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Article

GmDNAJC7 from Soybean Is Involved in Plant Tolerance to Alkaline-Salt, Salt, and Drought Stresses

by
Ting Jin
,
Zhong Shan
,
Shuang Zhou
,
Qianqian Yang
,
Junyi Gai
and
Yan Li
*
National Key Laboratory of Crop Genetics and Germplasm Enhancement, National Center for Soybean Improvement, Key Laboratory for Biology and Genetic Improvement of Soybean (General, Ministry of Agriculture), Jiangsu Collaborative Innovation Center for Modern Crop Production, Nanjing Agricultural University, Nanjing 210095, China
*
Author to whom correspondence should be addressed.
Agronomy 2022, 12(6), 1419; https://doi.org/10.3390/agronomy12061419
Submission received: 6 May 2022 / Revised: 3 June 2022 / Accepted: 7 June 2022 / Published: 13 June 2022
(This article belongs to the Special Issue Recent Progress in Molecular Investigations of Crop Plants and Crop Plant–Environment Interactions from Genetic and Genomic Perspectives: Applications and Challenges—Series II)
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Abstract

:
Soybean [Glycine max (L.) Merri.] is an important oilseed and food crop. In recent years, environmental degradation has accelerated soil alkalization, salinization, and water deficit, which have seriously threatened the soybean quality and yield. Chaperone DNAJ proteins play important roles in plant response to a number of abiotic and biotic stresses. Here, we investigated the function of a soybean DNAJ gene, GmDNAJC7, in plant tolerance to abiotic stresses. GmDNAJC7 gene expression was induced by alkaline-salt, salt, and drought treatments in soybean roots, suggesting its possible role in soybean response to these stresses. GmDNAJC7 overexpression improved the alkaline-salt tolerance of soybean composite plants, which showed a higher SPAD (Soil and Plant Analysis Development) value for chlorophyll content and leaf relative water content than the control plants after NaHCO3 treatment. Moreover, the GmDNAJC7 overexpressing Arabidopsis had a higher germination rate and average root length than the wild type and dnajc7 mutant, under NaHCO3, NaCl, and mannitol stresses, indicating that the ectopic expression of the GmDNAJC7 gene enhanced the alkaline-salt, salt, and drought tolerance in Arabidopsis. These findings suggest that GmDNAJC7 is involved in the alkaline-salt, salt, and drought tolerance in Arabidopsis and soybean. This study provides new insights into the role of DNAJ proteins in plant tolerance to abiotic stress.

1. Introduction

Soybean [Glycine max (L.) Merr.] is a major source of vegetable protein and edible oil as well as an important feedstock for livestock and as an industrial raw material [1]. Due to the unpredictability of environmental conditions and the inability of plants to move to avoid unfavorable conditions, the growth and production of plants are adversely affected by different abiotic stresses including alkalinity, salinity, and drought [2]. The greenhouse effect and decreases in the available water resources worldwide will exacerbate the negative impact of the alkali, salt, and drought stress on plants [3]. Soil alkalization, salinization, and water deficit have a serious impact on the soybean growth, development, and physiological metabolism, threatening the soybean quality and yield [4,5,6]. Because of their sessile nature, plants must sense and respond to changes in their environment [7]. One of the most common plant adaptations to environmental changes is differential regulation of growth, to grow either away from adverse conditions or toward more favorable conditions [7]. However, due to the limitation and variability of the environment [7], it is more feasible to cultivate abiotic stress tolerant plants to adapt to the environment. Therefore, breeding soybean varieties with abiotic stress tolerance is essential to develop marginal lands such as saline, alkaline, or dry land to promote soybean production and ensure global food security.
Chaperone DNAJ proteins were first identified in Escherichia coli in 1979 [8], and then found in various organisms including plants. DNAJ proteins, also known as heat shock protein 40 (Hsp40), are important partners of Hsp70 proteins. DNAJ proteins have been classified into three types based on sequence comparisons [9]. The A-type DNAJ protein was found to have a typical structure containing four parts: a highly conserved N-terminal (J region), a Gly/Phe rich region (GF region), a Cys rich Zn binding region, and a C-terminal containing 120–170 amino acids [9]. The B-type DNAJ protein was later identified as having three structural domains: the CTD domain, the G/F domain, and the DNAJ domain. The C-type DNAJ protein is homologous only to the DNAJ domain and does not possess the other typical region/domain of the A- and B-type members [9].
Several studies have shown that DNAJ proteins are involved in the plant response to abiotic stresses [10,11,12,13,14]. DNAJ protein ZjDjB1 of Zostera japonica was localized to the cytoplasm and nucleus. Ectopic expression of ZjDjB1 in Arabidopsis thaliana can improve thermo tolerance, reduce the accumulation of reactive oxygen species, and reduce the damage of membranes by maintaining a low activity of proteolytic enzymes [11]. The J3 (DNAJ homolog 3; heat shock protein 40-like) from A. thaliana activates H+-ATPase activity by physically interacting with and repressing PKS5 kinase activity. Plants lacking J3 are hypersensitive to salt at high external pH and exhibit decreased H+-ATPase activity [12]. The AtDjA3 gene is involved in seed development and tolerance to abiotic stresses such as NaCl [13]. The expression of OR (encoding DNAJ-like zinc finger protein) in A. thaliana was upregulated by drought treatment, and seedlings of the OR-overexpressing (OE) lines showed improved growth performance and survival rate under drought stress [14]. However, the functions of DNAJ genes in soybean are largely unknown.
In this paper, members of the soybean DNAJ gene family were identified by using an Hidden Markov model (HMM) profile (PF01556) from the ensemblPlants database. Based on previously published Affymetrix GeneChip datasets for alkali stress, salt stress, and drought stress, the expression profiles of the soybean DNAJ genes were investigated. A soybean DNAJ gene, GmDNAJ7 with multiple Gene Ontology (GO) terms, and induced by alkali, salt, and drought stresses, was selected for further study. The role of GmDNAJ7 in plant tolerance to abiotic stress was studied by transgenic soybean composite plants and Arabidopsis. The possible molecular mechanism of GmDNAJ7 in response to abiotic stress was discussed by protein–protein interaction prediction analysis and GO annotation analysis. This study aims to provide new insights into the role of GmDNAJ7 in soybean tolerance to alkaline-salt, salt, and drought stresses and the possible underlying mechanisms.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

The seeds of soybean cultivar “Tianlong 1” and wild-type (WT) Arabidopsis Col-0 used in this experiment were provided by the National Center for Soybean Improvement at Nanjing Agricultural University. The Arabidopsis dnajc7 T-DNA insertion mutant (stock, SALK_034886, dnajc, Col-0 background) used in this study was obtained from the Arabidopsis Biological Resource Center (http://www.arabidopsis.org, accessed on 26 May 2018).
“Tianlong 1” soybean seeds were surface-sterilized with 1% sodium hypochlorite for 30 s, and rinsed five times with deionized water. Ten seeds were sown in plastic pots (Φ10 × 8 cm) filled with clean quartz sand in a growth chamber under a photoperiod of 14/10 h (light/dark) at 28/24 °C. Seven days after germination, four plants were left in each pot. Then, 14-day-old soybean seedlings were subjected to different abiotic stresses including 90 mM NaHCO3, 200 mM NaCl, and 20% (w/v) polyethylene glycol (PEG) 6000, respectively. The root tips (2–3 cm) from all treatments and the control were sampled at 6 h, 12 h, 24 h, 36 h, and 48 h, respectively, and then frozen in liquid nitrogen immediately for RNA isolation.
Arabidopsis seeds were surface-sterilized with 15% clorox three times and rinsed five times with sterilized water, and then stratified at 4 °C for three days. Seeds were planted on 1/2 Murashige and Skoog (MS) medium (pH 5.8, MDBio, Qingdao, China) in a growth chamber under a photoperiod of 16/8 h (light/ dark) at 22 °C.

2.2. Phylogenetic Analysis of Arabidopsis and Soybean DNAJ Superfamily

The 204 sequences of the soybean DNAJ protein superfamily (Table S1) were identified using the DNAJ domain (PF01556) as queries in the ensemblPlants database (https://plants.ensembl.org/index.html, accessed on 30 March 2022) and downloaded from the phytozome database (https://phytozome-next.jgi.doe.gov/, accessed on 30 March 2022). The full protein sequences of the above DNAJ proteins and Arabidopsis DNAJ protein superfamily [15] were used for multiple sequence alignments by ClustalW2 (https://www.ebi.ac.uk/Tools/msa/clustalw2/, accessed on 30 March 2022) [16]. The unrooted phylogenetic tree was then constructed using MEGA 6.0 [17], based on the maximum likelihood (ML) algorithm with 1000 bootstraps.

2.3. RNA Extraction and Gene Expression Analysis

The expression profiles of the soybean DNAJ genes in response to abiotic stress were obtained using previously published Affymetrix GeneChip datasets [18,19,20] including soybean under alkaline salt (GSE17883, 50 mM/L NaHCO3 for 0 h and 6 h), saline (GSE41125, treated with 150 mM NaCl for 1 h, 3 h, 6 h, 12 h or 24 h, or mock treatments of ddH2O only), and drought (GSE50408, drought stress for 0 day and 25 days) stresses. RNA-Seq data from different soybean tissues were downloaded from Soybase (http://soybase.org, accessed on 27 April 2022).
Total RNA from soybean were extracted using an RNAprep Pure Plant Kit (Tiangen Biotech, Beijing, China). The cDNA was synthesized by the PrimeScript™ 1st Strand cDNA Synthesis Kit (TaKaRa, Gunma, Japan). The qRT-PCR was conducted using SYBR Premix ExTaqTM II Mix (TaKaRa, Gunma, Japan) on a Roche 480 Real-Time Detection System (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s instructions. Each experiment was performed in triplicate. The transcript levels in soybean plants were calculated relative to GmUKN1 [21] using the 2−△△CT methods [22]. Three biological replicates were conducted. The primers were designed using Primer Premier 5 software (http://www.premierbiosoft.com/primerdesign/, accessed on 10 March 2018) and are listed in Table S2.

2.4. Subcellular Localization of GmDNAJC7

The subcellular localization of the GmDNAJC7 protein was determined using Agrobacterium-mediated transient transformation of tobacco. The coding sequence (CDS) of GmDNAJC7 was cloned into the pBINGFP4 vector in fusion with the green fluorescence protein (GFP) gene, then transformed into Agrobacterium tumefaciens EHA105. The activated and monoclonal clone was transferred to yeast extract broth (YEB) medium containing antibiotics (Kana + Rif) and incubated for 12 h (250 rpm/28°). When the optical density (OD600) value was between 0.6 and 0.8, the Agrobacteria were collected by centrifugation at 6000 rpm/5 min. The Agrobacteria was resuspended with a resuspension solution (0.5 M MES, 1 M MgCl2, 100 mM AS) and then the centrifugation and resuspension were repeated, adjusting the OD value to about 0.5. The resuspension was injected by syringe into the back of 6~8-week-old leaves of Nicotiana Benthamiana, then incubated in the dark for 2 h and put back to the incubator. After 2~3 d, the fluorescence was examined using a laser scanning microscope (Zeiss LSM780 META, Jena, Germany) at a 488 nm wavelength.

2.5. Obtaining and Analysis of Transgenic Soybean Composite Plants

35S:GmDNAJC7 and the empty vector pBinGFP4 were separately transformed into the Agrobacterium rhizogenes strain K599 [23], then used to infect soybean hypocotyls of an alkaline-salt sensitive soybean variety Tianlong 1, to obtain transgenic soybean composite plants, according to the previously described methods [23]. After two weeks of plant growth, the positive hairy roots were identified by the green fluorescence signal of GFP using a stereoscopic fluorescence microscope (Mshot, Guangzhou, China) at a 488 nm wavelength, and the non-transgenic roots were cut off. The transgenic soybean composite plants were treated with 1/2 Hoagland solution containing 0 or 90 mM NaHCO3 [24] for five days, then the SPAD (Soil and Plant Analysis Development) value for the chlorophyll content [25] and leaf relative water content [26] were measured. Three biological replications were performed and five independent transgenic plants (n = 3 × 5 = 15) were measured for each repeat.

2.6. Ectopic Expression of GmDNAJC7 in Arabidopsis and Abiotic Stress Treatment

The full-length CDS of GmDNAJC7 was cloned into the pCAMBIA3301 vector under the control of the CaMV 35S promoter. The pCAMBIA3301–GmDNAJC7 vector was introduced into Agrobacterium rhizogenes EHA105 and Arabidopsis was transformed by the floral dip method using Col-0 wild type plants [27]. Putative transgenic plants were screened based on their resistance to 20 mg L−1 glufosinate (PhytoTech, Lenexa, KS, USA). Homozygous transgenic T3 plants were obtained through self-crossing and also selected by glufosinate.
To observe the effects of NaHCO3, NaCl, or mannitol on seed germination and root growth, three independent GmDNAJC7-overexpressing lines (#1, #2, #3), dnajc7 mutant, and WT Arabidopsis plants were selected for testing. In the abiotic stress plate tests for Arabidopsis, 150 mM NaCl [28], 6 and 8 mM NaHCO3 [29], and 150 and 300 mM mannitol [30] have often been used for the germination rates, and 150 mM NaCl [31], 3, 4, 5 mM NaHCO3 [32], and 200 mM mannitol [31] for the root lengths. Therefore, we carried out the plate tests using the following concentrations of abiotic stresses. For the seed germination analyses, plant seeds were sown on 1/2 MS medium plates with different concentrations of NaHCO3 (0, 6, 8, and 10 mM), NaCl (0, 125, 150, and 200 mM) or mannitol (0, 200, and 300 mM). The seed germination rates and cotyledon greening rates were recorded and calculated after 5 and 10 d. To measure the root lengths of Arabidopsis seedlings, seeds were spotted on 1/2 MS medium containing various concentrations of NaHCO3 (0, 3, 4, 5 mM), NaCl (0, 100, 125, 150 mM), or mannitol (0, 200, 300 mM). Photographs were taken 10 d after sowing, and root lengths were measured by image analysis using ImageJ software [33].

2.7. Statistical Analysis

Statistical analyses were performed with the SAS version 9.2 software package for Windows (SAS Institute Inc., Cary, NC, USA). Differences between two samples were analyzed by the Student’s t-tests, and comparisons among means were made using Duncan’s multiple range tests.

3. Results

3.1. Identification and Characterization of Soybean DNAJ Superfamily

In order to comprehensively identify the DNAJ members in soybean, we used the keywords of “DNAJ” and “Hsp40”, and the HMM profile of the DNAJ domain (PF01556) as queries in ensemblPlants database to search the soybean reference genome (Wm82.a2.v1) in the Phytozome v13.0 database. As a result, 204 putative soybean DNAJ candidates were identified (Table S1). The conserved DNAJ domain (PF01556) was confirmed with SMART (https://smart.embl.de/, accessed on 30 March 2022). Phylogenetic analysis of 204 soybean DNAJ proteins, together with 89 sequences of the Arabidopsis DNAJ superfamily, [15] was performed (Figure 1), and the results showed that GmDNAJC7 was closely related to AtDNAJC7.

3.2. Expression Profiling of Soybean DNAJ Genes in Response to Alkaline Salt, Salt and Drought Stresses

To further investigate the possible functions of soybean DNAJ genes, the expression profiles of the 204 soybean DNAJ genes in response to abiotic stresses including alkali, salt, and drought in soybean plants (Figure 2) were examined by exploring the previously published Affymetrix GeneChip datasets (GSE17883, GSE41125, GSE50408). A total of 71, 89, and 85 soybean DNAJ genes were upregulated by alkali, salt, and drought stresses compared with the control, respectively. Among the top 10 upregulated genes with the highest fold changes in response to abiotic stresses, GmDNAJC7 had the most GO function annotations (Table S3). The genomic DNA of GmDNAJC7 was 5703 bp in length and contained 10 introns and 11 exons. Its CDS was 2319 bp long, which encoded a protein of 772 amino acids, containing the five predicted TPR domains and one DNAJ domain.

3.3. The Expression of GmDNAJC7 Is Upregulated in Response to NaHCO3, NaCl, and PEG Stresses

The tissue expression pattern of GmDNAJC7 was analyzed using the expression data from the stems, leaves, flowers, pods, roots, root hairs, nodules, and seeds of the soybean variety Williams 82 from the Phytozome database (https://phytozome-next.jgi.doe.gov/, accessed on 27 April 2022). The highest expression of GmDNAJC7 was observed in the root hairs, followed by the stems, nodules, roots, and leaves (Figure 3A). To investigate the relative expression level of GmDNAJC7 in response to abiotic stresses, soybean seedlings were treated with NaHCO3 (90 mM), NaCl (200 mM), and PEG (20%) for 14 days. Roots of the soybean plants were sampled after 6 h, 12 h, 24 h, 36 h, and 48 h to determine the relative expression levels of GmDNAJC7 (Figure 3B–D). The results demonstrated that the mRNA levels of GmDNAJC7 in the soybean roots were upregulated by NaHCO3, NaCl, and PEG-mediated dehydration stresses, and reached the maximum at 36 h after these treatments (Figure 3B–D). These results suggest that the GmDNAJC7 gene is responsive to all three abiotic stresses including NaHCO3, NaCl, and PEG, therefore GmDNAJC7 may be involved in soybean tolerance to these abiotic stresses.

3.4. GmDNAJC7 Is Localized to the Nucleus and Cytoplasm

The pBinGFP4-35S:GmDNAJC7 vector was constructed and transformed into Agrobacterium tumefaciens strain EHA105 to locate the GmDNAJC7 protein by transient expression in tobacco leaves. The GFP fluorescence signal detected by laser confocal microscopy suggests that the GmDNAJC7 protein was localized to the nucleus and cytoplasm (Figure 4).

3.5. Overexpression of GmDNAJC7 Improved Soybean and Arabidopsis Tolerance to NaHCO3 Stress

Since the expression level of GmDNAJC7 was induced by NaHCO3 treatment, we next tested whether the overexpression of GmDNAJC7 could improve the soybean tolerance to alkaline-salt stress or not. The coding region of the GmDNAJC7 gene was expressed in fusion with GFP (while the empty vector 35S:GFP was used as the control), and transformed into soybean hypocotyls to obtain transgenic composite plants, in the genetic background of an alkaline-salt sensitive soybean variety Tianlong 1. The presence of fluorescence signals suggests that both empty vector pBinGFP4 and fusion expression vector pBinGFP4-35S:GmDNAJC7 were successfully transformed into transgenic soybean composite plants (Figure 5A). Under the normal condition (0 mM NaHCO3), all soybean composite plants grew well, with no obvious difference (Figure 5B). After five days of treatment with 90 mM NaHCO3, the 35S:GmDNAJC7-GFP transformed plants looked better than the 35S:GFP transformed soybean composite plants. The growth of the 35S:GmDNAJC7-GFP transformed plants was less affected by alkali stress, while the 35S:GFP transformed soybean composite plants showed severe chlorosis and wilting of the leaves (Figure 5B). The 35S:GmDNAJC7-GFP transformed soybean composite plants had significantly higher values of average SPAD for the chlorophyll content (Figure 5C) and the leaf relative water content (LRWC) than those of the 35S:GFP transformed soybean composite plants under 90 mM NaHCO3 treatment (Figure 5D). These results demonstrate that overexpression of GmDNAJC7 could reduce the water loss and maintain a relatively higher chlorophyll content after alkaline stress to some extent, in order to reduce the damage of NaHCO3 treatment on soybean plants.
We further investigated the role of GmDNAJC7 in response to alkaline-salt stress by transgenic Arabidopsis plants. GmDNAJC7 was overexpressed (OE) using the CaMV 35S promoter in Arabidopsis. The germination rates of Arabidopsis lines were compared in the absence or presence of NaHCO3, respectively. The results showed that there was no difference in the germination rates at 5 d and cotyledon greening rates at 10 d between the wild type (WT), GmDNAJC7 OE lines, and dnajc7 mutant under the control condition, but the germination rates and cotyledon greening rates of the GmDNAJC7 OE lines were significantly higher than those of the WT and dnajc7 mutant under NaHCO3 stress (Figure 6A–C). To further test the effect of NaHCO3 stress on Arabidopsis, we measured the root length of Arabidopsis. Under the normal condition, no significant difference was observed between the GmDNAJC7 OE lines, WT, and mutant (Figure 6D,E). After 10 d of NaHCO3 treatment, the average root lengths of three GmDNAJC7 OE lines were significantly greater than those of the WT and mutant (Figure 6D,E). The germination rates of WT Arabidopsis were significantly higher than that of the dnajc7 mutant under 6 and 8 mM of NaHCO3 stress (Figure 6A,B). These results suggest that ectopic expression of GmDNAJC7 enhanced the alkaline-salt tolerance in Arabidopsis.

3.6. Ectopic Expression of GmDNAJC7 in Arabidopsis Enhanced Salt Tolerance

The Arabidopsis WT, GmDNAJC7 OE lines, and dnajc7 mutant were planted on 1/2 MS medium or media with different concentrations of NaCl, then the seed germination rates at 5 d and cotyledon greening rates at 10 d were measured (Figure 7A–C). After 10 days of growth, the root lengths were measured (Figure 7D,E). There was no difference in the germination rates, cotyledon greening rates, or root lengths between the WT, GmDNAJC7 OE lines, and dnajc7 mutant on medium without NaCl, but the germination rates, cotyledon greening rates, and root lengths of the GmDNAJC7 OE lines were significantly higher than those of the WT and dnajc7 mutant under NaCl stress (Figure 7). The germination rates of WT Arabidopsis were significantly higher than that of the dnajc7 mutant under NaCl stress (Figure 7A,B). These results suggest that ectopic expression of GmDNAJC7 enhanced the salt tolerance in Arabidopsis.

3.7. Ectopic Expression of GmDNAJC7 in Arabidopsis Enhanced Drought Tolerance

We further investigated the function of GmDNAJC7 in response to mannitol stress by transgenic Arabidopsis plants (Figure 8). The germination rates (Figure 8A,B) and root lengths (Figure 8C,D) of the WT, GmDNAJC7 OE lines, and dnajc7 mutant did not differ in the absence of mannitol, while the GmDNAJC7 OE lines showed an advantage to that of the WT and dnajc7 mutant under mannitol stress. The germination rate and root length of WT Arabidopsis showed an advantage over those of the dnajc7 mutant under 200 mM mannitol stress (Figure 8C,D). Taken together, these results suggest that the ectopic expression of GmDNAJC7 enhanced the drought tolerance in Arabidopsis.

4. Discussion

4.1. GmDNAJC7 Overexpression Can Improve the Tolerance of Soybeans and Arabidopsis to NaHCO3, NaCl, and Mannitol Stresses

Salt, alkali, and drought are important abiotic stressors that threaten crop yield [4,5,6]. Soil salinization and alkalization can reduce soil osmotic potential, and cause ion imbalance, disrupt physiological processes, and inhibit the growth and development of plants, leading to a serious decline in its yield and quality, and even the death of plants [6,35]. It is also known that under drought stress, soybean yield will be significantly reduced [36]. In order to improve the ability of crops to resist abiotic stresses, various abiotic stress-related genes have been studied and molecular breeding was used to improve the crop resistance to abiotic stresses to prevent yield loss [37,38,39]. There has been lots of progress on soybean tolerance to salt and drought stresses. Recent studies have shown that genes such as GmMYB14 [38], GmNF-YC14 [40], and GsERD15B [6] are involved in salt or drought tolerance while there have only been a few reports on alkali salt tolerance. Here, we investigated the role of the soybean DNAJC7 gene in plant tolerance to not only salt (NaCl), drought (mannitol), but also alkaline-salt (NaHCO3), which would broaden our knowledge on alkali salt tolerance.
In this study, the expression profiles of the 204 soybean DNAJ genes in response to abiotic stresses including alkali, salt, and drought in soybean plants were examined (Figure 2). Based on the expression pattern and gene annotation, we hypothesize that GmDNAJC7 is likely to be involved in plant response to abiotic stress. We further characterized the candidate gene GmDNAJC7 by bioinformatics analysis, molecular biology, and genetic transformation. GmDNAJC7 expression can be induced by NaHCO3, NaCl, and PEG treatment (Figure 3), suggesting its possible role in soybean tolerance to alkali, salt, and drought stresses. Then, we employed transgenic soybean composite plants and Arabidopsis to investigate the role of GmDNAJC7 in alkaline-salt tolerance (Figure 5 and Figure 6). Transgenic soybean composite plants and Arabidopsis plants have previously been used to study gene functions in saline-alkaline tolerance [41]. We found that overexpression of GmDNAJC7 enhanced the alkaline salt tolerance of soybean and Arabidopsis. We also found that ectopic expression of GmDNAJC7 enhanced the salt and drought tolerance of Arabidopsis (Figure 7 and Figure 8). Taken together, these results revealed that GmDNAJC7 overexpression can significantly improve plant tolerance to abiotic stresses such as alkali, salt, and drought.

4.2. GmDNAJC7 Mediates Plant Tolerance to Abiotic Stresses Probably through Endoplasmic Reticulum Stress Response, Hydrogen Peroxide Response, and Cellulose Biosynthesis Pathways

The mechanisms of the DNAJ gene family in plant tolerance to abiotic stresses have previously been proposed in several studies. It was reported that the DNAJ gene could provide drought tolerance through its impact on proline biosynthesis in Arabidopsis [14]. The DNAJ homolog3 (J3) regulates H+-ATPase activity by inactivating the PKS5 kinase in response to salt [12]. ZjDjB1 increased the thermotolerance perhaps by maintaining a low activity of proteolytic enzymes [11]. In order to explore the possible mechanism of GmDNAJC7 response to abiotic stresses, protein–protein interaction network prediction analyses were performed (Figure 9). Based on the protein–protein interaction network prediction analyses, GmDNAJC7 might interact with nine other proteins (Figure 9, Table S4) including five heat shock proteins (Hsp70), two translocation protein sec63 homolog proteins (sec63), and two hypoxia upregulated proteins (HIF1A). Next, GO terms were further used to predict the possible roles of these nine proteins and their encoding genes in plant tolerance to abiotic stresses.
Five Hsp70 genes and two HIF1A genes were annotated as involved in the GO:0034976 (response to endoplasmic reticulum stress), and two sec63 genes were annotated as involved in the GO:0030968 (endoplasmic reticulum unfolded protein response) (Table S4). Endoplasmic reticulum (ER) stress is defined by a protracted disruption in protein folding and the accumulation of unfolded or misfolded proteins in the ER [42]. This accumulation of unfolded proteins can result from excessive demands on the protein folding machinery triggered by environmental, biotic, and abiotic stresses such as nutrient deficiencies, oxidative stress, pathogens, and heat [43]. In plant cells, ER stress is typically induced by adverse environmental conditions such as salt [44], drought [45], and heat [46]. Therefore, the plant cell response to ER stress is targeted to maintain cellular homeostasis by increasing the ER’s protein folding capacity, thus allowing plants to adapt to multiple environmental stresses [42].
Another group of genes include one GmHSP70 gene and two HIF1A genes annotated as involved in the response to hydrogen peroxide (GO:0042542) and GmDNAJC7 annotated as involved in the hydrogen peroxide biosynthetic process (GO:0050665) (Table S4). These four genes might be related to the oxidation–reduction or response to oxidative stress. Hydrogen peroxide (H2O2) is the most stable of the reactive oxygen species (ROS) and plays a crucial role as a signaling molecule in various physiological processes [47]. Abiotic stresses such as dehydration, low and high temperatures, and saline-alkali stress can disturb this balance [48]. In contrast, the antioxidant defense system protects plants from abiotic stress induced oxidative damage by detoxifying the ROS [49]. The 5-aminolevulinic acid induces H2O2 accumulation in strawberry roots and mediates Na+ transporter gene expression and more Na+ retention in the roots, thereby improving the plant’s salt tolerance [50]. MeRAV5 promoted the activities of MePOD to affect H2O2 accumulation, which is important in drought stress resistance in cassava [51].
There is one GmHSP70 gene and two HIF1A genes annotated as involved in the cellulose biosynthetic process (GO:0030244) and GmDNAJC7 annotated as involved in the cell wall organization (GO:0071555) (Table S4). The plants’ biomass is largely constituted by the plant cell wall [52]. Cellulose is a principal component of the cell wall and is synthesized by microtubule-guided cellulose synthase enzymes at the plasma membrane [52]. Cellulose synthesis enzymes and cellulose synthesis may be regulated by abiotic factors and react on stressors [7]. Ectopic expression of SOD and APX genes in Arabidopsis alters the metabolic pools and genes related to secondary cell wall cellulose biosynthesis and improves the salt tolerance [53]. Histone acetyltransferase GCN5 contributes to cell wall integrity and salt stress tolerance by altering the expression of cellulose synthesis genes [54]. Plant cell–wall-related remodeling is the primary response against abiotic stresses including drought [55]. Wheat VIH protein modulates the cell wall by affecting the changes in the cell–wall polysaccharide composition (AG, AX, and cellulose), and ultimately, improved the tolerance to drought in Arabidopsis [56].
The genes encoding these interacting proteins might be co-expressed with GmDNAJC7 at the transcriptional level. Therefore, we investigated their expression profiles in response to abiotic stresses using the Affymetrix GeneChip datasets including alkali (GSE17883), salt (GSE41125), and drought (GSE5040) in soybean plants to determine the relative expression of these nine genes that might be related to endoplasmic reticulum stress response, hydrogen peroxide response, or cellulose biosynthesis. It is interesting that these nine genes were significantly upregulated by at less one stress (Figure 9B). Thus, we propose that the overexpression of GmDNAJC7 enhanced the plant abiotic stress tolerance, probably by increasing the expression levels of genes related to endoplasmic reticulum stress response, hydrogen peroxide response, and cellulose biosynthesis.

5. Conclusions

In summary, 204 members of the soybean DNAJ gene family were identified, and their expression profiles in soybean response to abiotic stresses including alkali, salt, and drought were investigated. A soybean DNAJ gene, GmDNAJ7, with multiple GO terms and induced by alkali, salt, and drought stresses, was selected for further study. Overexpression of GmDNAJ7 enhanced the alkaline-salt tolerance of soybean composite plants and Arabidopsis as well as the salt and drought tolerance of Arabidopsis. The germination rate and average root length of the GmDNAJC7 OE lines were significantly higher than those of the WT and dnajc7 mutant under the NaHCO3, NaCl, and mannitol stresses. Taken together, these findings suggest that soybean GmDNAJ7 plays an important role in plant tolerance to alkaline-salt, salt, and drought stresses.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy12061419/s1, Table S1: The list of soybean DNAJ genes. Table S2: Primers used for qRT-PCR, gene cloning, and vector construction. Table S3: The GO annotations of the soybean DNAJ genes. Table S4: The annotations of genes encoding GmDNAJC7 and its predicted interacting proteins.

Author Contributions

Conceptualization, T.J. and Y.L.; Data curation, T.J. and Z.S.; Formal analysis, T.J.; Funding acquisition, Y.L.; Investigation, T.J., Z.S., S.Z. and Q.Y.; Methodology, T.J.; Project administration, Y.L.; Resources, J.G. and Y.L.; Supervision, Y.L.; Validation, T.J.; Visualization, T.J.; Writing—original draft, T.J.; Writing—review & editing, T.J. and Y.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Core Technology Development for the Breeding Program of Jiangsu Province (JBGS-2021-014).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in the Supplementary Materials.

Acknowledgments

We would like to thank Daolong Dou at Nanjing Agricultural University for kindly providing us with the vectors of pBinGFP4, and thank Peter Gresshoff’s laboratory (University of Queensland) who developed the A. rhizogenes strain K599 and share it freely around the world.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

CDSCoding sequence
EREndoplasmic reticulum
GOGene Ontology
GFPGreen fluorescent protein
H2O2Hydrogen peroxide
LRWCLeaf relative water content
MSMurashige and Skoog
MLMaximum likelihood
OEOverexpression
SPADSoil and plant analysis development
PEGPolyethylene glycol
ROSReactive oxygen species
WTWild type
YEBYeast extract broth

References

  1. Liu, Y.; Du, H.; Li, P.; Shen, Y.; Tian, Z. Pan-Genome of wild and cultivated soybeans. Cell 2020, 182, 162–176. [Google Scholar] [CrossRef] [PubMed]
  2. Araújo, S.; Beebe, S.; Crespi, M.; Delbreil, B.; González, E.; Gruber, V.; Lejeune-Henaut, I.; Link, W.; Monteros, M.J.; Prats, E. Abiotic stress responses in legumes: Strategies used to cope with environmental challenges. Crit. Rev. Plant Sci. 2015, 34, 237–280. [Google Scholar] [CrossRef]
  3. Gray, S.B.; Brady, S.M. Plant developmental responses to climate change. Dev. Biol. 2016, 419, 64–77. [Google Scholar] [CrossRef] [Green Version]
  4. Carrera, C.S.; Solís, S.M. Leaf structure and ultrastructure changes induced by heat stress and drought during seed filling in field-grown soybean and their relationship with grain yield. An. Acad. Bras. Cienc. 2021, 93, e20191388. [Google Scholar] [CrossRef] [PubMed]
  5. Mete, F.Z.; Mia, S.; Dijkstra, F.A.; Abuyusuf, M.; Hossain, A.; Agronomy, D.O.; University, S.T.; Science, D.; Sydney, T.U.O. Synergistic effects of biochar and NPK fertilizer on soybean yield in an alkaline soil. Pedosphere 2015, 25, 713–719. [Google Scholar] [CrossRef]
  6. Jin, T.; Sun, Y.; Shan, Z.; He, J.; Wang, N.; Gai, J.; Li, Y. Natural variation in the promoter of GsERD15B affects salt tolerance in soybean. Plant Biotechnol. J. 2021, 19, 1155–1169. [Google Scholar] [CrossRef] [PubMed]
  7. Wang, T.; Mcfarlane, H.E.; Persson, S. The impact of abiotic factors on cellulose synthesis. J. Exp. Bot. 2016, 67, 543–552. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  8. Georgopoulos, C.P.; Lam, B.; Lundquist-Heil, A.; Rudolph, C.F.; Yochem, J.; Feiss, M. Identification of the E. coli dnaK (groPC756) gene product. Mol. Gen. Genet. 1979, 172, 143–149. [Google Scholar] [CrossRef] [PubMed]
  9. Wang, G.; Cai, G.; Xu, N.; Zhang, L.; Sun, X.; Guan, J.; Meng, Q. Novel DnaJ protein facilitates thermotolerance of transgenic tomatoes. Int. J. Mol. Sci. 2019, 20, 367. [Google Scholar] [CrossRef] [Green Version]
  10. Wang, G.; Cai, G.; Kong, F.; Deng, Y.; Ma, N.; Meng, Q. Overexpression of tomato chloroplast-targeted DnaJ protein enhances tolerance to drought stress and resistance to Pseudomonas solanacearum in transgenic tobacco. Plant Physiol. Biochem. 2014, 82, 95–104. [Google Scholar] [CrossRef] [PubMed]
  11. Chen, S.; Qiu, G. Overexpression of seagrass DnaJ gene ZjDjB1 enhances the thermotolerance of transgenic Arabidopsis thaliana. Physiol. Mol. Biol. Plants 2021, 27, 2043–2055. [Google Scholar] [CrossRef]
  12. Yang, Y.; Qin, Y.; Xie, C.; Zhao, F.; Zhao, J.; Liu, D.; Chen, S.; Fuglsang, A.T.; Palmgren, M.G.; Schumaker, K.S.; et al. The Arabidopsis chaperone J3 regulates the plasma membrane H+-ATPase through interaction with the PKS5 kinase. Plant Cell 2010, 22, 1313–1332. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Salas-Muñoz, S.; Rodríguez-Hernández, A.A.; Ortega-Amaro, M.A.; Salazar-Badillo, F.B.; Jiménez-Bremont, J.F. Arabidopsis AtDjA3 null mutant shows increased sensitivity to abscisic acid, salt, and osmotic stress in germination and post-germination stages. Front. Plant Sci. 2016, 7, 220. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  14. Ali, F.; Wang, Q.; Fazal, A.; Wang, L.J.; Song, S.; Kong, M.J. The DnaJ-like zinc finger protein ORANGE promotes proline biosynthesis in drought-stressed Arabidopsis seedlings. Int. J. Mol. Sci. 2022, 23, 3907. [Google Scholar] [CrossRef] [PubMed]
  15. Miernyk, J.A. The J-domain proteins of Arabidopsis thaliana: An unexpectedly large and diverse family of chaperones. Cell Stress Chaperones 2001, 6, 209–218. [Google Scholar] [CrossRef]
  16. Larkin, M.A.; Blackshields, G.; Brown, N.P.; Chenna, R.; Mcgettigan, P.A.; Mcwilliam, H.; Valentin, F.; Wallace, I.M.; Wilm, A.; Lopez, R.; et al. Clustal W and Clustal X version 2.0. Bioinformatics 2007, 23, 2947–2948. [Google Scholar] [CrossRef] [Green Version]
  17. Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 2013, 30, 2725–2729. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  18. Ge, Y.; Li, Y.; Zhu, Y.M.; Bai, X.; Lv, D.K.; Guo, D.; Ji, W.; Cai, H. Global transcriptome profiling of wild soybean (Glycine soja) roots under NaHCO3 treatment. BMC Plant Biol. 2010, 10, 153. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  19. Song, Y.; Ji, D.; Li, S.; Wang, P.; Li, Q.; Xiang, F. The dynamic changes of DNA methylation and histone modifications of salt responsive transcription factor genes in soybean. PLoS ONE 2012, 7, e41274. [Google Scholar] [CrossRef] [PubMed]
  20. Carvalho, H.H.; Brustolini, O.J.; Pimenta, M.R.; Mendes, G.C.; Gouveia, B.C.; Silva, P.A.; Silva, J.C.; Mota, C.S.; Soares-Ramos, J.R.; Fontes, E.P. The molecular chaperone binding protein BiP prevents leaf dehydration-induced cellular homeostasis disruption. PLoS ONE 2014, 9, e86661. [Google Scholar] [CrossRef] [Green Version]
  21. Guan, R.; Qu, Y.; Guo, Y.; Yu, L.; Liu, Y.; Jiang, J.; Chen, J.; Ren, Y.; Liu, G.; Tian, L.; et al. Salinity tolerance in soybean is modulated by natural variation in GmSALT3. Plant J. 2014, 80, 937–950. [Google Scholar] [CrossRef] [Green Version]
  22. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−△△CT method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef]
  23. Kereszt, A.; Li, D.; Indrasumunar, A.; Nguyen, C.D.; Nontachaiyapoom, S.; Kinkema, M.; Gresshoff, P.M. Agrobacterium rhizogenes-mediated transformation of soybean to study root biology. Nat. Protoc. 2007, 2, 948–952. [Google Scholar] [CrossRef] [PubMed]
  24. Zhang, J.; Wang, J.; Jiang, W.; Liu, J.; Yang, S.; Gai, J.; Li, Y. Identification and analysis of NaHCO3 stress responsive genes in wild soybean (Glycine soja) roots by RNA-seq. Front. Plant Sci. 2016, 7, 1842. [Google Scholar] [CrossRef] [Green Version]
  25. Pérez-Patricio, M.; Camas-Anzueto, J.L.; Sanchez-Alegría, A.; Aguilar-González, A.; Gutiérrez-Miceli, F. Optical method for estimating the Chlorophyll contents in plant leaves. Sensors 2018, 18, 650. [Google Scholar] [CrossRef] [Green Version]
  26. Zhang, F.; Zhang, J. Bulk analysis by resequencing and RNA-seq identifies candidate genes for maintaining leaf water content under water deficit in maize. Physiol. Plant. 2021, 173, 1935–1945. [Google Scholar] [CrossRef] [PubMed]
  27. Clough, S.J.; Bent, A.F. Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1998, 16, 735–743. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  28. Ariyarathne, M.A.; Wone, B.W.M. Overexpression of the Selaginella lepidophylla bHLH transcription factor enhances water-use efficiency, growth, and development in Arabidopsis. Plant Sci. 2022, 315, 111129. [Google Scholar] [CrossRef] [PubMed]
  29. Yu, Y.; Duan, X.; Ding, X.; Chen, C.; Zhu, D.; Yin, K.; Cao, L.; Song, X.; Zhu, P.; Li, Q.; et al. A novel AP2/ERF family transcription factor from Glycine soja, GsERF71, is a DNA binding protein that positively regulates alkaline stress tolerance in Arabidopsis. Plant Mol. Biol. 2017, 94, 509–530. [Google Scholar] [CrossRef]
  30. Li, X.; Tang, Y.; Zhou, C.; Zhang, L.; Lv, J. A wheat WRKY transcription factor TaWRKY46 enhances tolerance to osmotic stress in transgenic Arabidopsis plants. Int. J. Mol. Sci. 2020, 21, 1321. [Google Scholar] [CrossRef] [Green Version]
  31. Harbaoui, M.; Ben Romdhane, W.; Ben Hsouna, A.; Brini, F.; Ben Saad, R. The durum wheat annexin, TdAnn6, improves salt and osmotic stress tolerance in Arabidopsis via modulation of antioxidant machinery. Protoplasma 2021, 258, 1047–1059. [Google Scholar] [CrossRef] [PubMed]
  32. Chen, J.; Li, X.; Ye, X.; Guo, P.; Hu, Z.; Qi, G.; Cui, F.; Liu, S. An S-ribonuclease binding protein EBS1 and brassinolide signaling are specifically required for Arabidopsis tolerance to bicarbonate. J. Exp. Bot. 2021, 72, 1449–1459. [Google Scholar] [CrossRef] [PubMed]
  33. Schindelin, J.; Rueden, C.T.; Hiner, M.C.; Eliceiri, K.W. The ImageJ ecosystem: An open platform for biomedical image analysis. Mol. Reprod. Dev. 2015, 82, 518–529. [Google Scholar] [CrossRef] [Green Version]
  34. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools: An integrative toolkit developed for interactive analyses of big biological data. Mol. Plant 2020, 13, 1194–1202. [Google Scholar] [CrossRef]
  35. Yang, J.Y.; Zheng, W.; Tian, Y.; Wu, Y.; Zhou, D.W. Effects of various mixed salt-alkaline stresses on growth, photosynthesis, and photosynthetic pigment concentrations of Medicago ruthenica seedlings. Photosynthetica 2011, 49, 275–284. [Google Scholar] [CrossRef]
  36. Arya, H.; Singh, M.B.; Bhalla, P.L. Towards developing drought-smart soybeans. Front. Plant Sci. 2021, 12, 750664. [Google Scholar] [CrossRef] [PubMed]
  37. Van Zelm, E.; Zhang, Y.; Testerink, C. Salt tolerance mechanisms of plants. Annu. Rev. Plant Biol. 2020, 71, 403–433. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  38. Chen, L.; Yang, H.; Fang, Y.; Guo, W.; Chen, H.; Zhang, X.; Dai, W.; Chen, S.; Hao, Q.; Yuan, S.; et al. Overexpression of GmMYB14 improves high-density yield and drought tolerance of soybean through regulating plant architecture mediated by the brassinosteroid pathway. Plant Biotechnol. J. 2021, 19, 702–716. [Google Scholar] [CrossRef] [PubMed]
  39. Bian, X.H.; Li, W.; Niu, C.F.; Wei, W.; Hu, Y.; Han, J.Q.; Lu, X.; Tao, J.J.; Jin, M.; Qin, H.; et al. A class B heat shock factor selected for during soybean domestication contributes to salt tolerance by promoting flavonoid biosynthesis. New Phytol. 2020, 225, 268–283. [Google Scholar] [CrossRef] [Green Version]
  40. Yu, T.F.; Liu, Y.; Fu, J.D.; Ma, J.; Fang, Z.W.; Chen, J.; Zheng, L.; Lu, Z.W.; Zhou, Y.B.; Chen, M.; et al. The NF-Y-PYR module integrates the abscisic acid signal pathway to regulate plant stress tolerance. Plant Biotechnol. J. 2021, 19, 2589–2605. [Google Scholar] [CrossRef]
  41. Ketehouli, T.; Zhou, Y.G.; Dai, S.Y.; Carther, K.F.I.; Sun, D.Q.; Li, Y.; Nguyen, Q.V.H.; Xu, H.; Wang, F.W.; Liu, W.C.; et al. A soybean calcineurin B-like protein-interacting protein kinase, GmPKS4, regulates plant responses to salt and alkali stresses. J. Plant Physiol. 2021, 256, 153331. [Google Scholar] [CrossRef] [PubMed]
  42. Singh, M.B.; Lohani, N.; Bhalla, P.L. The role of endoplasmic reticulum stress response in pollen development and heat stress tolerance. Front. Plant Sci. 2021, 12, 661062. [Google Scholar] [CrossRef] [PubMed]
  43. Wan, S.; Jiang, L. Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in plants. Protoplasma 2016, 253, 753–764. [Google Scholar] [CrossRef] [PubMed]
  44. Cakır Aydemir, B.; Yüksel zmen, C.; Kibar, U.; Mutaf, F.; Büyük, P.B.; Bakır, M.; Ergül, A. Salt stress induces endoplasmic reticulum stress-responsive genes in a grapevine rootstock. PLoS ONE 2020, 15, e0236424. [Google Scholar] [CrossRef] [PubMed]
  45. Xiang, Y.; Sun, X.; Gao, S.; Qin, F.; Dai, M. Deletion of an endoplasmic reticulum stress response element in a ZmPP2C-A gene facilitates drought tolerance of maize seedlings. Mol. Plant 2017, 10, 456–469. [Google Scholar] [CrossRef] [Green Version]
  46. Sandhu, J.; Irvin, L.; Liu, K.; Staswick, P.; Zhang, C.; Walia, H. Endoplasmic reticulum stress pathway mediates the early heat stress response of developing rice seeds. Plant Cell Environ. 2021, 44, 2604–2624. [Google Scholar] [CrossRef]
  47. Qureshi, M.K.; Gawroński, P.; Munir, S.; Jindal, S.; Kerchev, P. Hydrogen peroxide-induced stress acclimation in plants. Cell Mol. Life Sci. 2022, 79, 129. [Google Scholar] [CrossRef]
  48. Neill, S.; Desikan, R.; Hancock, J. Hydrogen peroxide signalling. Curr. Opin. Plant Biol. 2002, 5, 388–395. [Google Scholar] [CrossRef]
  49. Hasanuzzaman, M.; Raihan, M.R.H. Regulation of reactive oxygen species and antioxidant defense in plants under salinity. Int. J. Mol. Sci. 2021, 22, 9326. [Google Scholar] [CrossRef]
  50. Wu, W.W.; He, S.S.; An, Y.Y. Hydrogen peroxide as a mediator of 5-aminolevulinic acid-induced Na+ retention in roots for improving salt tolerance of strawberries. Physiol. Plant. 2019, 167, 5–20. [Google Scholar] [CrossRef] [Green Version]
  51. Yan, Y.; Wang, P.; Lu, Y.; Bai, Y.; Wei, Y.; Liu, G.; Shi, H. MeRAV5 promotes drought stress resistance in cassava by modulating hydrogen peroxide and lignin accumulation. Plant J. 2021, 107, 847–860. [Google Scholar] [CrossRef] [PubMed]
  52. Endler, A.; Kesten, C.; Schneider, R.; Zhang, Y.; Ivakov, A.; Froehlich, A.; Funke, N.; Persson, S. A mechanism for sustained cellulose synthesis during salt stress. Cell 2015, 162, 1353–1364. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  53. Shafi, A.; Gill, T.; Zahoor, I.; Ahuja, P.S.; Sreenivasulu, Y.; Kumar, S.; Singh, A.K. Ectopic expression of SOD and APX genes in Arabidopsis alters metabolic pools and genes related to secondary cell wall cellulose biosynthesis and improve salt tolerance. Mol. Biol. Rep. 2019, 46, 1985–2002. [Google Scholar] [CrossRef] [PubMed]
  54. Zheng, M.; Liu, X.; Lin, J.; Liu, X.; Wang, Z.; Xin, M.; Yao, Y.; Peng, H.; Zhou, D.X.; Ni, Z.; et al. Histone acetyltransferase GCN5 contributes to cell wall integrity and salt stress tolerance by altering the expression of cellulose synthesis genes. Plant J. 2019, 97, 587–602. [Google Scholar] [PubMed]
  55. Moore, J.P.; Vicré-Gibouin, M.; Farrant, J.M.; Driouich, A. Adaptations of higher plant cell walls to water loss: Drought vs. desiccation. Physiol. Plant. 2008, 134, 237–245. [Google Scholar] [CrossRef] [PubMed]
  56. Shukla, A.; Kaur, M.; Kanwar, S.; Kaur, G.; Sharma, S.; Ganguli, S.; Kumari, V.; Mazumder, K.; Pandey, P.; Rouached, H.; et al. Wheat inositol pyrophosphate kinase TaVIH2-3B modulates cell-wall composition and drought tolerance in Arabidopsis. BMC Biol. 2021, 19, 261. [Google Scholar] [CrossRef]
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Figure 1. The phylogenetic tree of the DNAJ superfamily proteins in soybean and Arabidopsis. The 204 soybean protein sequences and 89 Arabidopsis protein sequences [15] of the DNAJ superfamily were downloaded from the Phytozome database. The unrooted phylogenetic tree was constructed using MEGA6.0 based on the maximum likelihood (ML) algorithm with 1000 bootstraps, and displayed by iTOL v6 (https://itol.embl.de/, accessed on 7 April 2022).
Figure 1. The phylogenetic tree of the DNAJ superfamily proteins in soybean and Arabidopsis. The 204 soybean protein sequences and 89 Arabidopsis protein sequences [15] of the DNAJ superfamily were downloaded from the Phytozome database. The unrooted phylogenetic tree was constructed using MEGA6.0 based on the maximum likelihood (ML) algorithm with 1000 bootstraps, and displayed by iTOL v6 (https://itol.embl.de/, accessed on 7 April 2022).
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Figure 2. The heatmap of the expression profiles for the soybean DNAJ genes in response to alkali, salt, and drought stresses. The heatmap was constructed using the TBtools [34]. The DNAJ gene expression profiles in soybean under alkaline-salt, saline, and drought stresses were obtained by exploring the previously published Affymetrix GeneChip datasets: GSE17883, GSE41125, GSE50408. The color key represents the fold change (stress vs. control) scaled by row, with red to blue, indicating upregulation to downregulation.
Figure 2. The heatmap of the expression profiles for the soybean DNAJ genes in response to alkali, salt, and drought stresses. The heatmap was constructed using the TBtools [34]. The DNAJ gene expression profiles in soybean under alkaline-salt, saline, and drought stresses were obtained by exploring the previously published Affymetrix GeneChip datasets: GSE17883, GSE41125, GSE50408. The color key represents the fold change (stress vs. control) scaled by row, with red to blue, indicating upregulation to downregulation.
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Figure 3. The expression pattern of GmDNAJC7. (A) GmDNAJC7 expression in various tissues of soybean (Glycine max) variety Williams 82, using data downloaded from Phytozome (https://phytozome-next.jgi.doe.gov/, accessed on 27 April 2022). The relative expression of GmDNAJC7 was calculated using its expression level in seeds as the control. (B–D) Relative expression of GmDNAJC7 in response to alkali, salt, and drought stresses. Fourteen-day-old Tianlong1 soybean seedlings were subjected to NaHCO3 (90 mM), NaCl (200 mM), or PEG (20%) treatments for 6, 12, 24, 36 or 48 h. The roots of the soybean seedlings were sampled separately, and the relative expression levels of GmDNAJC7 were determined by qRT-PCR, and compared with the samples of the 0 mM treatment at each time point as the control. GmActin11 was used as the reference gene. Data represent the mean ± standard deviation of three biological replications with three repeats within each replication (n = 3 × 3 = 9). Data with the same letters in lowercases (a and b) above bars indicate no significant differences at the 0.05 level according to the Duncan’s multiple range test.
Figure 3. The expression pattern of GmDNAJC7. (A) GmDNAJC7 expression in various tissues of soybean (Glycine max) variety Williams 82, using data downloaded from Phytozome (https://phytozome-next.jgi.doe.gov/, accessed on 27 April 2022). The relative expression of GmDNAJC7 was calculated using its expression level in seeds as the control. (B–D) Relative expression of GmDNAJC7 in response to alkali, salt, and drought stresses. Fourteen-day-old Tianlong1 soybean seedlings were subjected to NaHCO3 (90 mM), NaCl (200 mM), or PEG (20%) treatments for 6, 12, 24, 36 or 48 h. The roots of the soybean seedlings were sampled separately, and the relative expression levels of GmDNAJC7 were determined by qRT-PCR, and compared with the samples of the 0 mM treatment at each time point as the control. GmActin11 was used as the reference gene. Data represent the mean ± standard deviation of three biological replications with three repeats within each replication (n = 3 × 3 = 9). Data with the same letters in lowercases (a and b) above bars indicate no significant differences at the 0.05 level according to the Duncan’s multiple range test.
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Figure 4. Subcellular localization of the GmDNAJC7–GFP fusion protein in the leaf cells of Nicotiana benthamiana. Bars, 10 μm.
Figure 4. Subcellular localization of the GmDNAJC7–GFP fusion protein in the leaf cells of Nicotiana benthamiana. Bars, 10 μm.
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Figure 5. Alkaline-salt tolerance analyses of transgenic soybean composite plants. (A) Identification of positive transgenic soybean plants by green fluorescence using a stereoscopic fluorescence microscope. Bar = 1 cm. (B) Phenotype of transgenic soybean composite plants under 0 mM or 90 mM NaHCO3 for five days. Bar = 5 cm. (C,D) The SPAD (Soil and Plant Analysis Development) value for the chlorophyll content and leaf relative water content (LRWC) of transgenic soybean composite plants under 0 mM or 90 mM NaHCO3 for five days, respectively. Data represent the mean ± standard deviation of three biological replications and each repeat contained four independent transgenic plants (n = 3 × 4 = 12). The soybean variety of “TianLong1” was used. Differences were evaluated using the two-tailed Student’s t tests (** p < 0.01).
Figure 5. Alkaline-salt tolerance analyses of transgenic soybean composite plants. (A) Identification of positive transgenic soybean plants by green fluorescence using a stereoscopic fluorescence microscope. Bar = 1 cm. (B) Phenotype of transgenic soybean composite plants under 0 mM or 90 mM NaHCO3 for five days. Bar = 5 cm. (C,D) The SPAD (Soil and Plant Analysis Development) value for the chlorophyll content and leaf relative water content (LRWC) of transgenic soybean composite plants under 0 mM or 90 mM NaHCO3 for five days, respectively. Data represent the mean ± standard deviation of three biological replications and each repeat contained four independent transgenic plants (n = 3 × 4 = 12). The soybean variety of “TianLong1” was used. Differences were evaluated using the two-tailed Student’s t tests (** p < 0.01).
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Figure 6. The effect of GmDNAJC7 ectopic expression on Arabidopsis tolerance to NaHCO3 treatment. (A) Seed germination assay of different Arabidopsis lines on 1/2 Murashige and Skoog (MS) agar plates and 1/2 MS supplied with various concentrations of NaHCO3. Photographs were taken 10 days after treatment. WT: wild type; #1, #2, #3: Arabidopsis lines overexpressing GmDNAJC7; dnajc7: Arabidopsis mutant. (B,C) Seed germination rates after five days and cotyledon greening rates after 10 days for different Arabidopsis lines as shown in A. (D) Phenotypes of Arabidopsis lines subjected to 0, 3, 4, and 5 mM NaHCO3 treatment for 10 days, respectively. (E) Root lengths of Arabidopsis lines as shown in (D). Root lengths were analyzed using ImageJ software. Bars represent the mean values ± standard deviations of three independent experiments and each repeat contained at least four independent plants per line for each treatment (n ≥ 12). Bars with the same letters in lowercase above bars indicate no significant differences at the 0.05 level according to the Duncan’s multiple range tests.
Figure 6. The effect of GmDNAJC7 ectopic expression on Arabidopsis tolerance to NaHCO3 treatment. (A) Seed germination assay of different Arabidopsis lines on 1/2 Murashige and Skoog (MS) agar plates and 1/2 MS supplied with various concentrations of NaHCO3. Photographs were taken 10 days after treatment. WT: wild type; #1, #2, #3: Arabidopsis lines overexpressing GmDNAJC7; dnajc7: Arabidopsis mutant. (B,C) Seed germination rates after five days and cotyledon greening rates after 10 days for different Arabidopsis lines as shown in A. (D) Phenotypes of Arabidopsis lines subjected to 0, 3, 4, and 5 mM NaHCO3 treatment for 10 days, respectively. (E) Root lengths of Arabidopsis lines as shown in (D). Root lengths were analyzed using ImageJ software. Bars represent the mean values ± standard deviations of three independent experiments and each repeat contained at least four independent plants per line for each treatment (n ≥ 12). Bars with the same letters in lowercase above bars indicate no significant differences at the 0.05 level according to the Duncan’s multiple range tests.
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Figure 7. The effect of GmDNAJC7 ectopic expression on Arabidopsis tolerance to NaCl treatment. (A) Seed germination assay of different Arabidopsis lines on 1/2 MS agar plates and 1/2 MS supplied with various concentrations of NaCl. Photographs were taken 10 days after treatment. WT: wild type; #1, #2, #3: Arabidopsis lines overexpressing GmDNAJC7; dnajc7: Arabidopsis mutant. (B,C) Seed germination rates after five days and cotyledon greening after 10 days for different Arabidopsis lines as shown in A. (D) Phenotypes of Arabidopsis lines subjected to 0, 100, 125, and 150 mM NaCl treatment for 10 days, respectively. (E) Root lengths of Arabidopsis lines as shown in (D). Root lengths were analyzed using ImageJ software. Bars represent the mean values ± standard deviations of three independent experiments and each repeat contained at least four independent plants per line for each treatment (n ≥ 12). Bars with the same letters in lowercase above bars indicate no significant differences at the 0.05 level according to the Duncan’s multiple range tests.
Figure 7. The effect of GmDNAJC7 ectopic expression on Arabidopsis tolerance to NaCl treatment. (A) Seed germination assay of different Arabidopsis lines on 1/2 MS agar plates and 1/2 MS supplied with various concentrations of NaCl. Photographs were taken 10 days after treatment. WT: wild type; #1, #2, #3: Arabidopsis lines overexpressing GmDNAJC7; dnajc7: Arabidopsis mutant. (B,C) Seed germination rates after five days and cotyledon greening after 10 days for different Arabidopsis lines as shown in A. (D) Phenotypes of Arabidopsis lines subjected to 0, 100, 125, and 150 mM NaCl treatment for 10 days, respectively. (E) Root lengths of Arabidopsis lines as shown in (D). Root lengths were analyzed using ImageJ software. Bars represent the mean values ± standard deviations of three independent experiments and each repeat contained at least four independent plants per line for each treatment (n ≥ 12). Bars with the same letters in lowercase above bars indicate no significant differences at the 0.05 level according to the Duncan’s multiple range tests.
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Figure 8. The effect of GmDNAJC7 ectopic expression on Arabidopsis tolerance to mannitol treatment. (A) Seed germination assay of different Arabidopsis lines on 1/2 MS agar plates and 1/2 MS supplied with various concentrations of mannitol. Photographs were taken 10 days after mannitol treatment. WT: wild type; #1, #2, #3: Arabidopsis lines overexpressing GmDNAJC7; dnajc7: Arabidopsis mutant. (B) Seed germination rates after five days for different Arabidopsis lines as shown in A. (C) Phenotypes of Arabidopsis lines subjected to 0, 200, and 300 mM mannitol treatment for 10 days, respectively. (D) Root length of Arabidopsis lines as shown in (C). Root lengths were analyzed using ImageJ software. Bars represent the mean values ± standard deviations of three independent experiments and each repeat contained at least four independent plants per line for each treatment (n ≥ 12). Bars with the same letters in lowercase above bars indicate no significant differences at the 0.05 level according to the Duncan’s multiple range tests.
Figure 8. The effect of GmDNAJC7 ectopic expression on Arabidopsis tolerance to mannitol treatment. (A) Seed germination assay of different Arabidopsis lines on 1/2 MS agar plates and 1/2 MS supplied with various concentrations of mannitol. Photographs were taken 10 days after mannitol treatment. WT: wild type; #1, #2, #3: Arabidopsis lines overexpressing GmDNAJC7; dnajc7: Arabidopsis mutant. (B) Seed germination rates after five days for different Arabidopsis lines as shown in A. (C) Phenotypes of Arabidopsis lines subjected to 0, 200, and 300 mM mannitol treatment for 10 days, respectively. (D) Root length of Arabidopsis lines as shown in (C). Root lengths were analyzed using ImageJ software. Bars represent the mean values ± standard deviations of three independent experiments and each repeat contained at least four independent plants per line for each treatment (n ≥ 12). Bars with the same letters in lowercase above bars indicate no significant differences at the 0.05 level according to the Duncan’s multiple range tests.
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Figure 9. The prediction and relative expression of the GmDNAJC7-interacting proteins/genes. (A) The predicted protein–protein interaction network of GmDNAJC7 using the STRING database and Cytoscape software. Nodes represent the proteins and edges represent interactions. (B) Heatmap of the expression profiles for the predicted GmDNAJC7-interacting proteins encoding genes in response to alkali, salt, and drought stresses. The heatmap was constructed using the TBtools. Gene expression profiles in soybean under alkaline salt, saline and drought stresses were obtained by using previously published Affymetrix GeneChip datasets: GSE17883, GSE41125, GSE50408. The color key represents the fold change (stress vs. control) scaled by row, with red to blue indicating upregulation to downregulation.
Figure 9. The prediction and relative expression of the GmDNAJC7-interacting proteins/genes. (A) The predicted protein–protein interaction network of GmDNAJC7 using the STRING database and Cytoscape software. Nodes represent the proteins and edges represent interactions. (B) Heatmap of the expression profiles for the predicted GmDNAJC7-interacting proteins encoding genes in response to alkali, salt, and drought stresses. The heatmap was constructed using the TBtools. Gene expression profiles in soybean under alkaline salt, saline and drought stresses were obtained by using previously published Affymetrix GeneChip datasets: GSE17883, GSE41125, GSE50408. The color key represents the fold change (stress vs. control) scaled by row, with red to blue indicating upregulation to downregulation.
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Jin, T.; Shan, Z.; Zhou, S.; Yang, Q.; Gai, J.; Li, Y. GmDNAJC7 from Soybean Is Involved in Plant Tolerance to Alkaline-Salt, Salt, and Drought Stresses. Agronomy 2022, 12, 1419. https://doi.org/10.3390/agronomy12061419

AMA Style

Jin T, Shan Z, Zhou S, Yang Q, Gai J, Li Y. GmDNAJC7 from Soybean Is Involved in Plant Tolerance to Alkaline-Salt, Salt, and Drought Stresses. Agronomy. 2022; 12(6):1419. https://doi.org/10.3390/agronomy12061419

Chicago/Turabian Style

Jin, Ting, Zhong Shan, Shuang Zhou, Qianqian Yang, Junyi Gai, and Yan Li. 2022. "GmDNAJC7 from Soybean Is Involved in Plant Tolerance to Alkaline-Salt, Salt, and Drought Stresses" Agronomy 12, no. 6: 1419. https://doi.org/10.3390/agronomy12061419

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