Loss of the Acetyltransferase NAA50 Induces Endoplasmic Reticulum Stress and Immune Responses and Suppresses Growth

Abstract

Stress signaling in plants is carefully regulated to ensure proper development and reproductive fitness. Overactive defense signaling can result in dwarfism as well as developmental defects. In addition to requiring a substantial amount of energy, plant stress responses place a burden upon the cellular machinery, which can result in the accumulation of misfolded proteins and endoplasmic reticulum (ER) stress. Negative regulators of stress signaling, such as ENHANCED DISEASE RESISTANCE 1 (EDR1), ensure that stress responses are properly suspended when they are not needed, thereby conserving energy for growth and development. Here, we describe the role of an uncharacterized N-terminal acetyltransferase, NAA50, in the regulation of plant development and stress responses in Arabidopsis (Arabidopsis thaliana). Our results demonstrate that NAA50, an interactor of EDR1, plays an important role in regulating the tradeoff between plant growth and defense. Plants lacking NAA50 display severe developmental defects as well as induced stress responses. Reduction of NAA50 expression results in arrested stem and root growth as well as senescence. Furthermore, our results demonstrate that the loss of NAA50 results in constitutive ER stress signaling, indicating that NAA50 may be required for the suppression of ER stress. This work establishes NAA50 as essential for plant development and the suppression of stress responses, potentially through the regulation of ER stress.

Figure 1.

NAA50 physically interacts with EDR1. A, Naa50 is conserved in Arabidopsis. Amino acid alignment depicting Arabidopsis NAA50 and human Naa50. This alignment was generated using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) and visualized in Jalview (Waterhouse et al., 2009). Individual residues are colored based upon the Clustal color scheme, which assigns color to residues where amino acid category is conserved. B, EDR1^ST interacts with NAA50 in yeast two-hybrid. AD, GAL4 activation domain fusion; BD, GAL4 DNA binding domain fusion; LAM, lamin; T, SV40 large T antigen. C, Immunoblot analysis of yeast strains from B. EDR1-BD accumulated poorly in yeast, and a significant accumulation of degraded EDR1-BD (*) was visible. D, NAA50 coimmunoprecipitates with EDR1. The indicated constructs were transiently expressed in N. benthamiana and then immunoprecipitated using GFP-Trap beads. sYFP-tagged MYC was used as a negative control. E, NAA50 colocalizes with the ER marker SDF2. mCherry-tagged NAA50 and GFP-tagged SDF2 were transiently coexpressed in N. benthamiana. Bars = 50 μm. These experiments were repeated three times with similar results.

Figure 2.

NAA50 is required for plant development. A, Loss of NAA50 results in dwarfed seedlings. Representative 7-d-old, MS-grown seedlings are depicted. B, NAA50-sYFP complements naa50-mediated dwarfism. Four-week-old adult plants are shown. NP, Native NAA50 promoter (includes the 297 nucleotides upstream of the NAA50 start site). C, naa50 plants can develop stems and flowers. A 5-week-old naa50-2 plant is shown. WT, wild type.

Figure 3.

Loss of NAA50 results in developmental changes. A, naa50 seedlings have altered root morphology. The seedling roots depicted are from 1-week-old seedlings. B, Vacuole and cell morphology are altered in naa50 seedling roots. Shown are fluorescence micrographs taken of 7-d-old wild type and naa50-1 seedlings expressing mCherry-tagged γTIP. Scale bars = 50 μm. C, Dexamethasone (DEX) treatment induces knockdown of NAA50 in DEX:NAA50-ami plants. qRT PCR was performed on cDNA generated from multiple adult DEX:NAA50-ami plants following dexamethasone treatment. Displayed are the averages of three replicates with error bars indicating SD. Asterisk denotes P < 0.05 (by Student’s t test). Expression values were normalized to ACTIN2. This experiment was repeated three independent times with similar results. D, NAA50 knockdown induces changes to root cell morphology. Five-day-old seedlings were transferred from MS plates to MS plates supplemented with DEX. Images were taken 3 d after dexamethasone exposure. E, NAA50 knockdown slows root elongation. Seven-day-old seedlings were transferred to MS plates supplemented with ethanol or dexamethasone. Images were taken 3 d after transfer to ethanol- or DEX-supplemented media. F, NAA50 knockdown induces stem bending. Images were taken 24 h after dexamethasone treatment. Numbers indicate proportion of all stems which displayed the given morphology. G, NAA50 knockdown stalls stem growth. Stem measurements were taken on DEX:Scrambled-ami (n = 8) and DEX:NAA50-ami (n = 10) immediately before and 6 d after dexamethasone treatment. No stem growth was detected in DEX:NAA50-ami plants subsequent to dexamethasone application. Error bars indicate SD. H, Removal of the apical meristem inhibits NAA50 knockdown-mediated stem bending. Adult DEX:NAA50-ami plants were sprayed with dexamethasone, and images were taken 24 h later. The shoot apical meristem was removed immediately before dexamethasone treatment.

Figure 4.

Loss of NAA50 induces cell death and senescence. A, NAA50 knockdown induces senescence in adult leaves. Four-week-old plants were sprayed with dexamethasone. Images were taken immediately before and 7 d after treatment. B, NAA50 knockdown induces senescence in seedlings. Seedlings were grown on MS plates for 7 d, and then transferred to MS plates supplemented with ethanol or dexamethasone. Images were taken 7 d after transfer to ethanol- or dexamethasone-supplemented media. C, naa50 seedling roots contain dead cells. Seven-day-old seedlings were stained with trypan blue dye. D, Cell death staining in naa50 roots is spotty and irregular. Images depict trypan blue-stained roots from 7-d-old seedlings. E, Loss of EDR1 does not alter senescence in NAA50 knockdown plants. Images were taken of 4-week-old plants immediately before and 7 d after dexamethasone treatment. WT, wild type.

Figure 5.

Reduction in NAA50 expression induces changes to growth and defense signaling. A, Reducing NAA50 expression results in a down-regulation of growth signaling, and an up-regulation of defense signaling. Significantly altered transcripts were identified by comparing expression levels in dexamethasone-treated DEX:NAA50-amiRNA plants at the given time point to Scrambled-amiRNA plants at the same time point. Furthermore, expression levels in DEX:NAA50-amiRNA plants at 12 and 24 h were compared with NAA50-amiRNA plants at 0 h after DEX treatment. To eliminate circadian effects, all samples were harvested at the same time (12 and 24 h samples were sprayed with dexamethasone 12 and 24 h before harvest). GO term enrichment analysis was performed using the BiNGO application to determine whether the DEX:NAA50-amiRNA transcriptome was enriched for specific biological processes. NS, not statistically significant. B, The DEX:NAA50-amiRNA transcriptome bears similarity to biotic and abiotic stress studies. The 330 most significantly altered transcripts were compared were previous studies using the Genevestigator Signature tool. The five most related transcriptomes based on the calculated Relative Similarity scores are shown. A heatmap was generated using Heatmapper (http://www2.heatmapper.ca/expression/) to display the relative log₂ fold-change for each of the 330 transcripts for each study. These 330 transcripts were selected based on log₂ fold-change when comparing expression levels in the DEX:NAA50-amiRNA 12-h dataset to the DEX:NAA50-amiRNA time 0 dataset; all 330 were also significantly different when comparing the DEX:NAA50-amiRNA 12-h dataset to the Scrambled-amiRNA 12-h dataset.

Figure 6.

Loss of EDR1 and NAA50 result in changes to ER stress signaling. A, edr1-1 mutants display heightened ER stress sensitivity. Leaves from 6-week-old plants were infiltrated with various concentrations of TM using a needleless syringe. Leaves were removed, and images taken 3 d after injection. B, ER stress induces naa50-like root dwarfism. Seedlings were germinated on MS plates or MS supplemented with TM or DTT. Representative 10-d-old seedlings are shown. C, ER stress induces naa50-like root cell morphology. Roots of 10-d-old seedlings are depicted. Seedlings were grown on regular MS plates or MS plates supplemented with TM or DTT. D, ER stress induces cell death in roots. Ten-day-old seedlings were stained with trypan blue after growth on MS or MS supplemented with TM or DTT. E, naa50-1 seedlings display heightened ER stress signaling in the absence of TM treatment. qRT-PCR was performed on cDNA generated from wildtype and naa50-1 seedlings. Seedlings were germinated on MS plates and 5 d later transferred to regular MS or MS supplemented with 1 μg/mL TM. RNA was collected 20 h after transfer to new plates. Gene expression values were normalized to ACTIN2. Values depict the averages of three biological replicates, each consisting of twenty individual seedlings. Error bars represent SD between three independent biological replicates. Asterisks denotes P-value < 0.05 (by Student’s t test). F, bZIP60 splicing is induced in naa50-1 seedlings. RT-PCR was performed on the same cDNA used in E. Each lane represents a unique biological replicate derived from 20 seedlings. WT, wild type.

Figure 7.

NAA50 enzymatic activity is required for plant development. A, Recombinant NAA50 displays auto-acetylation activity in vitro. Recombinant HIS-tagged NAA50 was expressed and purified from E. coli. In vitro reactions were performed at 30°C for the indicated time points. Samples were then boiled and subjected to gel electrophoresis and immunoblotting using an antiacetyl-Lys antibody. This experiment was repeated three times with similar results. This figure was generated from one immunoblot derived from a single experiment with irrelevant lanes removed. B, NAA50 colocalizes with NAA10. sYFP-tagged NAA50 was transiently coexpressed with mCherry-tagged NAA10 in N. benthamiana. Bars = 50 μm. C, Immunoblotting demonstrates that HA-tagged NAA50 mutant transgenes are expressed in transgenic plants. Leaf tissue from hygromycin-resistant T3 plants was subjected to gel electrophoresis and immunoblotting using an anti-HA antibody. D, Mutant NAA50 transgenes do not complement naa50 root dwarfism. Representative 10-d-old seedlings are depicted. E, Mutant NAA50 transgenes do not complement naa50 root cell morphology defects. Images were taken of representative 10-d-old seedlings. F, NAA50^I145A can complement naa50-mediated rosette dwarfism. The image depicts representative 6-week-old plants. G, NAA50^I145A and NAA50^Y34A do not complement naa50-mediated sterility. Stems were removed from 7-week-old plants. WT, wild type.