Studies On The Relationship Between H₂O₂ In Subsidiary Cells And The Stomatal Switch And The Source Of H₂O₂ In Subsidiary Cells In Maize

The stoma is the "window" for the gas exchange in plants, and its movement is not only related to their photosynthesis or transpiration status, but also closely related to the response to biotic and abiotic stresses. Current research about the mechanism of stomatal movement has been centered on guard cells rather than the subsidiary cells. The previous study of our lab has found that the stomatal movement is not only related to the H2O2 of guard cells, but also to the H2O2 in subsidiary cells. The subsidiary cells of maize can not produce H2O2 by themselves, so H2O2 in subsidiary cells of the maize leaf epidermis is exogenous, and it may be transported by the plant aquaporins (APQ). In this study, a popular maize cultivar "ZhengDan-958" was used as the material. This paper studied the cell localization of H2O2 by cytochemical fluorescent probe, combined with scanning electron microscopy to study the diurnal variation of stomata, analyzed the gene expression of ZmPIP2; 4, ZmPIP2; 5, ZmPIP2; 6by Q-RT-PCR technology, with Tubulin and GAPDHas reference genes, and used the cell immunohistochemical methods to examine the cell localization of aquaporins ZmPIP2; 5. The results and conclusions were as follows:(1)The diurnal variation of H2O2 distribution in maize stomata:At 8:00 am and 18:00 pm time points, when stomata not yet fully open, H2O2 was only the distributed in guard cells,but very little in subsidiary cells. At 10:00 am and 16:00 pm, when the stomata were open, H2O2 is mainly located in guard cells, no in subsidiary cells. But at 12:00 and 14:00 o’clock, when stomata closed, the distribution of H2O2 was significantly increased in guard cells and subsidiary cells, and the increment in subsidiary cells is significant more than that in guard cells. During diurnal variation, stomatal aperture became smaller with the H2O2 increasing in subsidiary cells. At 10:00 am the stomatal aperture reached the first peak, and at 16:00 pm the stomatal aperture achieved the second peak. Therefore, the H2O2 in subsidiary cells is involved in the regulation of stomatal switching, and the increment of H2O2 in subsidiary cell promoted the stomatal closure.(2)Aquaporin facilitates transmembrane diffusion of H202in maize subsidiary cells:Under light and dark treatments, compared with the control group suplemented with aquaporin inhibitor AgNO3, the accumulation of H2O2 in subsidiary cells was opposite to the control group.When epidermis was exposed to light treatment then aquaporin inhibitor AgNO3 was added and dark treatment began, almost no H2O2 distribution in subsidiary cells. But When epidermis was exposed to dark treatment then aquaporin inhibitor AgNO3 was added and light treatment began, H2O2 distribution in subsidiary cells was significantly higher, contrast with the normal processing conditions of light and dark treatments. Exogenous H2O2 caused H2O2 accumulation in subsidiary cells, but if aquaporin inhibitor AgNO3 was added in advance followed by addition of exogenous H2O2, it did not cause H2O2 accumulation in subsidiary cells. So H2O2 in subsidiary cells is exogenous, and aquaporins facilitates transmembrane diffusion of H2O2into maize subsidiary cells.(3)Q-RT-PCR analysis:After short cells appeared, H2O2 started to accumulate in subsidiary cells, at the same time the expression of ZmPIP2;5 was upregulated, in agreement with the changes of H2O2 distribution in the subsidiary cells. Bofore short cells appeared, the genes of ZmPIP2;4, ZmPIP2;5 and ZmPIP2;6were not responsive to the water stress and the gene expression was not upregulated. But after short cells appeared, as soil water shortages became more severe, the gene expression of ZmPIP2;4, ZmPIP2;5 and ZmPIP2;6 was upregulated. So the accumulation and purge of H2O2 in subsidiary cells are possibly transportated by aquaporins ZmPIP2;5.(4)The cellular localization of aquaporin ZmPIP2;5:Before and after the short cells appeared, membrane of guard cells and subsidiary cells had the distribution of aquaporin ZmPIP2;5; but after the short cells appeared, the distribution of aquaporin ZmPIP2;5 on the membrane of guard cells and subsidiary cells all increased.Based on the above experiment results, we can conclude as follows:During diurnal variation, stomatal aperture becomes smaller with the H2O2 increasing in subsidiary cells. The H2O2 in subsidiary cells is involved in the regulation of stomatal switching, and the increment of H2O2 in subsidiary cell promotes the stomatal closure. H2O2 in subsidiary cells is exogenous, which is accumulated and eliminated by the transportation mediated possibly by aquaporin ZmPIP2;5.