The Physiological And Molecular Mechanisms Of Silicon-enhancend Resistance To High Manganese Stress In Rice

This study aimed to explore the physiological and molecular mechanisms of Si-enhanced toleranceto Mn toxicity, and improve rice growth and yield to sustain rice production. The roles of Si inenhancing tolerance to manganese (Mn) toxicity were studied in two rice (Oryza sativa L.) cultivars: i.e.cv. Xinxiangyou640(XXY), a Mn-sensitive cultivar and cv. Zhuliangyou99(ZLY), a Mn-tolerantcultivar. Plants were cultured in nutrient solution containing normal Mn (6.7μM) or high Mn (2.0mM),both with or without Si supply at1.5mM Si. We investigated the effects of Si on plant growth, elementsuptake, transport, distribution, antioxidative defense capacity and photosynthetic parameters of bothcultivars under high Mn stress; Using high-throughput sequencing, we performed a comprehensiveanalysis of the influence of Mn on gene expression of the sensitive rice with or without Si. Using thesignificant enrichment analysis of Gene Ontology and Pathway combined with the analysis ofphysiological indicators, we obtained the key genes related to photosynthesis; Using real-timequantitative PCR technique, we further studied the relative expression levels of the key genes ofinterest.The main results are presented as follows:1. Plant growth was severely inhibited by high Mn stress in cv. XXY, but was enhanced by Sisupply. Mn toxicity symptoms observed in leaves were more severe in the Mn-sensitive cultivar (XXY)than in the Mn-resistant cultivar (ZLY) under high Mn stress. In the high Mn treatment, greater Mnconcentrations in shoots and roots respectively were found in the Mn-tolerant cultivar than in theMn-sensitive cultivar. In cv. XXY, Si-enhanced tolerance resulted from a restriction of Mn transport,whereas Mn uptake was depressed in cv. ZLY.2. Mn concentration in foliage and roots of Mn-tolerant cultivar was much greater than that ofMn-sensitive cultivar at the high Mn level. In cv. XXY, the suppression of Mn resulted from arestriction of K transport, whereas K uptake was depressed in cv. ZLY. Supply with Si significantlyincreased the relative contents of K, Fe and Zn in foliage at the high Mn level compared with theSi-untreated plants in the Mn-sensitive cultivar. Supply with Si significantly increased the relativecontents of K and Zn in foliage at the high Mn level, whereas considerably decreased the relativecontents of Ca and Fe compared with the Si-untreated plants in the Mn-sensitive cultivar. Supply withSi considerably decreased the relative content of K in roots of Mn-sensitive cultivar, but significantlyincreased it in foliage at the high Mn level. This suggests that Si can promote K transport from roots toshoots under high Mn stress. This indicates that Si plays an important role in keeping the relativebalance between elements in roots of Mn-tolerant cultivar.3. For the Mn–sensitive cultivar, high Mn significantly increased the activities of superoxidedismutase (SOD), the concentrations of ascorbate (AsA) and non-protein thiols (NPT), while itsignificantly decreased the activities of catalase (CAT) and ascorbate peroxidase (APX) and theconcentrations of glutathione (GSH), thus leading to accumulation of high levels of malondialdehyde(MDA) in root tissues and destruction of plasma membrane integrity. However, high Mn significantlyincreased the concentrations of GSH and AsA, but significantly decreased the activities of CAT and the concentrations of NPT, thus leading to accumulation of high level of MDA in the Mn-tolerant cultivar(ZLY). The addition of Si significantly decreased the concentrations of MDA and H2O2in Mn-stressedroot tissues, thereby mitigating the damage. Under high Mn stress, application of silicon enhances theantioxidant activities of rice roots, and reduces lipid peroxidation and damage to the integrity of theapical membrane, thereby enhancing rice resistance to manganese toxicity. This alleviative effect ismore pronounced in Mn-sensitive rice (XXY) than in Mn-tolerant rice (ZLY). In cv. XXY, high Mnsignificantly increased SOD, CATand APX activities but decreased NPT and GSH concentrations,leading to accumulation of H2O2and MDA in leaves of rice. The addition of Si significantlycounteracted high Mn-elevated MDA and H2O2concentrations and enhanced plant growth. In cv. ZLY,high Mn considerably raised SOD activities and GSH concentrations in leaves of rice, thus leading torelatively low oxidative damage. Silicon mainly influenced non-enzymatic antioxidants in Mn-sensitiverice cultivars under high Mn stress.4. High Mn stress considerably decreased the content of chlorophyll'a', chlorophyll'b',carotenoids and Chlorophyll'a+b' in cv. XXY, while only carotenoids content was decreased in cv. ZLY.Net photosynthetic rates (Pn) of the two rice cultivars tested were all decreased under high Mn stress.By scanning electron microscopy and transmission electron microscopy, high Mn stress made leafstomata closed and grana lamellae disorder in cv. XXY, while stomatal aperture was slightly decreasedand the chloroplast lamellar structure was not affected in cv. ZLY. By the addition of silicon thecontent of carotenoids and Chlorophyll'a+b' were significantly increased in cv. XXY, also thecarotenoid content was significantly increased in cv. ZLY. The addition of Si improved thephotosynthetic efficiency and alleviated the chloroplast ultrastructure under high Mn level in cv. XXY.5. There were about lots of differently expressed genes (The false discovery rate (FDR)≤0.001and|log2ratio(Mn/CK)|≥1) in which1336appeared to be up-regulated and1495appeared to bedown-regulated in rice treated with high level of Mn compared with the normal level of Mn. Under highMn stress, Si addtion induced647up-regulated genes,892down-regulated genes compared with theMn-treated plants. Under the normal Mn level, Si addtion induced1558up-regulated genes among16525genes, and2028down-regulated genes. In the high Mn-treated plants amended with Si,16273genes were expressed, with320genes up-regulated and172genes down-regulated compared with theSi-treated plants. The differentially expressed genes were relating to various transcription factors (TFs),large number of transporters, numerous transferase proteins, etc, involving in the major primary andsecondary metabolisms. Functional analysis showed that these differentially expressed genes wereinvolved in metabolism, ion transport, signal transduction, transcription regulation, and stress responsegenes etc. Manganese resistance mechanism in rice is very complex and is a consequence ofcoordinated expression of a large number of genes.6. Mn-induced inhibition of photosynthesis can be attributed to the suppressed chlorophyllbiosynthesis, light-harvesting process and ATP synthesis, the impaired stability of PSI structure and theimpaired regeneration of the acceptor molecule for CO2fixation of the Calvin cycle. Si apparentlyallows plants to respond to Mn toxicity more efficiently by increasing chlorophyll content, light-use-efficiency and ATP quantity, stabilizing the structure of PSI, and promoting CO2assimilation.Our findings suggest active involvement of Si in Mn detoxification ranging from physiologicalresponses to gene expression.