Heme Oxygenase/Carbon Monoxide System Regulates Wheat Seed Germination Under Osmotic Stress And Seedling De-Etiolation

In recent years, more and more attention has been paid to heme oxygenase (HO; EC 1.14.99.3) and one of its products carbon monoxide (CO). This system could confer beneficial cytoprotection against oxidative damage in mammalian cells. Accumulation reports have demonstrated that HO/CO system exhibited the significant effects in plant.In this paper, we first employed different concentrations of polyethylehe glycol (PEG) (12.5%,25%, and 50%) (W/V) pretreating wheat seeds for 1 d, found that 12.5% PEG pretreatment could promote wheat seeds to germinate much earlier,25% PEG pretreatment decreased the germination rate and 50% PEG stress completely inhibited wheat seeds germination. During the recuperating time, the germination rate of wheat seeds after 25% PEG pretreatment could achieve to the normal level, but 50% PEG could not. We also investigated the changes of thiobarbituric acid reactive substance (TBARS) contents, HO activities, and HO-1 gene expression level. The following results suggested that both 12.5% and 25% PEG pretreatment increased HO activities and endogenous HO-1 transcripts, however,12.5% PEG could obviously lead to the decrease of TBARS content in wheat seeds during the recuperation time, whereas treatment with 25% PEG could not.50% PEG stress not only increased the content of TBARS but also inhibited the HO activity and endogenous HO-1 transcripts. Therefore, HO-1 inducer Hematin was used against 25% PEG stress. Data showed that Hematin could dramatically stimulate wheat seed germination and root elongation under 25% PEG stress in dose-dependent manner. With respect to the 25% PEG stress treatment alone, the effects of 1.0μmol·L-1 Hematin treatment was most obviously, which could up-regulate HO activities as well as HO-1 gene expression. At the same time, we also discovered that the inducible effects exerted Hematin might through enhancing the activities of total amylase to accelerate the degradation of starch and inducing the increase of antioxidant enzymes activities to decrease the content of TBARS consequently. Such above results were also confirmed by using semi-quantitative RT-PCR, for instance, the transcripts of catalase (CAT), ascorbate peroxidase (APX), Mn-superoxide dismutase (Mn-SOD), and Cu,Zn-superoxide dismutase (Cu,Zn-SOD) were up-regulated.To open out whether endogenous HO-derived CO modulated the germination of wheat seeds resisted to osmotic stress, different concentrations of CO saturated aqueous solution were also used to treat wheat seeds under 25% PEG stress and we found that it acted as the same function as Hematin in relieving wheat seeds germination inhibition. Afterward, by the way of determining CO content of wheat seeds with different treatments described above, we discovered that Hematin could rapidly induce the endogenous CO release under 25% PEG stress. The changes of CO content were consistent with that of HO activities. Employment of HO-1 inhibitor Zinc protoporphyrin (ZnPPIX) elicited the positive effects exerted by Hematin, which also comfirmed that CO participated in regulating the germination of wheat seeds under osmotic stress, endogenous HO/CO system play an important role in responses of wheat seeds to osmotic stress.Furthermore, this phenomenon might contribute to Hematin-induced nitric oxide (NO). Since Hematin could even induce the generation of NO during the early period of seed germination subjected to 25% PEG stress. After adding NO specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide potassium salt (cPTIO), these obvious effects of Hematin were also reversed to different extent. Taken together, it is speculated that NO might participate in HO/CO signal system transduction process in attenuating the inhibition of wheat seed germination caused by osmotic stress.Wheat seedlings will become etiolating when grown in the darkness. It was proved that NO could increase the chlorophylls content of etiolated wheat seedling leaves in prevenient reports. Our study found that during the process of light-induced recuperation, the endogenous HO activities, HO-1 gene transcript level, and CO release were all increased. Addition of HO-1 inhibitor ZnPPIX reversed these positive effects caused by light, which demonstrated that endogenous HO/CO system was involved in light-induced wheat seedling de-etiolation. Meanwhile, we also discovered that exogenous HO-1 inducer Hematin and CO saturated aqueous solution could accumulate the chlorophylls content of etiolated wheat seedlings in dose-dependent manner. Among them,10.0μmol·L-1 Hematin and 1.0% CO saturated aqueous solution were the most effective concentration. Besides, with respect to dark-grown plants alone, Hematin and CO saturated aqueous solution not only boost HO activities and total phytochrome content, but also up-regulate HO-1 and PhyA, PhyB, and PhyC transcripts level of etiolated wheat seedlings. SNP acts as the same role as Hematin and CO saturated aqueous solution. In contrast, adding nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) used in animals or NO specific scavenger 2-(4-carboxyphenyl)-4,4,5, 5-tetramethylimidazoline-l-oxyl-3-oxide potassium salt (cPTIO) blocked the positive effects induced by SNP, Hematin, and CO saturated aqueous solution. In this study, we also discovered that Hematin and CO saturated solution treatment could also induce the endogenous generation of NO, which suggested that NO participated in the greening process of wheat seedlings controlled by HO/CO system.Based on these results, it is proposed that exogenous HO-1 inducer Hematin and CO saturated aqueous solution would activate endogenous HO/CO system to magnify signal cascade or exogenous CO itself could act as an signal molecular to accomplish phytochrome-controlled wheat seedling de-etiolation, which might have a "Crosstalk" with NO.In order to investigate the function of heme oxygenase, the cDNA encoding HO-1 from Arabidopsis was used for searching the EST pool to contig the full length of wheat HO-1. So far, we acquired HO-1 sequences by RT-PCR. Alignment analysis showed that HO-1 of wheat had high homology to other plants. Under NaCl, PEG-6000, and H2O2 stresses, we tested the expression of HO-1 gene in wheat leaves by using semi-quantitative RT-PCR and found that it was increased in short time in stress of NaCl.