Introduction

Drought is one of the main environmental constraints to crop productivity worldwide by affecting various plant physiological and biochemical processes, such as photosynthesis, respiration, translocation, ion uptake, carbohydrates and nutrient metabolism [1–3]. Detrimental impact of drought on soybean has been a matter of great concern since this important legume is considered as one of the world’s leading economic oilseed crops, providing the largest source of vegetable oil, proteins, macronutrients and minerals for human consumption and animal feed with increasing demands year by year [4].

In the past two decades, great efforts have been made to elucidate the mechanisms of drought tolerance in plants through various molecular and genomic approaches, and a number of genes, which respond to drought, have been identified [5]. Occupying approximately 7 % of the total genes found in a plant genome, transcription factors (TFs) are important regulators of gene expression, thus controlling diverse biological processes including growth, development and responses to environmental stimuli [6]. Thanks to the completion of the soybean genome sequencing project in 2010, more than 55 TF families have been identified by various TF databases, among which the NAC (NAM—no apical meristem, ATAF—Arabidopsis transcription activation factor, and CUC—cup-shaped cotyledon) TF family was estimated to consist of more than 180 members [7–11]. The NAC TFs, which share a preserved DNA-binding domain located at the N-terminal end, and a variable domain at the C-terminal end, were first described in Petunia more than a decade ago [12]. They are now known to comprise a large family of plant-specific TFs identified in many plant species, including crops, and have been reported to be involved in regulation of a number of biological processes, including plant responses to different environmental stresses [13–17].

Meng et al. [18] identified the first six soybean GmNAC genes called GmNAC1-6, and the expression of these genes in soybean under osmotic stress was thoroughly examined [6]. Later on, Tran et al. [19] proved that nine out of 31 GmNAC genes studied in soybean seedlings were induced by dehydration, high salinity, cold and/or ABA treatments. More recently, a comprehensive analysis of GmNAC family by Le et al. [20] identified 152 full-length GmNAC TFs in soybean, of which 11 members were found to be membrane-associated proteins. Expression analyses using real-time quantitative PCR (RT-qPCR) verified that out of 38 predicted abiotic stress-related GmNAC genes, twenty-five and six GmNACs were induced and repressed, respectively, by twofold or more in roots and/or shoots of soybean seedlings subjected to a dehydration treatment [20]. In addition, a number of these genes displayed differential drought-responsive expression profiles in various tissues at the same development stage, or in the same tissue but at different development stages [21].

In the present study, we aimed to examine whether there was correlation between differential expression of GmNAC genes in the shoots and the differential drought-tolerant ability of two soybean cultivars, DT51 (a drought-tolerant variety) and MTD720 (a drought-sensitive variety), displaying contrasting drought-tolerant phenotypes that were observed in a previous study [22]. The results obtained in this work have enabled us to propose a list of GmNAC candidate genes that are deserved for further in-depth analyses, leading to development of drought-tolerant soybean varieties by improving shoot-related traits through genetic engineering.

Materials and methods

Plant growth and drought experiments

Growing conditions and the drought treatment of the drought-tolerant DT51 and drought-sensitive MTD720 soybean cultivars were described in a previous study [22]. Plants were well-watered and grown in plastic tubes (80 cm in height and 10 cm in diameter), containing a mixture of soil, coconut fiber and cow pat (6:2:2 w/w) from Southern Fertilizer Company, for 12 days under greenhouse conditions (30/28 °C day/night temperatures, photoperiod of 12/12 h, and 60–70 % humidity). The 12-day-old plants were subsequently subjected to a non-irrigation period of 15 days until the soil moisture content (SMC) was decreased to 5–6 %. Control plants were regularly watered once per day to maintain SMC at 65–70 %. After the drought treatment, control and drought-treated plants were carefully removed from soil by longitudinally cutting the plastic tubes. The shoot tissues (vegetative developmental stage V6), including leaves and stem, were collected from well-watered and drought-stressed plants in three biological replicates, and immediately frozen in liquid nitrogen for subsequent expression analyses using RT-qPCR.

Total RNA isolation and RT-qPCR

Total RNA isolation, cDNA synthesis and RT-qPCR were carried out as previously described [23]. Twenty-three dehydration-responsive GmNAC genes along with their specific primers were selected from the study conducted by Le et al. [20]. Detailed information about the selected GmNAC genes and their specific primers was provided in Table S1. Fbox was used as reference gene and 2−ΔΔCt method was used in analysis of expression levels of the GmNAC genes [24]. The amplification efficiencies of 24 primer pairs in the shoot tissues (23 selected GmNAC and Fbox reference genes) were calculated using LinRegPCR software (version 2012.0).

In silico expression analysis

Expression data of GmNAC genes obtained under a wide variety of growth conditions, including biotic stress, alkaline stress and photoperiod change, as well as in different shoot growth stages, were extracted from Genevestigator (https://www.genevestigator.ethz.ch/) [25].

Statistical analysis

A GmNAC gene was considered as drought-responsive if its expression changed by at least twofold (P < 0.05) by drought treatment. For comparative expression analysis of GmNACs between DT51 and MTD720, differential expression ratio with at least twofold was regarded as significant. Statistical significance of differential expression within a cultivar, or between two cultivars under either normal or drought treatment was assessed by Student’s t test (one tail, unpaired, equal variance) with the P-value < 0.05.

Results

In this study, we examined the potential contribution of GmNAC genes to shoot-related traits, which affect drought-tolerant capacity of soybean, by comparing differential expression of 23 drought-responsive GmNAC genes in shoots of two contrasting drought-responsive cultivars DT51 and MTD720. The contrasting drought-responsive phenotypes of DT51 (drought-tolerant) and MTD720 (drought-sensitive) were determined based on the shoot-related data and drought-tolerant index as summarized in Table S2 [22]. Seventeen (group A) up- and six (group B) down-regulated GmNAC genes, which exhibited the highest expression change in shoots and/or roots of soybean seedlings by dehydration [20], were selected for comparative expression analysis in shoots of unstressed and drought-stressed DT51 and MTD720 plants using RT-qPCR (Table S1). In DT51, out of 17 GmNAC genes of group A, we found 6 genes, including GmNAC006, 038, 043, 085, 092 and 101, which were induced more than twofold in the shoots under water deficit (Table 1). We recorded no significant alteration in expression of the remaining genes belonged to group A. As for the 6 GmNAC genes of group B, drought treatment significantly repressed the expression of two genes, GmNAC027 and 083, in DT51 shoots, but did not alter the expression of the 4 remaining GmNAC genes (Table 1).

Table 1 Expression of 23 GmNAC genes in shoots of DT51 and MTD720 upon 15-day-drought treatment
Full size table

In comparison with the drought-tolerant DT51, in the drought-sensitive MTD720, six genes of group A (GmNAC038, 043, 044, 085, 099 and 101) was up-regulated in shoots by drought stress (Table 1). Hence, four genes (GmNAC038, 043, 085 and 101) shared similar response in both cultivars upon the drought treatment. The expression of the rest of the GmNAC genes belonged to group A did not significantly alter in MTD720 shoots under drought stress. Regarding the GmNAC genes in group B, data in Table 1 indicated that out of six GmNACs, GmNAC071 and 113 were significantly induced in the shoots of MTD720 by drought treatment, while the remaining four GmNACs displayed insignificant change in drought-stressed MTD720 shoots.

The RT-qPCR data also allowed us to compare the expression levels of the selected GmNAC genes between DT51 and MTD720 shoots under the same conditions. Data analysis indicated that under normal growing condition, eight GmNAC genes (GmNAC019, 043, 071, 083, 085, 095, 101 and 148) had higher expression levels in DT51 shoots than in MTD720 shoots (Table 2). Meanwhile, only two genes, GmNAC006 and 052, exhibited significantly higher expression levels in MTD720 shoots relative to DT51 shoots (Table 2). Under drought stress, we also detected more GmNAC genes with greater expression levels in DT51 shoots than in MTD720 shoots. Specifically, six genes, namely GmNAC019, 043, 062, 085, 095 and 101, expressed more highly in DT51 shoots relative to MTD720 shoots, whereas five genes, GmNAC038, 044, 099, 083 and 113, accumulated higher amounts of transcript in MTD720 shoots in comparison with DT51 shoots (Table 2). These results together indicated that GmNAC043, 085 and 101 were induced by drought in both culivars and had higher induction levels in DT51 shoots than in MTD720 shoots under both normal and drought conditions.

Table 2 Comparison of the expression levels of 23 GmNAC genes in shoots of DT51 and MTD720
Full size table

In addition, in silico analysis using expression data housed in Genevestigator [25] demonstrated that among 23 examined GmNACs, twelve genes, including GmNAC011, 019, 022, 043, 052, 071, 085, 092, 101, 102, 109 and 148, showed altered expression in soybean under various stressful conditions, such as biotic stress, alkaline stress, photoperiod change, and in different growth stages, suggesting their involvement in soybean responses to various types of stresses, as well as in regulation of soybean growth and development (Fig. S1).

Discussion

It is well established that breeding or genetic engineering for appropriate shoot-related traits could enhance tolerance of crops to drought [26]. NAC TFs have been shown to control many shoot-related traits in plant responses to drought. For example, in rice, SNAC1 was induced mainly in guard cells by drought treatment, and its overexpression resulted in an increase in drought tolerance under field conditions which was associated with improved shoot-related traits. Specifically, transgenic SNAC1 rice plants showed delayed leaf-rolling and reduced rate of water loss resulted from increased stomatal closure as compared with the wild-type plants [27]. In another research, drought tolerance of transgenic rice overexpressing OsNAC6 was significantly enhanced at least by restricting shoot growth rate when only limited resource was available during stress [28]. Our previous morphological and physiological investigations demonstrated that the higher drought-tolerant degree of DT51 versus MTD720 was associated with improvement of both shoot-related and root-related traits [22], whose regulations are quite complex and involve many molecular and biochemical processes [26, 29]. In present study, to examine whether drought-responsive GmNAC genes might be involved in improvement of drought tolerance of DT51 versus MTD720 in association with shoot-related traits, we performed a differential expression analysis of the selected 23 drought-responsive GmNAC genes in DT51 and MTD720 under both well-watered and drought conditions. Our results revealed that there were more GmNAC genes induced by drought treatment in MTD720 than in DT51 (Table 1). This observation might indicate that more GmNAC genes should be induced and/or activated in response to stress in susceptible plants to enable them to adapt better to adverse environmental conditions.

In addition, we found that in the shoot tissues, there were eight and six GmNAC genes with higher expression levels in DT51 than in MTD720 under non-stressed and stressed conditions, respectively, whereas there were only two and four genes, respectively, exhibiting higher expression levels in MTD720 than in DT51 under well-watered and drought conditions (Table 2). This result implies that the higher number of GmNAC genes, which showed higher expression levels in the shoots of DT51 versus MTD720, under both normal and water deficit conditions, might contribute to better shoot-related traits of DT51, and thus higher drought-tolerant capacity, in comparison with MTD720 (Table S2). Thus, the results of our study suggest a positive correlation between the number of GmNAC genes, which showed higher expression levels, and the improved drought-tolerant degree, at least in the case of these two contrasting drought-responsive cultivars. In fact, this correlation was not only observed in shoots but also in roots of DT51 and MTD720 as shown by our previous study [23]. These two studies together have clearly shown that several GmNAC genes act in a tissue-specific manner and/or genotype-dependent under both normal and drought conditions [23]. Specifically, our results revealed that (i) the numbers of drought-inducible GmNAC genes in roots and shoots of each cultivar are different. In DT51, eleven genes (GmNAC011, 022, 027, 043, 085, 092, 095, 099, 101, 102 and 109) were up-regulated in drought-treated roots, whereas only six genes showed induction in drought-stressed shoots (GmNAC006, 038, 043, 085, 092 and 101). In MTD720, nine genes (GmNAC022, 038, 043, 062, 085, 095, 099, 101 and 109) and eight genes (GmNAC038, 043, 044, 071, 085, 099, 101 and 113) were up-regulated by drought in roots and shoots, respectively; (ii) in both two cultivars, several GmNAC genes were induced in shoots, but not in roots, and vice versa some GmNACs are induced in roots but not in shoots by drought. For instance, GmNAC011 was found to be induced in roots but not in shoots of DT51, whereas GmNAC006 was of DT51 by water withholding treatment; (iii) different GmNAC genes were found to be repressed in shoots and roots of each cultivar by drought. For instance, only GmNAC148 was down-regulated in drought-treated DT51 roots, whereas GmNAC027 and 083 were repressed in drought-stressed DT51 shoots. On the other hand, eight drought-repressive genes (GmNAC006, 027, 011, 019, 071, 083, 113 and 148) were found in MTD720 roots, while none was found in MTD720 shoots during drought treatment; and (iv) our data also showed that the numbers of GmNAC genes, which exhibited higher expression levels in DT51 roots and shoots under both normal and drought conditions relative to MTD720, were different. Specifically, seven (GmNAC017, 085, 092, 095, 101, 109 and 148) and thirteen (GmNAC006, 011, 017, 019, 022, 027, 071, 083, 085, 092, 095, 101 and 109) GmNAC genes displayed higher transcript levels in DT51 roots than in MTD720 roots under normal and drought treatments, respectively. On the other hand, with respect to well-watered and drought-treated shoots, eight (GmNAC019, 043, 071, 083, 085, 095, 101 and 148) and six (GmNAC019, 043, 062, 085, 095 and 101) GmNAC genes showed higher expression levels in DT51, respectively, than in MTD720.

Our findings revealed that the drought-responsive GmNAC genes might play particular role in regulating drought responses in different soybean cultivars that might have their unique defense strategies against drought. In agreement with this observation, a number of GmNAC genes also displayed different stress-responsive expression patterns in W82 [20] when compared with DT51 and/or MTD720 ([23] and this work). Alternatively, the difference in age of the plants and/or in treatment methods applied might result in the difference in gene expression profile found in W82 versus DT51 and/or MTD720. In study of Le et al. [20], 12-day-old W82 seedlings were dehydrated on a filter paper for 10 h, whereas in our study, drought treatment was performed on 12-day-old soil-grown plants for a period of 15 days (thus, the age of the plants used in expression analysis in this study was 27-day-old) by withholding water. Taken together, our data suggested that drought-responsive expression of a significant number of GmNAC genes is genotype- and/or tissue-dependent.

Among the examined genes, three genes, GmNAC043, 085 and 101, were in our particular interest since these genes showed (i) a similar up-regulation tendency in both cultivars under drought, and (ii) exhibited higher expression levels in DT51 shoots than in MTD720 shoots not only under stressed but also non-stressed conditions (Tables 1, 2). In a phylogenetic analysis, GmNAC043 (previously named GmNAC3) was assigned to the stress-responsive NAC subgroup [6], containing three well-known positive regulators ANAC019, ANAC055 and ANAC072 that have been demonstrated to enhance drought tolerance in Arabidopsis transgenic plants [30]. GmNAC085 is another attractive candidate gene, which encodes a protein sharing 39 % amino acid identity with SNAC1, one of the most well-known NAC genes subjected to field test in transgenic rice as previously discussed [27]. Additionally, a search in Genevestigator database indicated that all three genes GmNAC043, 085 and 101 were inducible by various treatments, such as biotic stress, alkaline stress and photoperiod change, as well as showed development stage-related expression (Fig. S1). These findings suggest that these three genes play roles not only in soybean responses to drought but also to other kinds of stresses, broadening their potential use in genetic engineering for improved tolerance to various types of stresses.