The dual‐action evolutionarily conserved NatB catalytic subunit NAA20 regulates poplar root development in response to salt and osmotic stresses

In Populus simonii, the N-terminal acetyltransferase subunit gene PsiNAA20 was induced by salt stress and osmotic stress and regulates root development. The spatiotemporal specificity of PsiNAA20-interacting gene expression and translation efficiency suggested dual functions in poplar root development under salt stress and osmotic stress. Salt stress is an increasingly severe problem worldwide, resulting in production losses, developmental changes, and ion transport anomalies (Munns & Gilliham, 2015). The extent of salinization damage has escalated globally as a result of unsustainable irrigation practices and climate change (van Zelm et al., 2020). Populus simonii is used as a model forest species in studies of abiotic stress tolerance, due to its extraordinary capacity to adapt to salt, drought, cold, and heat stress (Song et al., 2014). In fact, some P. simonii varieties persist in soils with up to 5% salt content; however, the genetic basis of salt tolerance in this species has yet to be explored. In this study, the genes conferring salt tolerance in poplar were identified by performing a genome-wide association study of 505 P. simonii accessions (Figure S1). An assessment of the relative survival rates (RSR) of P. simonii accessions after 21 d of 3% NaCl stress showed that ~17% of the accessions had a high salt stress tolerance (RSR ≥ 80%), whereas ~21% were highly sensitive to NaCl stress (RSR ≤ 20%). The strongest association between natural allelic diversities and RSR was detected for the single-nucleotide polymorphism Chr02:20765457 (P = 3.64 × 10−10), located in the fourth exon of the PsiNAA20 gene, which encodes the catalytic subunit of NatB (Figure 1A). The C allele is derived and encodes a nonsynonymous mutation that results in the replacement of a valine residue with an alanine residue. The CC genotype results in a higher expression level of PsiNAA20 and in a higher relative survival rate (Figure S2). In addition, 51 haplotypes were identified according to the PsiNAA20 gene, among which five were found in more than 15 varieties; these five haplotypes were divided into two major groups (A and B) (Figure 1B). Group A included three haplotypes (Hap02, 03 and 05), 71.4% of which originated from Northwest China. Hap03 accessions (T–T–T–C) had higher PsiNAA20 transcript abundance levels and salt tolerance (Figures 1C, D, S3). These results suggested that PsiNAA20 is a positive regulator of poplar salt tolerance. PsiNAA20 was found to be highly expressed in endodermis of root and lateral root cap (Figure S4) and its transcript abundance was induced by abscisic acid (ABA), cold, and heat stress (Figure S5). PsiNAA20 may thus be an ideal target for the regulation of poplar root development in response to abiotic stress. PsiNAA20 confers salt tolerance in poplar (A) Manhattan plot of the genome-wide association analysis results of salt tolerance in 505 Populus simonii individuals. (B) Median-joining network of the haplotype distribution based on the PsiNAA20 coding region. (C) Genetic effect of Hap01 and Hap03 on PsiNAA20 transcript abundance. (D) Genetic effect of Hap01 and Hap03 on survival rate under salt stress. (E) Performance of wild type, PsiNAA20 overexpression (OE) lines and NAA20 knockout (KO) lines under normal growth conditions. n = 6. Bars, 10 cm. (F) Performance of wild type, PsiNAA20 OE lines and NAA20 KO lines under salt stress. n = 6. (G) Fv/Fm value of wild type, PsiNAA20 OE lines and NAA20 KO lines under salt stress. n = 6. Fv, variable fluorescence in the dark-adapted state; Fm, maximum fluorescence in the dark-adapted state. (H) Histogram of Fv/Fm value of wild type, PsiNAA20-OE lines and NAA20-KO lines under salt stress. n = 6. (I) Effects of overexpressed PsiNAA20 on net Na+ fluxes in poplar root. n = 6. (J) Effects of overexpressed PsiNAA20 on net H+ fluxes in poplar root tip. n = 6. Vanadate, a plasma membrane (PM) H+-ATPase inhibitor. (K) Development of wild type, PsiNAA20 OE lines and NAA20 KO lines under osmotic stress (1% ~ 5% mannitol). n = 6. Bars, 5 cm. (L) Histogram of Fv/Fm value of wild type, PsiNAA20-OE lines and NAA20-KO lines under osmotic stress. n = 6. (M) Performance of wild type, PsiNAA20 OE lines and NAA20 KO lines under osmotic stress. n = 6. (N) Subcellular localization of PsiNAA20 in Nicotiana benthamiana leaves is shown. Empty vector was used as a control. Bars, 20 μm. (O) Yeast one-hybrid assay of PsiMYB43 binding to PsiNAA20 promoter fragments. The full-length construct of PsiMYB43 was fused to GAL4 DBD and expressed in the yeast strain AH109 Gold. Transformed yeast was grown in either synthetic dropout (SD)/-Ura-leu or Trp or SD/-Ura-leu+AbA media. pGAD-p53 + p53-pAbAi and pGADT7-AD+Pro PsiNAA20–pAbAi were carried out in the same manner as positive and negative controls, respectively. n = 6. (P) Dual-LUC (firefly luciferase) assays of the effect of PsiMYB43 on PsiNAA20 promoter. n = 6. Asterisk indicates the significance level at 0.01. For statistical analysis of experimental data, One-way analysis of variance was performed and significant differences between different groups were determined through Fisher's Least Significant Difference (LSD) test. Error bars represent SE. Different letters on error bars indicate significant differences at P < 0.01. The mechanism by which PsiNAA20 confers salt tolerance in poplar was explored in salinity stress experiments performed in PsiNAA20 overexpression (PsiNAA20-OE) and bi-allelic knockout (NAA20-KO) lines of the hybrid Populus alba × Populus glandulosa cv. "84K" (Figure S6). No obvious phenotypic differences were observed between the wild-type (WT) and PsiNAA20-OE lines under normal growth conditions (Figure 1E). However, both plant height and net photosynthesis rates were significantly reduced in the NAA20-KO lines, by 42.3% and 51.7%, respectively (Figure S7). After 21 d of salt stress treatment, root number, root weight, root diameter, root length density and salt tolerance were higher in the PsiNAA20-OE lines than in the WT and NAA20-KO lines (Figures 1F, S8). Fv/Fm (variable fluorescence in the dark-adapted state/maximum fluorescence in the dark-adapted state) and peroxidase activity were significantly higher, and malondialdehyde (MDA) and H2O2 levels significantly lower, in PsiNAA20-OE than in the other lines (Figures 1G, H, S9). Together, these results indicated a role for PsiNAA20 in promoting salt stress tolerance in poplar without a growth penalty. To identify Na+/H+ flux in PsiNAA20-OE under salt stress, a non-invasive microtest system was used to record Na+/H+ flux in the WT and PsiNAA20-OE lines following their exposure to 85 mmol/L NaCl (~3‰ NaCl). Sodium orthovanadate, a specific inhibitor of plasma membrane (PM) H+-ATPase, was used to repress PM H+-ATPase activity. Salt treatment caused pronounced Na+ and H+ effluxes across the PM of the PsiNAA20-OE lines, induced by activated PM H+-ATPase (Figure 1I, J). PsiHA1 transcript levels and ATPase activity were induced under salt and osmotic stress, implying that activated PsiHA1 transcription and PM H+-ATPase activity maintain the PM H+ gradient and are the driving force for Na+ extrusion and K+ retention, thereby avoiding the excessive accumulation of Na+ by PsiNAA20-OE lines (Figure S10). This would explain the significantly lower sodium content of those lines than of the WT and NAA20-KO lines. The significantly higher potassium content of the PsiNAA20-OE lines than of the WT and NAA20-KO lines indicated the repression of K+ efflux in response to the upregulation of PsiNAA20 (Figure S11). A high capacity to retain K+ under salt stress is therefore crucial for the salt adaption of poplar. Changes in soil salinity may also induce osmotic stress in plants by rapidly reducing the amount of water available to roots (van Zelm et al., 2020). Whether PsiNAA20 confers osmotic tolerance in poplar was therefore explored in osmotic stress experiments. Root development and the Fv/Fm were significantly lower in the WT and PsiNAA20-OE lines than in NAA20-KO lines under 14 d of osmotic stress (Figure 1K, L), indicating significantly lower MDA and H2O2 levels in the NAA20-KO lines as well. Peroxidase activity was significantly higher in the NAA20-KO lines (Figure S12). In addition, under osmotic stress, root weight, root diameter, root length density, root hydraulic conductivity, and the root respiration rate were significantly higher, and root relative leakage conductance was significantly lower (Figure S13), in the NAA20-KO lines than in the WT and PsiNAA20-OE lines (Figure 1M). These results demonstrated the dual-action of PsiNAA20 in response to salt stress and osmotic stress in poplar. The dual action of PsiNAA20 in response to both stresses was further examined in a yeast two-hybrid sequencing (Y2H-seq) analysis of PsiNAA20 using an osmotic- and salt-responsive complementary DNA (cDNA) yeast library. Under salt stress, 76.6% of the genes interacting with PsiNAA20 were upregulated in contrast to only 42.1% of these genes under osmotic stress (Table S1; Figure S14). The upregulated genes were mainly enriched in the functions of root morphogenesis, root phototropism and lateral root development, indicating changes in the root architecture of poplar under salt and osmotic stresses. Among the genes induced in the latter, PsiABAH, which functions in ABA degradation, was found to interact with PsiNAA20, implying that PsiNAA20 transcript regulation is coupled with ABA signal transduction under osmotic stress. To elucidate the bifunctional mechanism of PsiNAA20 in response to osmosis and salt stress, tissue-specific gene expression patterns and translation efficiency were analyzed. The results showed that genes responsive to osmotic pressure are preferentially expressed in mature leaves and salt responsive genes in roots (Figure S15). Moreover, the translation efficiency of genes responsive to salt stress was significantly higher than that of genes responsive to osmotic stress (Figure S16), pointing to an important role for the spatiotemporal specificity of interacting genes in maintaining dual-function PsiNAA20. A subcellular localization analysis revealed the nuclear localization of PsiNAA20-GFP (green fluorescent protein) (Figure 1N). A potential upstream regulator of PsiNAA20 was identified using a salt stress response cDNA library together with a one-hybrid assay (Y1H). PsiMYB43, which is mainly expressed in roots and cambium, was identified as a putative candidate regulator as it was induced by both salt stress and osmotic stress (Figure S5). PsiMYB43 may thus bind directly to the PsiNAA20 promoter to activate its expression under salt stress (Figures 1O, P, S17). N-terminal acetyltransferases (NATs) are a highly conserved family of proteins that includes six conserved members (Nat A–F) (Rathore et al., 2016). NAA20 belongs to the NatB complex, which includes the major NATs in eukaryotic cells (Huber et al., 2020). The major clades of NATs in the eukaryote lineage are represented by 34 species: seven holozoans, four fungi, two amoebozoa, 12 plants, four excavates, and five chromalveolatans. Among NAT members present in all 34 species, only NAA20 is present in all eukaryote lineages and its protein sequence similarity is the highest (E < 10−8) (Figure S18). To test the function of NAA20 in eukaryotic salt tolerance, PsiNAA20 was overexpressed in the yeast salt sensitive strain "INVSCI." The results showed that PsiNAA20 overexpression significantly promoted salt tolerance following treatment of the yeast with 2 mmol/L NaCl (Figure S19). Thus, NAA20 shows potential as a conserved target that could be used to improve salt tolerance in plants. This work was supported by STI2030—Major Projects (No. 2023ZD040710508) and the Project of Youth talent program of Forestry and Grassland Science and Technology Innovation (No. 2020132606) and the 111 Project (No. B20050). The authors declare no conflict of interest. Y.S. designed the experiments; Y.G., C.B, P. C., X.H., R.Z. and M.W. performed the research; Y.G. wrote the paper. Y.S. revised the manuscript; All authors read and approved the final manuscript. Additional Supporting Information may be found online in the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.13835/suppinfo Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.