As a newly discovered gas signal molecule,hydrogen gas(H2)is also a potential biological stimulant,which has attracted more and more attention due to its unique safety and the great changes that may bring to agricultural production.Considering safety and economy,hydrogen rich water(HRW)has always been the main way of H2 supply in agricultural application.However,due to the low solubility and high fugacity of H2 in water,it is difficult to realize the continuous supply of hydrogen.A new kind of nano material,ammonia borane loaded hollow mesoporous silica nanoparticle(AB@hMSN),shows high hydrogen loading capacity and continuous hydrogen release behavior,which makes up for the deficiency of HRW to a certain extent.At present,these hydrogen releasing nanomaterials are mainly used in the medical field,especially in the direction of tumor and cancer treatment,but there is little research on their botanical effects.Therefore,in order to further verify the feasibility of this kind of hydrogen releasing nano materials in agricultural application,"Lufeng"cucumber(Cucumis sativus L.)were used as model plant,through a series of pharmacology,anatomy and molecular biology experimental methods to explore the molecular mechanism of AB@hMSN induced adventitious root(AR)formation in Cucumber,the main results are as follows:1.The characterization of AB@hMSN was successfully analyzed,transmission electron microscope(TEM)images showed that the particle size of the synthesized AB@hMSN was uniform(particle diameter was about 150 nm)and also had a better dispersion;The data of fourier transform infrared spectroscopy(FTIR)showed that ammonia borane(AB)was well encapsulated and embedded in the carrier hMSN;At the same time,thermogravimetric analysis(TGA)datas showed that the loading capacity of AB was more than 40%.In addition,the presence of nanoparticles in the hypocotyls of cucumber explants treated by AB@hMSN was observed in transmission electron microscopy images,suggesting that AB@hMSN can enter hypocotyl cells through the apoplast pathway.2.The results showed that AB@hMSN induced cucumber adventitious rooting in a concentration dependent manner,and the best induction effect concentration was 5 mg/L AB@hMSN.Then,the contents of nitric oxide(NO)and cyclic guanosine monophosphate(c GMP)were determined by Gliss reagent method,confocal laser microscopy fluorescence staining and enzyme-linked immunosorbent assay(ELISA),respectively.Compared with the control,the contents of endogenous NO and c GMP were significantly increased by AB@hMSN treatment,and reached the peak after 24 hours of treatment,indicate NO and c GMP maight be the downstream signals of AB@hMSN induced adventitious rooting.It is well known that NO-c GMP signaling pathway is an important part of IAA’s regulation of root morphogenesis network.To further explore whether it is involved in the formation of AB@hMSN induced AR,cucumber explants were further treated with NO scavenging agents(c PTIO and PTIO)and guanylate cyclase(GC)inhibitors(ODQ and LY83583)alone or in combination as to reduce the level of endogenous NO and c GMP.Sodium nitroprusside(SNP,a NO donor)and 8-Br-c GMP(a membrane permeable c GMP derivative)were treated as positive controls.It was found that the above scavengers or inhibitors could significantly inhibit the AR formation induced by AB@hMSN and SNP in cucumber,while the addition of 8-Br-c GMP could restore the original induced effect.At the same time,the anatomical evidence of AR primordium development showed that AB@hMSN treatment significantly increased the number of AR primordia in 5 stages,especially in V primordia,which could also be reversed by ODQ and LY83583.Molecular evidence also showed that AB@hMSN,SNP and 8-Br-c GMP could significantly increase the expression of adventitious root formation-related genes,including auxin response genes(Cs Aux22B-Like、Cs Aux22D-Like)and cell cycle circulating genes(Cs CDC6)and so on.However,combined application with ODQ and LY83583 could disrupt these promoting effects.In conclusion,AB@hMSN can enhance the expression of adventitious root formation-related genes through NO-c GMP signaling pathway and thus promote the formation of AR in cucumber.This study not only attempts to expand the downstream signal network of H2 induced AR,but also provides a possibility for the application of nano hydrogen releasing materials in agriculture. |