Gene identification and sequence analysis of ThSOS genes
In total, 5 candidate SOS genes (ThSOS1–ThSOS5) were selected and identified. The proteins of 5 ThSOS ranged from 213 to 1165 amino acids (aa) in length (Table 2). Large variations were found in theoretical pI values (ranging from 4.76 to 6.42) and MW (from 22.42-128.83 kDa) among the 5 ThSOS genes. The prediction results showed that 5 ThSOS genes were localized in the plasma membrane, cytoplasm, extracellular space or chloroplasts, respectively (Table 2).
To determine the subclass of ThSOS genes, a phylogenetic analysis was performed using the sequences of ThSOS and SOS proteins from other species (Fig. 1A; Tab.S1). The results revealed that ThSOS1, 2, 3 genes were closely related to the SOS1, 2, 3 subfamilies of Arabidopsis. ThSOS3 belonged to the SOS3 (CBL4) subfamily, being clustered into the same clade with PtrSOS3, MnSOS3 and AtSOS3. Multiple sequence alignment analysis showed that ThSOS3 was closely related to PtrSOS3 (XP-002318422.1) from P. trichocarpa, MnSOS3 (XP-010100753.1) from Morus notabilis and AtSOS3-1 (AT5G24270) from Arabidopsis thaliana (Fig. 1B).
Characterization of cis-elements in ThSOS gene promoters
The upstream sequence ~2 kb promoter regions of each of the 3 ThSOS genes were cloned and used to identify the cis-elements using the PlantCARE database. Numerous stress related consensus cis-acting elements were detected, including abscisic acid responsiveness (ABRE), antioxidant response element (ARE) and TC-rich repeats. Moreover, ThSOS1 included one abiotic stress related element, MYB binding site (MBS); nine hormone stress related elements, such as salicylic acid responsiveness element (TCA-element), MeJA-responsiveness element (CGTCA-motif or TGACG-motif), gibberellin-responsiveness element (TATC-box), and auxin-responsiveness element (TGA-element); and one development related endosperm expression element (GCN4-motif). ThSOS3 contained MBS and LTR abiotic stress response elements, consisting of 5 hormone stress related elements, auxin-responsive elements (TGA-element or AuxRR-core) and salicylic acid responsiveness elements (TCA-element). Similarly, ThSOS4 contained many abiotic stress and hormone stress related elements, specifically as shown in Fig. 2. This result indicated that ThSOS might be involved in stress responses (abiotic stresses and hormone treatment) as well as plant development.
Expression of ThSOS genes under abiotic stresses and ABA treatment
To analyze the relative abundance of ThSOS genes, the expression profiles of 5 ThSOS genes under different stresses (NaCl, PEG6000), or hormone treatment (ABA), were measured using qRT-PCR.
In roots under NaCl stress, most ThSOS gene expressions were upregulated. Notably, ThSOS4 and ThSOS5 exhibited upregulated expression at all study stress points. The highest expression levels of ThSOS4 and ThSOS5 were induced 8.02-fold and 4.86-fold, respectively. The other three ThSOS genes, ThSOS1, ThSOS2 and ThSOS3, were downregulated at the initial stress time point and upregulated t later stages. The lowest expression levels of these three ThSOS genes in roots all occurred at 6 h: the expression levels of ThSOS1, ThSOS2 and ThSOS3 were 3.55%, 6.11% and 0.20%, respectively. These results indicate that these 3 genes can respond rapidly to salt stress in T. hispida roots. In leaves, ThSOS gene expression was mainly downregulated during the stress period. ThSOS2 and ThSOS3 reached their lowest expression levels, 3.78% and 1.56%, respectively, in the control at 6 h. The relative abundance of ThSOS1, ThSOS4 and ThSOS5 were similar to those of ThSOS2 and ThSOS3 but achieved their lowest expression levels at 24 h (Fig. 3A).
Under PEG6000 stress, most of the ThSOS genes were significantly upregulated, and all ThSOS genes achieved their highest expression levels at 72 h. Interestingly, the expression levels of ThSOS1, ThSOS2 and ThSOS3 in the roots were significantly downregulated under PEG6000 stress at 6 h (5.39%, 16.67% and 0.16% of the control, respectively). In leaves, the expression levels of ThSOS1, ThSOS2 and ThSOS3 genes were mainly downregulated throughout the period of stress, and ThSOS1 and ThSOS2 achieved their lowest levels of expression at 24 h. However, ThSOS3 reached its lowest expression level during the early stages of stress (6 h). In contrast to the gene expression patterns of these three ThSOS genes, the relative expression of ThSOS5 was significantly upregulated at almost all stress points (besides 24 h) and reached its highest expression at 72 h. The expression of ThSOS4 didn’t changed significantly under salt stress (Fig. 3B).
The relative abundance of ThSOS1, ThSOS4 and ThSOS5 were significantly upregulated in roots. In addition, the gene with the highest percentage of induction was ThSOS1, with a peak expression level at 6 h that was 161.28-fold that of the control. Except at 12 h, when expression was only 24.3% of the control, the relative expression of ThSOS2 was mainly upregulated. However, the expression of ThSOS3 was clearly downregulated at 6 h (0.4% of the control) and showed no significant changes at any other stress points. In the leaves, no significant changes were found in the expression of any ThSOS genes except for ThSOS2 from 12-72 h. All ThSOS genes (ThSOS1, ThSOS2, ThSOS3, ThSOS4, ThSOS5) under ABA stress reached their lowest levels of expression at 6 h (0.55%, 0.83%, 0.07%, 1.31% and 3.17% of the control, respectively) (Fig. 3C).
Transient expression of ThSOS3 in T. hispida
To ascertain whether the ThSOS3 gene in T. hispida was transiently overexpressed and suppressed, the transcription levels of ThSOS3 in the control (empty pROK2), OE (35S::SOS3) and SE (pFGC::SOS3) plants were examined by using qRT-PCR. Compared to Con plants, ThSOS3 expression was significantly increased in OE plants and significantly decreased in SE plants under salt stress condition (Fig. 4), indicating that the gain- or loss-of-function of ThSOS3 in T. hispida plants was successfully generated.
ThSOS3 confers salt stress tolerance to transgenic plants
To preliminarily explore the function of the ThSOS3 gene, diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining and related physiological indexes of three types of plants with different transformed T. hispida were studied. DAB and NBT staining showed that levels of H2O2 and O2- accumulation in transgenic (OE, SE) and control plants. Under salt stress, the staining strength in OE plants were lower than in Con; however, SE plants were higher than in Con (Fig. 5A-B). In addition, H2O2 and MDA contents were measured in different transgenic T. hispida. The results failed to demonstrate a difference in H2O2 and MDA contents among three kinds transient transgenic plants under normal conditions. However, under salt stress, SE plants showed the highest H2O2 and MDA contents, followed by the Con plants, the OE plants had the lowest H2O2 and MDA contents. The levels of H2O2 and MDA in SE plants were 1.27 and 1.53 times those of Con plants, respectively. However, the H2O2 and MDA contents in OE plants were the lowest, with values of only 82.02% and 85.2% of the contents in the Con plants, respectively, at 24 h (Fig. 5D-E).
To further explore the transient expression of ThSOS3 in T. hispida, ThSOS3 was overexpressed in Arabidopsis. Two independent T3 homozygous transgenic lines (OE1 and OE2) overexpressing ThSOS3 were selected and studied. Under salt treatment, the H2O2 and MDA levels in both OE lines were lower than those of WT plants, although they had similar H2O2 and MDA contents under normal conditions (Fig. S1A, B, D). In addition, OE1 and OE2 had prominently higher root growth and fresh weights than the WT (Fig. S2A-C). Analysis of the growth phenotypes of the Arabidopsis plants grown in soil showed that OE lines grew much better than the WT under salt treatment (Fig. S2D).
ThSOS3 increases improves ROS scavenging capability
Antioxidant enzymes (SOD and POD) are the two most important ROS scavenging enzymes, influencing cellular ROS levels. Thus, we further studied peroxidase (POD) and superoxide dismutase (SOD) activities. Without stress conditions, there were no significantly altered in SOD and POD activities among Con, OE and SE plants. However, SOD and POD activities were raised gradually with the salt treatment time extended. At 24 h, the antioxidant enzymes (SOD and POD) activities in OE plants were 1.35 and 1.24 times those of Con plants, while in SE plants were only 80% and 83.73% those of Con plants, respectively (Fig. 5F-G).
Similarly, in Arabidopsis the activities of SOD and POD exhibited no obvious differences among all the studied lines without stress conditions. Under salt stress, two OE lines showed the highest antioxidant enzymes (SOD and POD) activities compared with WT plants (Fig. S1E-F), which is consistent with the results obtained from T. hispida; therefore, overexpression of ThSOS3 significantly increased SOD and POD activity under salt stress.
Cell death and electrolyte leakage analysis
Evans blue staining was used to establish a cell membrane damage by the intensity of the stain under salt stress. Evans blue staining indicated that in OE plants presented light blue points of less area in comparison with Con plants under salt stress; while in SE plants were the opposite (Fig. 5C). We then measured electrolyte leakage. There was no significantly altered in three types of plants with different transformed T. hispida under normal conditions. The relative electrolyte leakage rates of SE plants were the highest at 24h which was 1.14 times that of Con plants, meanwhile those of OE were 0.822 folds those of Con plants under salt stress (Fig. 5H). Meanwhile, we further detected changes in the content of corresponding physiological indicators in Arabidopsis. The results showed that Arabidopsis and T. hispida were consistent (Fig. S1C, G).