Effects of exogenous GABA on the phenotype of tomato seedlings under salt stress
Extensive damage was apparent in the roots and leaves of seedlings cultivated under salt stress (Fig. 1). The roots of the Na-treatment group exhibited discoloration and atrophy at 1 d after salt stress, and the new leaves wilted at 2 d. The seedling heights were significantly shortened, while the leaves and roots were severely shrunken after salt stress for 4 d. At 6 d of NaCl stress, the seedlings were relatively weak, which manifested as nearly half of the leaves turning yellow and wilting, and the roots lost viability. However, exogenous GABA significantly alleviated the plant phenotypic symptoms of salt injury. At the initial stage of salt stress, the seedlings subjected to GABA treatment did not show root discoloration and their leaves did not wilt, while a small number of aerial roots appeared at 2 d. At 4 d of NaCl and GABA treatment, the seedlings were obviously shorter than those of the control. The leaves remained stretched and did not exhibit obvious wilting and there were more aerial roots than those under NaCl stress. At the end of the experiment (6 d), most of the leaves and roots of seedlings treated with NaCl+GABA remained clearly healthier than the seedlings under salt stress alone.
The salt damage index of the seedlings treated with NaCl increased with extension of treatment time, but GABA application significantly decreased the salt damage index of the seedlings (Fig. 2A). The salt damage index of the seedlings treated with NaCl+GABA decreased by 54.5%, 38.6%, 41.5% and 22.5% compared with that of the Na-treatment group. The growth rate in terms of plant height was investigated during the same experimental period (Fig. 2B). The rate of increase in seedling height was approximately 3.2% under normal growth conditions, while NaCl significantly inhibited the height growth of the seedlings; in addition, the rate of increase in seedling height gradually decreased with prolonged treatment time. Although GABA treatment did not enhance plant height under control conditions, GABA alleviate the inhibitory effect of salt stress on seedling height growth under NaCl treatment. The plant height growth rate of the Na+G-treatment group was dramatically higher than that of the Na-treatment group, showing 39.2%, 63.6%, 126.3% and 92.0% improvement at 1, 2, 4 and 6 d, respectively.
The fresh weight of tomato seedlings treated with NaCl and NaCl+GABA was significantly lower than that of C- and G-treatment groups (Fig. 3). Compared with the fresh weight of the NaCl treatment group, that of the NaCl+GABA treatment group was significantly increased by 23.7%, 28.8% and 37.9% at 2, 4 and 6 d, respectively, after treatment. The dry weight of the NaCl-treatment group was decreased significantly compared with that of the control treatment group but was not significantly different from that of the Na+G-treatment group. Chlorophyll content was greatly reduced in the leaves of seedlings under NaCl treatment compared with control treatment and decreased gradually with prolonged salt stress (Fig. 4). Exogenous GABA could delay the decrease in chlorophyll a and chlorophyll b levels under NaCl treatment. During the whole salt stress period, the levels of chlorophyll a and b in the Na+G-treatment group were significantly higher than those in the Na-treatment group, with a range of promotion of 132.1% and 50.0%, respectively, at 6 d after salt stress.
Effects of exogenous GABA on Na+ flux and Na+ content in leaves and roots under salt stress
To further clarify the process of Na+ transport from roots to shoots, non-invasive micro-test technology (NMT) was used to measure Na+ flux in leaves and roots after salt treatment for 2 d (Fig. 5A and 5B). Net Na+ efflux and influx were measured in leaves and roots, respectively. Under normal conditions, net Na+ efflux in leaves and Na+ influx in roots were very low (close to 0) with or without GABA treatment. NaCl stress significantly increased the net Na+ efflux in leaves and net Na+ influx in roots, with averages of 2309 pmol·cm–2·s–1 and 305.08 pmol·cm–2·s–1 in leaves and roots, respectively. However, the net Na+ efflux in leaves and net Na+ influx in roots were obviously reduced in GABA-treated seedlings under NaCl treatment, with an approximately 43.2% decline in leaves and a 50.2% decline in roots under NaCl treatment.
Na+ accumulation in leaves and roots is the main symptom of salt stress and the results showed that the roots accumulated more Na+ than the leaves under all treatments (Fig. 6). There was no significant difference in Na+ content in leaves and roots between the control and GABA treatments. The Na+ content in the leaves and roots of NaCl-treated seedlings was markedly higher than that of the control. However, exogenous GABA significantly inhibited Na+ accumulation in leaves and roots under salt stress, yielding reductions of 28.6% and 32.4%, respectively, relative to control levels at 4 d after treatment.
Effects of exogenous GABA on the expression levels of four GAD genes and GAD activity in leaves under salt stress
We cloned the four GAD genes, and the conserved region of the sequences showed high homology, with a sequence alignment consistency of 83.73% (Fig. S1). The initial relative expression pattern of all four GAD paralogues in the leaves of tomato seedlings was analysed under normal culture conditions, and the results showed that the four GAD gene transcripts exhibited significant expression differences (Fig. 7). Among these genes, SlGAD2 was the most highly expressed, with approximately 10.7-, 47.8- and 69.6-fold higher expression than SlGAD1, SlGAD3 and SlGAD4, respectively. Among the genes, SlGAD4 exhibited the lowest expression.
Salt stress significantly increased the expression of SlGAD1-3 compared with the control (Fig. 8). SlGAD1-3 showed the same expression trend across the treatments (Na+G > Na > C+G and C). NaCl+GABA treatment induced a greater amount of SlGAD1-3 expression than NaCl-treatment alone. In contrast, SlGAD4 expression levels were in the order C+G > Control > Na > Na+G, with the lowest expression level, only 0.039-fold that of the control, observed in the NaCl+GABA treatment at 12 h. Among the four genes, SlGAD1 exhibited the largest change in transcription level, reaching the highest level after 6 h of Na+G treatment, approximately 19.4-fold that of the control; in the Na-treatment group at 6 h, it had reached 14.5-fold that of the control. The change range of SlGAD2 and SlGAD3 was substantially lower than that of SlGAD1. At 12 h after Na treatment, the maximum variation in SlGAD2 and SlGAD3 was only 2.45-fold and 3.64-fold higher than that of control treatment. SlGAD4 expression decreased significantly under salt treatment, thus showing the lowest expression among the four genes. However, this expression pattern had little effect on the change of the general trend of the upregulation of SlGAD genes.
The GAD activity of plants treated with NaCl+GABA or NaCl was significantly higher than that of the control (Fig. 9) and showed an increasing trend for 6-48 h followed by a decreasing trend for 96 h. The GAD activity of the Nal+G-treatment group was the highest during the entire processing period and was markedly higher than that of the Na-treatment group, with an increasing of 20.3% to 61.3%.
Effects of exogenous GABA on amino acid content in leaves under salt stress
To analyse the changes in amino acid content, 16 amino acids in leaves from the different treatment groups were detected (Fig. 10A and 10B). Based on the general trend, the levels of most of the amino acids increased to varying degrees under salt stress compared with the control treatment. Among these amino acids, the levels of methionine (Met), GABA, alanine (Ala), proline (Pro) and glycine (Gly) were significantly higher than those in the control treatment after 2 d of salt stress, and the levels of llysine (Lys), leucine (Leu), GABA, Ala, Pro, threonine(Thr), glutamate (Glu) and aspartic acid (Asp) were significantly higher than those in the control treatment after 4 d of salt stress. GABA, Glu and Pro showed prominent variations among all the amino acids induced by NaCl stress. The levels of GABA, which this article focuses on, were significantly increased by 1.5- and 1.3-fold after 2 d and 4 d of salt stress, respectively. The GABA levels showed the following trend: NaCl+GABA > C+G> NaCl > control, and the levels in the Na+G- and C+G-treatment groups were significantly higher than those after salt stress by 1.6- and 1.3-fold, respectively, 2 d after treatment. Pro levels, which is representative of stress characteristics, significantly increased 1.38-fold under salt stress. The addition of exogenous GABA led to an 18.9% in Pro compared to the level under salt stress (Fig. 10C). However, the addition of exogenous GABA had no significant effect on the Pro level under normal treatment. Glu exhibited a notable increase in the Na-treatment group compared with the control group. Glu levels under salt stress have been further improved by exogenous GABA, with the increases of 16.3% and 15.7%,respectively.
Effects of exogenous GABA on the activity of antioxidant enzymes under salt stress
Superoxide dismutase (SOD) activity in leaves treated with Na, C+G or Na+G gradually increased with increasing treatment time, and all treatment groups showed significantly higher SOD activity than the control (Fig. 11A). The SOD activity in the Na+G-treatment group was the highest and significantly higher than that in the Na-treatment group during the entire treatment process, with increases of 25.1%, 22.1%, 23.4% and 18.6% at 1, 2, 4 and 6 d, respectively. The NaCl treatment ranked second, with increase of 19.0%-35.4% compared with the control.
With increasing treatment time, the peroxidase (POD) activity in leaves treated with NaCl+GABA, C+G or NaCl significantly increased and was obviously higher than that in the control group (Fig. 11B). The Na+G-treatment group showed the highest POD activity, followed by the GABA treatment group and the Na-treatment group, while the Na-treatment group had the lowest POD activity. The POD activity of the NaCl+GABA-treatment group significantly increased by 22.5%, 18.7%, 28.3% and 49.2% compared with the Na-treatment group and that of the Na-treatment group significantly increased by 11.0%-56.5% compared with the control.
During the entire treatment period, the catalase (CAT) activity in tomato leaves showed the following expression trend: Na+G > C+G > Na > C (Fig. 11C). Exogenous GABA treatment significantly increased CAT activity under salt stress compared with that under salt stress alone; the activity was 33.9%, 25.9%, 50.1% and 30.2% higher than that under NaCl treatment alone.
Effects of exogenous GABA on reactive oxygen production in leaves under salt stress
Under salt stress, the production rate of superoxide anion (O2) in tomato leaves was markedly higher than that in the control group (Fig. 12A). When GABA was added under salt stress, the production rate of O2 in leaves was significantly lower than that in the Na-treatment group, with a reduction proportion of closer to 11%. In Fig. 12B, the blue spots indicate the amount of O2. The number of blue spots under salt stress was significantly higher than that under the control and GABA treatment conditions, , but the number of blue spots under Na+G treatment was markedly lower than that under NaCl treatment.
Hydrogen peroxide (H2O2) content was determined using the DAB staining method (Fig. 13A). The H2O2 content increased with prolongation of salt treatment. The levels of H2O2 increased significantly under salt treatment, while GABA application significantly inhibited H2O2 accumulation under NaCl treatment, with reduction of 21.9%-23.5%. Under salt stress, the brown spots in tomato leaves indicated the amount of H2O2 (Fig. 13B). The amount of H2O2 in leaves under NaCl+GABA treatment was significantly lower than that under NaCl treatment. Exogenous GABA treatment significantly alleviated the active oxygen-related injury of seedlings under salt stress.
As shown in Fig. 14, malondialdehyde (MDA) content in leaves treated with NaCl was markedly higher than that in the control treatment across treatment time. After adding GABA into the nutrient solution of seedlings treated with NaCl, the MDA content decreased significantly, with reductions of 15.8%, 15.1%, 22.8% and 14.0%. This result indicated that exogenous GABA could significantly reduce the MDA content in leaves under salt stress to alleviate the damage caused by active oxygen in tomato seedlings under salt stress.
Relationships between phenotypic indexes and levels of reactive oxygen species under salt stress
Correlation analysis of phenotypic and physiological indexes revealed several significant correlations (Table 1). The salt damage index negatively correlated with the rate of increase in plant height, total chlorophyll content and fresh weight and positively correlated with leaf sodium ion content. The sodium ion content significantly correlated with all phenotypic indexes and the levels of all active oxygen species. The levels of ROS significantly correlated with the salt damage index, all phenotypic indicators and sodium ion content.
Table 1 Correlation analysis between phenotype and physiological index
Index
|
Salt damage
index
|
Growth rate
of height
|
Chla+b
Content
|
Fresh weight
|
Na+
content
|
O2·-
productive rate
|
H2O2
content
|
MDA content
|
Salt damage index
|
1.000
|
-0.885**
|
-0.962**
|
-0.799**
|
0.979**
|
0.828**
|
0.918**
|
0.876**
|
Growth rateof height
|
|
1.000
|
0.913**
|
0.863**
|
-0.942**
|
-0.837**
|
-0.920**
|
-0.820**
|
Chl(a+b) content
|
|
|
1.000
|
0.871**
|
-0.974**
|
-0.861**
|
-0.979**
|
-0.897**
|
Fresh weight
|
|
|
|
1.000
|
-0.924**
|
-0.781**
|
-0.911**
|
-0.758**
|
Na+content
|
|
|
|
|
1.000
|
0.824**
|
0.997**
|
0.921**
|
O2·-productive rate
|
|
|
|
|
|
1.000
|
0.850**
|
0915**
|
H2O2content
|
|
|
|
|
|
|
1.000
|
0.886**
|
MDA content
|
|
|
|
|
|
|
|
1.000
|
Note:‘*’shows significant correlation at the level of p<0.05, ‘**’shows extremely significant correlation at the level of p<0.01.