| Approximately 20% of worldwide arable land is affected by salt stress. Excess salinity reduces crop growth and yield and is becoming one of the most important factors restricting agricultural production. Usually, salt stress causes ion imbalance and osmotic stress in plants and leads to oxidative damage as a secondary stress. Therefore,it is important to develop effective strategies to improve plants' salt tolerance in agricultural production.Bacillus amyloliquefaciens SQR9 is a beneficial bacterium isolated from the rhizosphere soil of a healthy cucumber, has been used as an exogenous strain in a commercial bio-organic fertilizer for plant growth promotion and the suppression of soil-borne diseases in the field.This study revealed that SQR9 not only promoted plant growth but also enhanced plant salt tolerance. We studied plant salt pathways induced by SQR9 through analyzing the physiological indexes related to plant salt tolerance affected by inoculation of SQR9. The main plant salt pathways induced by SQR9 were confirmed by the study of the molecular weight of SQR9 signals for inducing plant salt tolerance and Arabidopsis mutants.Meanwhile, we found that spermidine is responsible for enhancing plant salt tolerance. The followings are the main results of this study.In this study,the effect of SQR9 on the salt tolerance of plant was investigated in hydroponic systems and agar culture. The biomass of maize,rice and Arabidopsis inoculated with SQR9 was enhanced under different concentration of NaCl. Under 100 mM NaCl, SQR9 significantly increased the shoot fresh weight and root fresh weight of maize by 2.69 and 1.78 times, and the shoot fresh weight and root fresh weight of Arabidopsis by 2.85 and 1.9 folds compared to the control,respectively. The effect of SQR9 on plant root is enhancement of lateral root development.Under salinity, SQR9 regulated some genes expression level to affect the related compounds content in plants, such as the chlorophyll, photosynthetic rate, abscisic acid(ABA),total soluble sugar (TSS),reduced glutathione (GSH),H2O2, Na+ content and antioxidant enzyme activity. RBCS and RBCL are key genes involved in photosynthesis,SQR9 upregulated transcription of ZmRBCS, ZmRBCL in maize and AtRBCS, AtRBCL in Arabidopsis to enhance photosynthesis, respectively. Meanwhile, the chlorophyll and carotenoid content of maize inoculated with SQR9 was 1.84 and 1.44 fold of control,respectively. In Arabidopsis, SQR9 significantly increased the chlorophyll and carotenoid content by 3.39 and 3.35 fold relative to the control, respectively. The inoculation of SQR9 enhanced photosynthetic rate significantly. ABA content was enhanced by salinity, but SQR9 counteracted the increase of ABA in response to salt stress though downregulating ZmNCED and AtNCED3 genes expression in maize and Arabidopsis, respectively. SQR9 significantly enhanced TSS content in maize and Arabidopsis by 2.29 and 1.47 fold relative to the control, respectively. The peroxidase (POD) activity was enhanced by SQR9 in maize,but not in Arabidopsis. The catalase (CAT) activity of maize and Arabidopsis inoculated of SQR9 was 1.75 and 2.19 fold of control, respectively. The GSH content of maize and Arabidopsis inoculated of SQR9 was 2.43 and 2.49 fold of control, respectively. Under salt stress, the H2O2 content in plant was decreased by inoculation of SQR9· The Na+ content in maize and Arabidopsis was reduced by SQR9 significantly. The Na+ content in the SQR9-treated rhizosphere was much higher than control, suggesting that the inoculation of SQR9 would help the plant refuse and expel Na+ to enhance plant salt tolerance. Meanwhile,SQR9 upregulated ZmNHMX1,ZmANHX2,ZmNHX3,ZmH+-PPase genes transcription in maize and AtNHXl, AtNHX7 genes transcription in Arabidopsis, which are related to Na+transport, to reduce Na+ content in plant.Plants were treated with SQR9 wrapped in dialysis bags with different molecular weight cutoffs (MWCO). We found that the molecular weight of the SQR9-derived signal that effectively confers plant salt tolerance is between 100 and 500 Da. In order to analyze which plant salt tolerance pathway induced by 100?500 Da SQR9 secretions,the effect of 100?500 Da SQR9 secretions on plant physiology was tested. SQR9-secreted signal with molecular weight between 100 and 500 Da confers maize salt tolerance by enhancing the GSH content and reducing the Na+ content. 100?500 Da SQR9 signal induces Arabidopsis salt tolerance by enhancing CAT activity and GSH content, as well as decreasing Na+content. To confirm the roles of GSH, CAT and ion homeostasis in SQR9-induced plant salt tolerance, Arabidopsis mutants deficient in GSH, CAT metabolic pathway and the SOS pathway were inoculated with SQR9. The gs and gr mutations deficient in glutamate-cysteine ligase and glutathione reductase, respectively, were sensitive to salt stress in presence of SQR9. In addition, sosl, sos2, sos3 and nhxl mutations deficient in SOS pathway lose SQR9-induced plant tolerance to salt stress. The results of related Arabidopsis mutants confirmation suggest that the main plant salt tolerance pathways induced by SQR9 are through accumulating of GSH to scavenge ROS content and protect plants from oxidative damage,and regulating SOS proteins to sequester Na+ into vacuoles and expel Na+ from the cell to alleviate Na+ toxicity.In order to analyze signal produced by SQR9 for enhancing plant salt tolerance, the organic compounds produced by SQR9 were detected. VOCs emitted by SQR9 did not significantly improve plant salt tolerance. This result means VOCs is not SQR9-derived signal for enhancing plant salt tolerance. AysnE strain was the mutant of SQR9, whose indole-3-acetic acid (IAA) production was reduced by 86%. AysnE still enhanced maize growth under salt stress, although growth range induced by AysnE strain was less than that induced by SQR9. This result indicated that IAA is not the primary signal for conferring plant salt tolerance. To identify the signal responsible for conferring plants tolerance to saltstress,the 100?500 Da SQR9 secretions were extracted with different organic solvents.Under salt stress, the shoot fresh weight treated with whole SQR9 secretions and SQR9 secretions from the aqueous, ethyl ether, and chloroform extracts by 75.75%, 34.68%,53.21% and 95.43%. The root fresh weight treated with whole SQR9 secretions and SQR9 secretions from the ethyl ether and chloroform extracts by 35.75%, 20.75% and 70.9%.However, the plant fresh weight had no significant difference and even significantly decreased when treated with the SQR9 secretion extracted with ethyl acetate and n-pentane,respectively. These results reveal that, the key signal molecule displayed the highest solubility is in chloroform phase, but was insoluble in ethyl acetate and n-pentane. The SQR9-derived molecules between the molecular weights of 100 and 500 Da were classified.Among these chemicals, the potential signals, including IAA, gibberellin acid 3 (GA3),trans-zeatin (ZT),trehalose, and some types of polyamines,were tested for solubility in the aforementioned extraction reagents. Only polyamines displayed the greatest solubility in chloroform compared with the other reagents; as a consequence, they are considered to be potential signals secreted by SQR9 for conferring plant salt tolerance.Polyamines produced by SQR9 were detected by high performance liquid chromatography (HPLC). Spermidine was detected in SQR9 secretion,while spermine was not detected. The optimal concentration of spermidine that effectively enhances plant salt tolerance is between 1 and 10?M. When the concentration of spermidine is more than 500 ?M, it would cause inhibition of plant growth and even cell death. The speB gene was found to be the key gene in spermidine biosynthesis and speB was completely deleted by double cross-over, and the c-AspeB strain was complemented with the entire speB gene.Spermidine content produced by SQR9, ?speB, c-AspeB was detected by HPLC.The extracellular spermidine contents for SQR9 and c-AspeB were 26.40 and 22.20 ?M in the bacterial culture (0D600=1), respectively. And intracellular spermidine contents for SQR9 and c-AspeB were 29.93 and 26.59 ?M in the bacterial culture (OD600=1), respectively. The mutation of speB caused a deficit in extra- and intracellular spermidine production. The growth curve of AspeB and c-AspeB has no significant difference with wide type SQR9.This means knocking out speB and complementing the entire speB gene didn't affect bacteria growth. Inoculation of ?speB didn't induce plant salt tolerance, but inoculation of c-AspeB still conferred plant salt tolerance. These results indicate that spermidine is significant signal produced by SQR9 for inducing plant salt tolerance. Spermidine produced by SQR9 upregulated the expression of GS and GR genes, which in turn enhanced the GSH content in plants to alleviate ROS damage. Spermidine may regulate the two-pore Ca2+channel (TPC1) and control compartmentation of Ca2+, which would stimulate the SOS signaling pathway. The enhanced transcription of NHX1 and NHX7 by SQR9-derived spermidine would sequester Na+ into vacuoles and expel Na+ from cell to reduce Na+toxicity.In summary, SQR9 induced several pathways in plant to enhance plant salt tolerance.The primary pathways induced by SQR9-derived spermidine are through enhancing GSH biosynthesis to alleviate ROS damage and regulating SOS pathway to sequester Na+ into vacuoles and expel Na+ from plant cell. |
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