Studies On The Mechanism Of Hydrogen Sulfide In Response To Low-Oxygen Stress In Plants

Global climate change alters frequency causing unexpected flooding stress in worldwide. In addition, most crop species including pea are highly intolerant of low oxygen conditions, prolonged flooding stress results in serious loss of grain yield. This is the consequence of slow rates of aerobic respiration and photosynthesis due to restricted access to atmospheric O2 and CO2 to cells of underwater organs. What’s more, higher concentrations of toxic secondary metabolite accumulation also leads to cell death. Hydrogen sulfide (H2S) is a highly diffusible, gaseous signal molecule. A number of studies in animal and plant cells have centered upon its effects on many biological processes. It is notably that H2S is involved in hypoxia signaling in animal cells. However, there have been no previous studies of H2S in plant cells in response to hypoxia. In the present study we analyzed whether H2S was involved in low oxygen sensing and alleviated hypoxia-induced plant cell death by regulating the contents of ROS, NO as well as the biosynthesis of ethylene, using pea and Arabidopsis mutants desl-1(L-cysteine desulfhydrase) of H2S metabolic pathways and through various physiological, and biochemical and molecular techniques. Moreover, the cellular and molecular mechanisms of H2S involved in hypoxia stress response were revealed finally. The main results are as follows.1) The Evans Blue staining results confirmed that cell death usually began in root apex transition zone (TZ) under hypoxia condition. ROS, which is detected by DAB (displaying H2O2) and NBT (displaying O2-) staining, accumulated in this area firstly. The results suggested that TZ is the most sensitive zone in sensing hypoxia stress.2) Pea seedlings pretreatment with NaHS (an H2S donor) followed by hypoxia treatment were then analyzed for the extent of root tip death by Evans Blue staining, results showed that exogenous H2S pretreatment dramatically reduced root tip death of pea seedlings under hypoxia. DAB and NBT staining analysis also showed that NaHS pretreatment dramatically reduced ROS accumulation in root tips.3) The results showed that root tip death induced by hypoxia was strongly enhanced by hydroxylamine (HA), which is inhibitor of the key enzymes responsible for endogenous H2S biosynthesis. The Evans Blue staining results also showed that the extent of root tip death in seedlings was strongly linked with endogenous H2S content, suggesting that H2S might involve in hypoxia signaling in plant cells.4) NaHS pretreatment did not increase the activities of PDC (pyruvate decarboxylase) and ADH (alcohol dehydrogenase), but dramatically increase the content of GSH (glutathione) and activity of APX (ascorbate peroxidase), suggesting that NaHS pretreatment promotes the hypoxia tolerance mainly through promoting the antioxidant capacity of plant cells.5) Using specific fluorescent probe, we showed that the contents of H2S and NO (nitric oxide) were increased dramatically in NaHS pretreated wild-type Arabidopsis (Col) compared to that in control. However, the contents of H2S and NO were reduced significantly in des1-1 relative to Col. The Evans Blue staining results also showed that des1-1 is very sensitive to hypoxia stress compare to Col, suggesting that the response of plants to hypoxia stress is regulated by cross talk between H2S and NO.6) The mutation of DES1 gene significantly affects marker genes expression involved in N-end rule, including ATE1 (Arg-tRNA transferase), ATE2, PRT6 (proteolysis), HRE1 (hypoxia responsive), HRE2, RAP2.12 (related to AP2.12), PCO1 (plant cysteine oxidase) and PCO2, combined with the Evans Blue staining results, indicating that H2S might involve in low oxygen sensing by N-end rule pathway; The mutation of DES1 gene also significantly affects marker genes expression involved in ER stress, including bZIP17 (basic domain/leucine zipper), bZIP28, bZIP60, BiP (binding protein), TIN (tunicamycin induced) and ERO (ER oxidoreductase), suggesting that H2S might participate in hypoxia stress response by affecting protein-folding.7) NaHS pretreatment inhibited ethylene production by inhibiting the activity of ACO (ACC oxidase). In addition, the mutation of DES1 gene significantly up-regulated gene expression of ETR2 (ethylene receptor), which is a marker gene in ethylene perception. Combined with the Evans Blue staining and average root length results, indicating that NaHS pretreatment inhibited ethylene biosynthesis and might stimulate a quiescence strategy to reduce energy consumption under hypoxia stress.8) To test the cellular and molecular mechanisms of H2S involved in hypoxia stress response, we constructed OAS-A1 (O-acetyl-L-serine(thiol)lyase) and DES1 over-expression vector (including the 35S promoters and promoters themselves) and RNAi vector, and transferred into Agrobacterium strain GV3101 successfully. In the hope of obtaining transgenic plant which over-expressed or RNAi the OAS-AI and DESl genes, and then study their tolerance in response to hypoxia stress.All together, these results suggested that H2S regulates plants in response to hypoxia stress by interacting with NO. ROS, as well as ethylene. Therefore, enhances the tolerance of the plant to hypoxic stress. This research has not only the important scientific significance, but also the potential application in improving the hypoxia tolerance of crops.