Physiological And Molecular Mechanisms Of Alternate Wetting And Drying Induced Formation Of Iron Plaque On Root Surface Of Rice (Oryza Sativa L.) Seedlings

Alternate wetting and drying（AWD） is one of most important agronomic practice by supplying water and oxygen in the paddy soil. Application of AWD can elevate rice root activity, promote the formation of high-efficiency, high-yield, high-quality rice significantly. Iron plaque on root surface（IP） is a natural barrier adapting to stress conditions, which plays important roles in promoting absorption of nutrient elements and blocking heavy metal transport from root to rice grain. During AWD irrigation, rice root forms IP on root surface frequently. However, it is little reported that physiological and molecular mechanisms of AWD induced IP formation. In this study, the components of IP and impact factors were clarified firstly. Then physiological mechanism of AWD on reddish brown IP, gene expressional profile analysis and molecular mechanism of reddish brown IP formation were discussed. The results of the studies are listed as follows:（1） Chemical component analysis revealed that IP mainly consisted of amorphous and crystalline iron, which accounted for over 84% of total iron on root surface. Fe3+ was the main iron speciation of rice root surface, which accounted for over 70%. Iron mineral composition on rice root surface with P-sufficient treatment was iron phosphates, while that with P-deficient treatment was ferric（hydr）oxide. IP on root surface consisted of reddish brown IP and non-reddish brown IP. Phosphorus level（P）, especially ratio of phosphate and ferrous（P/Fe） affected formation of non-reddish brown and reddish brown IP on rice root surface significantly. When P/Fe was less than 1:3, reddish brown IP increased gradually with the increase of P/Fe.（2） AWD treatment promoted oxidation reduction potential（Eh）, the value varied from 314.3 m V to 529.5m V. AWD decreased p H value, which varied from 5.53 to 3.43. AWD declined Fe2+ concentration, which varied from 310.6 μmol/L to 31.5 μmol/L.（3） Results from different Intensity and times of AWD showed that root activity of rice seedlings reached the maximum when AWD intensity is 12 h drain / 12 h flooded treatment and 5 times of AWD treatment, and the value was 1036.73 μg/h/g DW, which was increased by 16.8% compared with the control. Antioxidase activities of rice root were increased obviously by AWD treatment. Compared with the control, the activities of SOD, POD, CAT and APX were increased by 24.6-38.4%, 22.2-27.1%, 21.2-21.5% and 3.6-10.4% respectively, the concentration of superoxide anion（O2-·） and hydrogen peroxide（H₂O₂） were increased by 14.5-30.0% and 29.0-35.7%, respectively.（4） To investigate the role of H₂O₂ in IP formation, the formation of IP on root surface was studied by H₂O₂ inhibitors（DMTU: dimethyl thiourea, a H₂O₂ scavenger; CAT: catalase, H₂O₂ hydrolase; AT: 3-amino-1,2,4-triazole, a catalase inhibitor）. Compared with the treatment without inhibitors, root H₂O₂ concentration, root oxidation activity and DCB-Fe concentration of rice seedling with DMTU treatment were reduced by 27.2%, 19.8% and 18.4% respectively; while those with AT treatment were increased by 31.8%, 22.8% and 26.7% respectively; Application of exogenous CAT lowered oxidizing substances on root surface, and decreased DCB-Fe concentration by 24.5%.（5） To study potential sources of H₂O₂, gene expressional profile of rice root with flooding（CK）, AWD, CK+Fe and AWD+Fe treatments were analyzed. Up to 3822 differential expression genes（DEG） were found in four libraries（AWD versus CK, AWD+Fe versus CK+Fe, CK+Fe versus CK and AWD+Fe versus AWD）. Among them, DEG in AWD versus CK（506 up- and 687 down-regulated） was more than that in AWD+Fe versus CK+Fe（308 up- and 179 down-）. It is indicated that IP formation could recover the expressional level of partial genes. CK+Fe versus CK（728 up- and 1175 down-） and AWD+Fe versus AWD（1252 up- and 1189 down-） had more DEG in comparation to AWD versus CK and AWD+Fe versus CK+Fe.（6） Functional classification of DEG in four libraries based on Gene Ontology indicated that 270 genes were involved in oxidation-reduction process and 165 genes were involved in oxidoreductase activities. Analysis of KEGG（Kyoto Encyclopedia of Genes and Genomes） showed that DEG was distributed mainly in metabolic pathway, biosynthesis pathway, photosynthetic pathway, signal stimulation pathway and organelles pathway. Analysis of Venn diagram indicated that 38 genes shared DEG coding proteins associated with disease resistance, drought resistance, cell wall and cell membrane, oxidation-reduction, protein kinase and transfer, metabolic. In AWD versus CK, 102 genes of DEG were involved oxidation-reduction process, which accounted for 8.25% of total DEG, particularly 30 DEG of which were up-regulated expression, and 72 of them were down-regulated expression. Results of real-time fluorescent PCR confirmed the ones of gene expression profile. The correlation coefficient of linear regression analysis reached 0.90. The expressions of OsGLO3 and OsGLO4 with AWD treatment were 2.2 times and 2.7 times of that of the control treatment. OsGLO might be a candidate gene response to AWD induced IP formation.（7） Bioinformatic analysis of OsGLO indicated that OsGLO family had six genes. The conserved domains of amino acids were coded by OsGLO1, OsGLO3, OsGLO4 and OsGLO5, which showed similar patterns. Apart from OsGLO5, the similarity of both amino acid and nucleotide sequences of OsGLO1, OsGLO3 and OsGLO4 were over 70%, and possessed high homology and close evolution. Physico-chemical properties of protein coded by OsGLO1 and OsGLO4 were similar. Besides, tissue-specific analysis of OsGLO indicated that expression of OsGLO1 and OsGLO4 genes were relatively high in all tissues. Interestingly, the expressions of OsGLO4 in root and stem were the highest, which was 1.36 times of that of OsGLO1. Thus OsGLO4 gene was chosen for the following experiments.（8） To elucidate the effect of OsGLO4 on the formation of IP on root surface, OsGLO4 was interfered and overexpressed in Zhonghua 11 rice cultivar, then interference（OsGLO4-RNAi） and overexpression（OsGLO4-OX） materials were acquired from our collaborators. Results indicated that compared with WT, OsGLO4 expression, glycolate oxidase activity and DCB-Fe concentration of OsGLO4-RNAi were decreased by 35.6%, 70.9% and 16.5% respectively; while those of OsGLO4-OX were increased by 103.4%, 101.6% and 15.2% respectively. Effects of GLC（Glycolic acid, a promoter of glycolate oxidase） and HPMS（alpha hydroxyl-2-pyridine mesylate, a inhibitor of glycolate oxidase） on formation of IP on root surface of OsGLO4-OX were also investigated in the normal condition. The results indicated that GLC treatment increased IP amount by 10.9% while HPMS treatment decreased it by 29.7%.（9） To explore the effect of H₂O₂ on formation of IP on rice root surface, IP quantities on rice root surface of WT and OsGLO4-OX were studied using DMTU and AT regulators. Compared with treatment without inhibitors, DMTU treatment declined DCB-Fe concentration of WT and OsGLO4-OX by 17.5% and 18.4% respectively; while AT treatment increased it by 23.4% and 25.7% respectively. Under the normal conditions of rice, H₂O₂ concentration of rice basal root was increased by 72.6% in comparation to root tip, while CAT activity of rice basal root was 67.7% of that of root tip. A large amount of reddish brown IP was observed on basal root while little IP on root tip. AT treatment decreased H₂O₂ concentration of rice basal root by 28.0% compared with that of root tip of rice seedlings. A large amount of reddish brown IP accumulated on root tip more than basal root obviously. With the increment of AWD times, OsGLO4 expression in rice root was enhanced gradually. The activity of glycolate oxidase was increased up to 55.9%. Results from fluorescent staining of H₂O₂ showed that a large amount of H₂O₂ of rice root with AWD treatment was secreted on root surface, and large amount of H₂O₂ accumulated in root tip and pericyle cells.Taken together, our results suggested that AWD could elevate root activity and Eh of rhizosphere, decline p H value and Fe2+ concentration of solution, increase the oxidation of Fe2+ to Fe3+, thus promote reddish brown IP formation. These results further indicate that reddish brown IP formation was a rhizosphere physiological and biochemical comprehesive process, which were mediated by organism and microorganism. By increasing OsGLO4 expression, elevating activity of glycolate oxidase, accumulating a large amount of H₂O₂, OsGLO4 was involved in the formation of reddish brown iron plaque on root surface of rice seedlings.