GmDNJ1, a type-I heat shock protein 40 (HSP40), is responsible for both Growth and heat tolerance in soybean

Abstract

Global warming poses severe threats to agricultural production, including soybean. One of the major mechanisms for organisms to combat heat stress is through heat shock proteins (HSPs) that stabilize protein structures at above-optimum temperatures, by assisting in the folding of nascent, misfolded, or unfolded proteins. The HSP40 subgroups, or the J-domain proteins, functions as co-chaperones. They capture proteins that require folding or refolding and pass them on to HSP70 for processing. In this study, we have identified a type-I HSP40 gene in soybean, GmDNJ1, with high basal expression under normal growth conditions and also highly inducible under abiotic stresses, especially heat. Gmdnj1-knockout mutants had diminished growth in normal conditions, and when under heat stress, exhibited more severe browning, reduced chlorophyll contents, higher reactive oxygen species (ROS) contents, and higher induction of heat stress-responsive transcription factors and ROS-scavenging enzyme-encoding genes. Under both normal and heat-stress conditions, the mutant lines accumulated more aggregated proteins involved in protein catabolism, sugar metabolism, and membrane transportation, in both roots and leaves. In summary, GmDNJ1 plays crucial roles in the overall plant growth and heat tolerance in soybean, probably through the surveillance of misfolded proteins for refolding to maintain the full capacity of cellular functions.

Keywords: GmDNJ1; J domain; co‐chaperone; heat shock protein; heat stress; soybean.

FIGURE 1

GmDNJ1 is a typical type‐I HSP40. (a) A cartoon depicting the protein structure of GmDNJ1. The various domains were determined by Pfam (El‐Gebali et al., 2019). The nuclear localization signal (NLS) and nuclear export signal (NES) were determined by LOCALIZER (Sperschneider et al., 2017) and NetNES 1.1 Server (la Cour et al., 2004), respectively. (b) Phylogenetic tree of characterized HSP40s. The tree was built with MEGA X (Kumar et al., 2018) using the Maximum Likelihood method and the JTT matrix‐based model with 1,000 bootstrap replications for phylogeny test. Bootstrap values were labeled as percentages on the branches. The accession numbers of the HSP40 protein sequences included in this tree are GmDNJ1: NP_001238341.1; AtJ3: NP_189997.1; AtDjB1: NP_195759.1; AtDjC1: NP_187752.2; HsDNAJA1: NP_001530.1; HsDNAJB1: NP_006136.1; HsDNAJC9: NP_056005.1; ScYdj1: NP_014335.1; ScSis1: NP_014391.1; EcDnaJ: NP_414556.1; OsDjA5: XP_015632121.1; OsDjB7: XP_015637533.1; OsDjC12: EEE55258.1; AtHSP70: NP_001328002.1; and EcDnaK: WP_102804582.1. (c) Subcellular localization study of GmDNJ1. A total of >50 cells were observed for each line with similar results. (d) Luciferase refolding assay for testing GmDNJ1 co‐chaperone activity. The luciferase activity in each reaction was normalized to that containing E. coli DnaJ–DnaK‐GrpE in combination (lane 3), which was set at 100%. Each bar represents the average of at least three technical replicates with the error bar representing standard error

FIGURE 2

Expressions of GmDNJ1 were induced under abiotic stress treatments. First‐trifoliate seedlings of G. max cultivar C01 were treated with (a–e) 9% NaCl for 4 hr, (f–j) 5% PEG for 24 hr to induce osmotic stress, (k–o) 50 mM NaHCO₃ at pH 8.5 for 24 hr, (p–t) 10 mM paraquat for 4 hr to induce oxidative stress, and (u‐y) heat stress at 42°C for 4 hr. Expressions of GmDNJ1 and other stress‐responsive genes in leaves and roots were analyzed by RT‐qPCR. The expressions of GmDNJ1 in the treated tissues were normalized to those in the respective untreated tissues. α‐tubulin was used as the housekeeping gene for normalizing RNA input. Relative gene expression was calculated by the 2‐^ΔΔCT method. The error bar represents the standard deviation of three technical repeats. Two‐tailed student's t test was adopted to compare the expressions between untreated and treated samples. *, **, and ***indicates a significant difference at p < .05, p < .01, and p < .001, respectively. ns means that there was no statistically significant difference. A biological repeat of this experiment can be found in Figure S3

FIGURE 3

Growth performance and chlorophyll contents of Gmdnj1 mutant lines. (a) Photographs showing 2‐week‐old Gmdnj1 mutant plants treated at 45/28°C (heat‐treated) and 28/28°C (untreated) following the 16/8 hr light–dark cycle for 4 days. (b) Chlorophyll contents of the mutant lines with or without heat treatment. Data were assessed with one‐way ANOVA followed by Tukey's post hoc test. Different letters above the bars indicate means that were significantly different at p < .05. N ≥ 4. Errors bars: SEM. The experiment was performed twice with similar results. Replicate of the experiment can be found in Figure S5

FIGURE 4

Fresh weights and dry weights of Gmdnj1 mutant lines under normal and heat treatment conditions. (a) Shoot fresh weights, (b) root fresh weights, (c) shoot dry weights, and (d) root dry weights of wild‐type Williams 82 and two Gmdnj1 mutant lines after 4 days of heat treatment at 45/28°C under the 16/8 hr light–dark cycle. Data were assessed with one‐way ANOVA followed by Tukey's post hoc test. Different letters above the bars indicate means that were significantly different at p < .05. N ≥ 4. Errors bars: SEM. Replicate of the experiment can be found in Figure S6

FIGURE 5

ROS contents and expressions of genes encoding ROS‐scavenging enzymes in Gmdnj1 mutant lines. The ROS contents in leaf (a) and root (b) of untreated plants, and in leaf (c) and root (d) of heat‐treated plants were compared by measuring the H₂DCFCA fluorescence per unit protein in the extract. Expressions of HsfA2 (e), heat shock element containing superoxide dismutase‐encoding genes (f), and heat shock element containing ascorbate peroxidase‐encoding genes (g) in root of Gmdnj1 mutant were monitored. The data in a–d were analyzed with one‐way ANOVA followed by LSD test. Different letters above the bars indicate means that are significantly different at p < .05. N ≥ 4. Error bar: SEM. The data in (e–g) were analyzed with one‐way ANOVA followed by Tukey's test. Different letters above the bars indicate means that are significantly different at p < .05. N ≥ 3. Error bar: standard deviation. Replicate of the experiment can be found in Figure S7