Exogenous Hydrogen Peroxide Inhibits Root Gravitropism And Induces Horizontal Curvature Of Primary Roots During Gras Pea Germination

Hydrogen peroxide (H2O2), a Reactive Oxygen Species (ROS), plays many crucial roles as a signalling molecule in various physiological and biochemical processes, such as stomatal closure, root gravitropism, lateral root development, cell wall development, programmed cell death and pollen-stigma interactions and development. Although a large number of studies have focused on the roles of endogenous H2O2as a signalling molecule, reports on the effects of exogenously applied H2O2 on the growth and development of plants are scarce. Recently, it is interesting to find that application of exogenous H2O2 reversibly inhibits root gravitropism and induces horizontal curvature of primary root during grass pea germination in our lab. To further explore both the universality of this phenomenon and the possible underlying mechanism, physiological and biochemical methods, proteomics, histochemical localization and high performance liquid chromatography (HPLC) were used here. Simultaneously the changes of endogenous auxin and H2O2 levels were also analyzed. The results obtained in this study are as followed:(1) Exogenous application of H2O2 inhibited root gravitropism and induced curvature of primary root during the grass pea seed germination. The effects of H2O2 on primary root curvature were enhanced with growing of H2O2 concentrations during the grass pea seed germination. It was interesting to find that the curvature phenomenon was not observed in soybean, mung bean, rice, tobacco, maize or arabidopsis and only took placed in the members of the Vicieae tribe, which included Lens M., Lathyrus L., Pisum L. and Vicia L., suggesting that these tribe Vicieae species might have a common, unique mechanism regarding the formation of horizontal curvature.(2) The effect of H2O2 was the strongest when applied prior to the emergence of primary root, which is before 36 h after sowing. Conversely, H2O2 failed to induce the curvature of root when applied post-germination after radicle breaking though testa. These roots began to restore gravitropic growth along the gravitational axis at 24 h after the removal of H2O2, and almost all roots displayed gravitropic elongation within 48 h.(3) The horizontal-curvature of roots occured in elongation zones (EZ) by cytohistological observations. Compared to roots without curvature, cells in EZ exhibited asymmetry elongation. The volumes of inward cells were smaller than those of outside cells. This different degree of cell expansion between inside and outside cells caused asymmetric growth of roots and finally resulting in root horizontal bending.(4) Application of exogenous H2O2 decreased endogenous H2O2 level of roots and the root vitality, and increased the activities of several H2O2-scavenging enzymes and the level of antioxidants such as catalase (CAT), ascorbate peroxidase (APX), peroxidase (POD) and ascorbic (ASC) through histochemical localization and quantitative measurements of H2O2. Interestingly, the H2O2 localization in the roots showed an asymmetrical distribution and it mainly distributed at the inside of curvature.(5) Exogenous application of 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-butyric acid (IBA) and a-Naphthaleneacetic acid (NAA) could absolutely inhibit the horizontal-curvature of roots even when treated by H2O2, indicating that exogenously applied auxin was likely to disturb asymmetric auxin distribution of roots. Application of H2O2 decreased the normal level of auxin by detection of HPLC, which might be contributed to form asymmetric auxin distribution. On the contrary, 1-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA) had no inhibitory effect on horizontal curvature, suggesting that the horizontal curvature of primary root were not related to auxin polar transport.(6) Application of dimethylthiourea (DMTU), a scavenger of H2O2, together with H2O2 on grass pea roots, fully or partly inhibited the curvature of primary roots. The inhibitors of H2O2 production, diphenylene iodonium (DPI) and pyridine, was also applied to observe their effects on the curvature of primary roots. It was found that the horizontal curvature of primary roots still took place. The results above suggest that root horizontal curvature resulted from exogenous H2O2 rather than from excessive accumulation of H2O2 in root cells. It was found that H2O2 was mainly localized in xylem, pericycle, cortex tissues and epidermis, when seeds were imbibed in only dH2O by DAB staining and paraffin sections. The H2O2 was distributed only in cortex and epidermis tissues in cross-sections of primary roots treated with H2O2 Additionally, the degree of DAB staining in the epidermis from H2O2-treated roots was significantly greater than that of control, demonstrating that exogenous H2O2 only entered the epidermis and not the inner root tissues.(7) About 850-900 spots/gel were aquired by 2-DE gels, of which 71 spots (6 new-induced, one missed,44 up-regulated and 20 down-regulated spots) changed in expression levels were observed.15 differentially expressed proteins were selected, excised for tryptic digestion and analyzed through MALDI-TOF/TOF-MS/MS. Many important proteins which showed a significant change in protein expressions were found, such as NADH-ubiquinone oxidoreductase subunit, NADH dehydrogenase-ubiquinone iron-sulfur protein 1, mitochondrial-like, phosphoenolpyruvate carboxykinase, heat shock protein, ferritin, volin and proteasome subunit alpha type. And these proteins were involved in several categories based on their functions, including carbohydrate and respiration metabolism, cell defense, protein metabolism, energy metabolism and signal transduction.The results above indicated that exogenous application of H2O2 could induce the curvature of primary root via disturbing endogenous hydrogen peroxide and auxin levels and causing different degree of cell expansion between inside and outside cells and finally resulting in asymmetric growth of roots during grass pea seed germination. These studies revealed their relations among the treatment of exogenous H2O2, endogenous H2O2 level, antioxidant system, auxin level and some expression of important proteins. It also has very important significances for increasing our understanding that the effects of signalling molecule H2O2 on the development of primary roots of plants.