, Overlay of the ribosome profiles of Col and the rack1a rack1b mutant (rack1ab) without ABA treatment. B, Overlay of the ribosome profiles of Col with or without ABA treatment. C, Overlay of the ribosome profiling of Col and the rack1a rack1b mutant after ABA treatment. The positions of 40S ribosomal subunits, 60S ribosomal subunits, and 80S ribosomes were located based on the A₂₆₀ peaks and are indicated with arrows. Profiles are averages of four independent experiments with se indicated by error bars. Asterisks indicate significant differences using paired t tests (P < 0.05). Shown is Suc density gradient analysis of polysomes extracted from 4.5-d-old Col seedlings with or without 50 μm ABA treatment for 8 h. [See online article for color version of this figure.]

Figure 9.

The regulation of RACK1 and eIF6 expression by ABA. A, Quantitative RT-PCR analysis of RACK1 and eIF6 gene expression. The transcript levels of RACK1 and eIF6A genes were normalized against the transcript level of ACTIN2 for each sample. Total RNA was extracted from 4.5-d-old Arabidopsis seedlings and used for quantitative RT-PCR analysis. Shown are averages of three biological replicates ± se. B, Promoter::GUS assay. Seedlings at 4.5 d old were incubated in one-half-strength MS liquid medium with or without 50 μm ABA for 6 h and then subjected to GUS staining.