MC-LR-induced Phytotoxicity In The Roots Of Oryza Sativa

Eutrophication of fresh water bodies caused by the elevated nutrients inputs coming from sewage discharges has been becoming one of the most severe environmental problems worldwide. One of the most harmful consequences of water eutrophication is the visible cyanobacterial blooms on water surface. In recent years, Taihu Lake has been suffering severe cyanobacterial blooms. Many of the bloom-forming cyanobacteria are known to produce microcystins-LR (MC-LR), which can be released into water bodies. Irrigation with MC-LR-contaminated water poses a threat to the growth of agricultural plant species. Oxidative stress has been associated with the MC-LR-induced phytotoxicity in plants, which has been widely reported in many plant species. However, as a universal response to environmental stress on plants, oxidative stress is not unique to MC-LR-induced phytotoxicity. Freshwater from Taihu Lake is the main water source of agricultural irrigation in the surrounding region, which is one of the most intensive rice areas in China. Therefore, it is very important and essential to investigate the unique mechanism of MC-LR-induced toxicity in rice root growth. The cross-talk between auxin and nitric oxide (NO) is involved in the regulation of lateral root growth in dicots, but whether and how the synergic action of these two signaling molecules modulates the morphogenesis of crown root in monocots remains unclear. The aim of this study is to explore the molecular mechanism of MC-LR-induced inhibition of crown root development in rice seedlings, which mainly focus on the investigation of the interplay between auxin and NO.(1) Treatment with MC-LR significantly impeded the rice root morphogenesis by inhibiting root elongation, crown root formation, and lateral root development from primordial in dose-dependent manners. Treatment with MC-LR remarkably inhibited the expression of CRL1and OsWOXll, both of which has been identified as the genes controlling rice crown root growth specifically. Since the expression of CRL1and OsWOX11are induced by auxin during the growth of rice crown root, it was of interest to test whether MC-LR stress affected auxin level in rice roots.(2) Treatment with MC-LR was able to induce the decrease in IAA concentration in rice roots. Treatment with MC-LR stimulated the expression of CRL4, which disturbed the correct polar localization of PINs that are responsible for the polar transport of IAA to roots. Exogenous supplement of IAA homologue NAA attenuated MC-LR-induced growth stunt of rice roots. However, the application of NPA, an inhibitor of auxin polar transport, could abolish the effect of NAA, suggesting that MC-LR inhibited the growth of rice root by affecting auxin polar transport.(3) Treatment with MC-LR significantly induced the decrease in concentration of endogenous NO, which was detected by using specific fluorescent probe DAF-FM DA. Pretreatment with NO donor SNP showed the effect of restoring the formation of rice crown root and the expression of CRL1and OsWOX11in comparison with the inhibitory effect conferred by MC-LR treatment alone. Treatment with NO scavenger cPTIO was able to eliminate the effect of SNP. MC-LR exposure remarkably reduced endogenous NO generation by inhibiting the transcription of OsNial, which resulted in the decrease in NR activity. These results suggested that MC-LR inhibited the growth of rice root by inhibiting OsNial-dependent NO production.(4) Treatment with NAA stimulated the generation of endogenous NO in MC-LR-treated rice roots. However, the application of NPA could abolish the effect of NAA. Interestingly, treatment with cPTIO was able to abolish the attenuated effect of NAA on MC-LR-induced growth inhibition of rice roots, suggesting that NO acts downstream in regulating rice root development under MC-LR stress. In addition, treatment with SNP induced the increase in endogenous IAA concentration in MC-LR-treated rice roots, suggesting that the positive feedback regulation of auxin signaling by NO was possible in MC-LR-treated rice root.Conclusion, MC-LR-induced growth inhibition of rice crown root was closely associated with the decrease in OsNial-dependent NO production and the inhibition of auxin polar transport. In addition, NO acts both upstream and downstream of auxin in regulating the expression of CRL1and OsWOX11, which in turn manipulated the morphogenesis of rice crown root under MC-LR stress. This study may not only shed a new light to better understand the unique mechanism of MC-LR-induced phytotoxicity in rice, but also provide new insight to reveal the molecular process of crown root development in monocot cereals.