| Cereal aleurone cells can be used as a stand-alone system to study various applied signals. Because aleurone cells have different response to GA and ABA, it has become as a model system for the study of phytohormone-regulated PCD as well as corresponding gene expression. Heme oxygenase (HO, EC1.14.99.3) degrade heme to produce carbon monoxide (carbon monoxicide, CO) and HO/CO have the similar effects like nitric oxide (NO) in regulation of plant growth and development as well as resistence to biotic and abiotic stress. Recent study shows that in animals hydrogen (H2) act as an effective anti-oxidant in alleviating ROS caused damage. However, the effects of HO/CO on the a-Amylase gene expression, as well as HO/CO and H2on the PCD is not well understood. So this study investigated the molecular mechanisms of HO/CO and H2in regulation of a-amylase gene expression and PCD.The main results are shown as follows:1. a-Amy2/54gene expression was used as a molecular probe to investigate the interrelationship between nitric oxide (NO)/cyclic GMP (cGMP) and heme oxygenase-1(HO-1) in GA-treated wheat aleurone layers. The inducible expressions of a-Amy2/54and a-amylase activity were respectively amplified by two NO-releasing compounds, sodium nitroprusside (SNP) and spermine NONOate, in a GA-dependent fashion. Similar responses were observed when an inducer of HO-1, hemin-or one of its catalytic products, carbon monoxide (CO) in aqueous solution-was respectively added. The SNP-induced responses, mimicked by8-bromoguanosine3’,5’-cyclic monophosphate (8-Br-cGMP), a cGMP derivative, were NO-dependent. This conclusion was supported by the fact that endogenous NO overproduction was rapidly induced by SNP, and thereafter induction of a-Amy2/54gene expression and increased a-amylase activity were sensitive to the NO scavenger. We further observed that the above induction triggered by SNP/8-Br-cGMP was partially prevented by the addition of zinc protoporphyrin IX (ZnPPIX), an inhibitor of HO-1. These blocking effects were clearly reversed by CO, confirming that the above response was HO-1-specific. Further analyses showed that both SNP and8-Br-cGMP rapidly up-regulated HO-1gene expression and increased HO activity, and SNP responses were sensitive to cPTIO and the guanylate cyclase inhibitor6-anilino-5,8-quinolinedione (LY83583). Molecular evidence confirmed that GA-induced GAMYB and ABA-triggered PKABA1transcripts were up-regulated or down-regulated by SNP,8-Br-cGMP or CO cotreated with GA. Contrasting changes were observed when cPTIO, LY83583or ZnPPIX was added. Together, our results suggested that HO-1might be involved in NO/cGMP-induced a-Amy2/54gene expression in GA-treated aleurone layers.2. Heme oxygenase-1(HO-1) confers protection against a variety of oxidant-induced cell and tissue injury in animals and plants. In this report, we confirmed that programmed cell death (PCD) in wheat aleurone layers is stimulated by GA and prevented by ABA. Meanwhile, HO activity and HO-1protein expression exhibited lower levels in GA-treated layers, whereas hydrogen peroxide (H2O2) content was apparently increased. The pharmacology approach illustrated that scavenging or accumulating H2O2either delayed or accelerated GA-induced PCD. Furthermore, pretreatment with HO-1specific inhibitor zinc protoporphyrin IX (ZnPPIX), before exposure to GA, not only decreased HO activity but also accelerated GA-induced PCD significantly. The application of HO-1inducer hematin and the enzymatic reaction product of HO carbon monoxide (CO) aqueous solution, both of which brought about noticeable induction of HO expression, prevented GA-induced PCD substantially. These effects were reversed when ZnPPIX was added, suggesting that HO in vivo played a role in delaying PCD. Meanwhile, catalase (CAT) and ascorbate peroxidase (APX) activities or transcripts were enhanced by hematin, CO, or bilirubin (BR), the catalytic by-product of HO. This enhancement resulted in the decrease of H2O2production and delay of PCD. Additionally, antioxidants butylated hydroxytoluene (BHT), dithiothreitol (DTT), and ascorbic acid (AsA) were able to not only delay PCD, but also mimic the effects of hematin and CO on HO up-regulation. Overall, the above results suggested that up-regulation of HO expression delays PCD through the down-regulation of H2O2production. 3. In our experiments, we found that pretreatment with CoCl2and CoPPⅨ, which usually used as the HO inducer in animals, can delay GA-induced PCD in wheat aleurone layer. Further observation showed that ZnPPⅨ pretreatment not only decreases HO activity, but also accelerates PCD in the aleurone layers. Application of HO-1enzymatic reaction products BR and CO aqueous solution have the similar effects like CoCl2and CoPPⅨ in delaying GA-induced PCD, and it was also found that CO could upregulate HO activity. Further study showed that CoCl2, CoPPⅨ, CO, and BR pretreatment can down-regulate the gene expression of NADPH oxidase, and up-regulate SOD, CAT and APX gene expression and enzyme activities, while down-reculation superoxide anion (O2’) production and H2O2content. Overall, the above results suggested that up-regulation of HO expression in delaying of PCD by pretreatment with CoCl2or CoPPⅨ may be assiciated with the down-regulation of ROS accumulation.4. In our experiments, we found that endogenous H2content show a declining trend in GA treated wheat aleurone layer. H2aqueous solution cotreated with GA can delay the GA-induced PCD. Further research showed that H2can reduce the ROS content in ABA and GA treated aleurone protoplasts. It was also found that H2was able to alleviate superoxide anion (O2·-) and hydrogen peroxide (H2O2) accumulation and TBARS content. Above effects may associate with the up-regulation of APX, CAT, SOD gene expression and enzyme activity; and the down-regulation of NADPH oxidase gene expression by cotreatment with H2. H2could also delay the strongest oxidizing ROS-hydroxyl radical (·OH)-induced PCD, because H2can directly react with-OH. H2also delay ONOO--induced PCD in wheat aleurone layer. In short, as an effective anti-oxidant, H2can rapidly diffuse through the cell membrane, and could directly react with·OH or through regulating antioxidant enzyme down-regulation O2·-and H2O2content, thereby delaying the occurrence of aleurone cell PCD. |
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