The Physiological Mechanism Of Abscisic Acid Mediating Salicylic Acid-induced Chilling Tolerance Of Grafted Cucumber

Grafting is the simplest and most effective technology to improve the resistance against biotic and abiotic stress of vegetable crops,which is widely used in used the production of cucurbits,solanaceous,and other vegetables.Salicylic acid（SA）as an important signaling molecule.It plays an important role in regulating plant responses to biological and abiotic stresses.However,the interaction between SA and other signaling molecules,such as ABA and ROS,et al.under stress condition is still unclear.In the present study,we investigated the mechanism of ABA and H₂O₂are involved in SA-regulated chilling tolerance of grafted cucumber.The pumpkin（Cucurbita moschata D.cv.‘Jinmama 519’,Cm）and cucumber（Cucumis sativus L.,cv.‘Jinyou 35’,Cs）was used as rootstock,and’Jinyou 35’cucumber（Cucumis sativus L.）were used as rootstock and scion,respectively.The self-grafted cucumber（Cs/Cs）and grafted cucumber（Cs/Cm）were treated with the aerial chilling（5/25℃）,root-zone chilling（25/5℃）,the combined chilling（5/5℃）stress in an artificial climate room.The self-grafted cucumber at normal temperature（25/25℃）was used as control.Then the Cs/Cs and Cs/Cm seedlings were sprayed with SA,abscisic acid（ABA）,hydrogen peroxide（H₂O₂）and their inhibitors or scavengers.The deionized water（control）was used as the control.The main results are as follows:1.Under chilling stress,the grafted cucumber seedlings showed obviously minor chilling injury symptoms and lower chilling injury index（CI）,while significantly higher net photosynthec rate（Pn）,stomatal conductance（Gs）,transpiration rate（Tr）,activities of Ribulose 1,5-bisphosphate carboxylase（Ru BPCase）and Ru BPCase activase（RCA）,as well as the relative m RNA expression of rbc L and rbc S,compared the self-grafted seedlings.The RCA protein level in grafted seedlings were also significantly up-regulated than in self-grafted seedlings.Grafted seedlings exhibited an obvious increase in the actual photochemical efficiency of photosystemⅡ（PSⅡ）in light(Φ_PSⅡ),maximum photochemical efficiency（F_v/F_m）of PSⅡin darkness,photosynthetic performance index(PI_ABS),efficiency of exciton captured by the reaction center after transferring electrons to QA（φ₀）,reaction center activity of photosystem I（PSI）（ΔI/I₀）,as well as the relative expression of cold-responsive genes COR47 and CBF1,but a marded decrease inΦ_D0,and reactive oxygen（ROS）accumulation relative to the self-grafted seedlings.These data suggest that grafting alleviate the damage of the PSⅡand PSI response centers by reduce the ROS accumulation and upregulate the expression of cold-responsive genes under chilling stress,and improve the chilling tolerance of cucumber seedlings.2.Exogenous SA and ABA increased the Pn,activities of Ru BPCase and RCA,relative m RNA expressions of rbc L and rbc S,F_v/F_m,Φ_PSⅡ,PI_ABSandΨ₀,but decreased theΦ_D0,relative to the H₂O treatment.Compared with the self-grafted seedlings,the grafted seedlings revealed significantly higher Pn,activities of Ru BPCase and RCA,m RNA levels of rbc L and rbc S,F_v/F_m,Φ_PSⅡ,PI_ABSandΨ₀,but distinctly lowerΦ_D0.The results indicate that SA and ABA play an active role in the grafting-induced photosynthetic carbon assimilation and photoprotection in cucumber plants.3.Grafting can significantly improve the chilling-tolerance of cucumber and promote the synthesis and accumulation of endogenous ABA and H₂O₂.Under chilling stress,the exogenous SA treated-seedlings showed markedly minor chilling syptome and lower electrolyte leakage（EL）and CI,while higher m RNA expression of CBF1,COR47,9-cis-epoxycarotenoid dioxygenase gene（NCED）and respiratory burst oxidase homologue gene（RBOH1）,as well as the accumulation of ABA and H₂O₂compared with the H₂O treatment.However,2-aminoindan-2-phosphonicacid（AIP）and L-α-aminooxy-phenylpropionic acid（AOPP）,the SA synthesis inhibitors treated-seedlings revealed significantly serious chilling injury syptome,higher EL and CI,and lower m RNA expression of CBF1,COR47,NCED and RBOH1,as well as the accumulation of ABA and H₂O₂,relative to the H₂O treatment.Compared with the self-grafted seedlings grafted seedlings exhibited a remarkabl decrease in EL and CI,but an increase in m RNA expression of CBF1,COR47,NCED and RBOH1,and the accumulation of ABA and H₂O₂.These data suggest that ABA and H₂O₂may be involved in SA-induced chilling tolerance in grafted cucumber.4.ABA and H₂O₂decreased EL and CI,however,the ABA biosynthesis inhibitors sodium tungstate（Na₂WO₄）and fluridone（Flu）or H₂O₂scavenger N,N’-dimethylthiourea（DMTU）and diphenyleneiodonium chloride（DPI）increased the EL and CI,and these were significantly lower in grafted seedlings than in self-grafted seedlings.Na₂WO₄or DMTU abolished SA-induced chilling tolerance of grafted cucumber.ABA significantly increased RBOH1 m RNA expression and endogenous H₂O₂production.DMTU and DPI abolished ABA-induced chilling tolerance of cucumber.However,ABA had little effect on the phenylalanineammonialyase gene（PAL）m RNA expression and SA production.Application of SA inhibitors AIP and AOPP,ABA-induced plant response to chilling stress also had no significant change.H₂O₂had no significant effect on the SA and ABA contents and m RNA expression of their key genes.Na₂WO₄and Flu diminished grafting-or SA-induced RBOH1m RNA expression and H₂O₂biosynthesis,but DMTU and DPI did not affect ABA production induced by SA under chilling stress.These results suggest that H₂O₂,as a downstream signaler of ABA,mediates SA‐induced chilling tolerance in grafted cucumber plantsIn summary,the current study demonstrates that pumpkin as rootstock quickly accumulates SA in the roots under chilling stress and then transports it to the scion leaves and simultaneously induces the biosynthesis of SA in the scion leaves.The rootstock-induced SA accumulation in leaves promotes ABA production and further stimulates H₂O₂accumulation,and this subsequently activates the ICE-CBF-COR signal to mitigate the chilling damage of membrane lipid peroxidation and photosynthetic apparatus,and improve the chilling tolerance of grafted cucumber seedlings.