Role Of Endogenous Oxide In Lead Accumulation Of Pogonatherum Crinitumroot Cells

Lead (Pb), one of the most heavy metals in the environment. Excess accumulation of Pb can seriously inhibit plant growth and development, and reduce the quantity and quality of crops. Therefore, investigation of the signaling transduction chains (pathways) related to Pb uptake is helpful for elucidating the mechanism of Pb uptake in plants, preventing high accumulation of Pb in crops, inhibiting the involvement of Pb in food chains. However, there have been no detailed studies to address the signaling mechanism involved in Pb bioaccumulation of plants so far. NO has emerged as a key signalling molecule in plant defense responses to both biotic and abiotic stresses recently. NO was involved in plants development and seed germination and the regulation of defense reponses to biotic and abiotic stresses. Recently, there is evidence to support a role for exogenously applied NO released by a NO donor such as sodium nitroprusside (SNP) in alleviating Pb toxicity in plants. However, information is still lacking regarding the possibility of NO might be endogenously produced in response to Pb and exert specific roles in Pb uptake and toxicity of plants. Pogonatherum crinitum root cell cultures were chosen as an experimental system because the homogeneity and undifferentiated state of the cells, combined with the uniform delivery of the treatments, allow a clear and sensitive response to stresses. In this paper, the effects of Pb on endogenous NO generation, the role of NO in Pb uptake and the origin of Pb-induced NO production of Pogonatherum crinitum root cells were evaluated. The followings are the main results of the experiments:1. High Pb(NO3)2is toxicity in Pogonatherum crinitum root cells. High Pb exposure has inhibitory effects on the viability and weight of the Pogonatherum crinitum root cells. Our data show that the viability and weight of the cells treated with500μM Pb are significantly lower than those of the control cells, showing that high Pb exposure has inhibitory effects on the cells.2. Pb exposure triggered endogenous NO signaling of the Pogonatherum crinitum root cells. In this study, we examined the NO levels of Pogonatherum crinitum root cells exposed to Pb. The data indicate that NO levels of the cells treated with Pb are increased immediately after Pb treatment. Pb treatment induced NO generation of the cells is time-dependent and dose-dependent. Thus, results clearly indicate that Pb exposure triggers NO generation in the cells.3. The endogenous NO signaling triggered by Pb is essential for Pb uptake of Pogonatherum crinitum root cells. To further evaluate the role of NO in Pb uptake, we assayed the effects of NO specific scavenger cPTIO(2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl3-oxide) on Pb accumulation of the cells. The results show that pretreatment of the cells with cPTIO not only abolishes Pb-triggered NO burst but also reduced Pb contents of the cells. Thus, our results clearly demonstrate that Pb-triggered NO generation was necessary for Pb uptake of Pogonatherum crinitum root cells.4. NR activity and the expression of NIA1are inducible by Pb in Pogonatherum crinitum root cells during Pb-triggered NO generation. To investigate the origin of Pb-triggered NO, we assayed nitrate reductase (NR) activities and the expression levels of NIA1and NIA2genes in the cells exposed to Pb. The results showed that Pb exposure enhanced NR activity and stimulated NIA1expression during the period of Pb-triggered NO burst. Together, our results indicated that the expression of NNIA1in Pogonatherum crinitum root cells could be induced by Pb and the Pb-induced NIA1expression might result in the increase of NR activity.5. NR was responsible for Pb-triggered endogenous NO burst in Pogonatherum crinitum root cells. In order to further examine whether NR is involved in Pb-triggered NO generation, we assayed the effects of NR inhibitors tungstate (TUN) and Gin (glutamine) on Pb-induced NO generation. The results show that pretreatment of the cells with TUN and Gin not only restrained the Pb-enhanced NR activities but also reduced the Pb-triggered NO generation. Thus, our data suggest that NR is involved for Pb-induced NO generation. We also examined the effects of NOS inhibitors L-NAME (NG-nitro-L-Arg methylester) and PBITU (S,S'-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea) on Pb-triggered NO generation of the cells. The data show that treatment of the cells with NOS inhibitors only has slight effect on Pb-triggered NO generation. Together with the results of present work, it is strongly suggested that NR activity is the main source of NO generation in Pb-exposed Pogonatherum crinitum root cells. In addition to NR-mediated NO generation, several other NO synthesis pathways is considered to be the possible source of NO in cells.6. Pb-triggered NR activity is involved in Pb uptake of Pogonatherum crinitum root cells. To investigate whether NR plays role in Pb uptake, we examined the effect of NR inhibitors on Pb uptake of Pogonatherum crinitum root cells. The data show that pretreatment of NR inhitors not only suppresses the Pb-induced NR activity but also reduces Pb contents of the cells. Thus, the results demonstrate that the Pb-triggered NR activity plays critical role in Pb accumulation in Pogonatherum crinitum root cells. Furthermore, our data show that the inhibition of NR inhibitors on Pb uptake can be reversed by application of NO via its donor SNP. Together, the results demonstrate that the NR-mediated NO signaling triggered by Pb in Pogonatherum crinitum root cells is involved in Pb uptake.7. High NO via its donor SNP (5.0mM) increases Pb accumulation in Pogonatherum crinitum root cells. The effects of exogenous NO on Pb uptake of Pogonatherum crinitum root cells have been evaluated in this work. The results show that application of low dose of NO via its donor SNP (0.1and1.5mM) has no effect on Pb contents of the cells, while treatment of the cells with high SNP (5.0mM) increases Pb accumulation in the cells. Thus, our results suggest that the effects of exogenous NO on Pb uptake are dose-dependent. Taken together, the results of our present work first demonatrate that Pb exposure triggers NO-dependent signaling pathway in plant cells and the endogenous NO signaling triggered by Pb is essential for Pb uptake of Pogonatherum crinitum root cells, our results also suggest that NR is the main source of NO generation in Pb-exposed Pogonatherum crinitum root cells. The results of our research have scientific significance in understanding the mechanism of Pb uptake in plant cells and suggest a new strategy to reduce Pb content in crops and enhance Pb accumulation in plants in phytoremediation of contaminated environments by regulating NR activity.