Studies On The Mechanism Of The Alternative Pathway In Nitrogen Deprivation Induced Cross-tolerance To Salt Stress In Poa Annua Callus

The impact of salinity on agricultural land has brought widespread concerns in the whole society. Studies on mechanisms of plant resistance to salt stress have become a hot area. Alternative pathway is an important pathway that can be induced by environment stresses in plants. In the present study, we showed a new mechanism involving the alternative pathway in nitrogen deprivation-induced tolerance to salt stress in Poa. annua callus.The main results were summarized as follows:1. The seeds of P. annua were used as explants and cultured in vitro to induce callus. The experimental results showed that B5+2.4-D 5 mg.L-1 was an optimal medium for callus induction. The callus induction rate was about 11.00%.2. The malondialdehyde(MDA) content increased by 91.13%, 81.66%, 74.94%, 36.56%, 52.9%, 16.49%, and 16.05%, respectively; and the capacity of the alternative pathway increased by 59.62%, 56.23%, 48.67%, 20.11%, 58.13%, 8.26%, and 15.74%, respectively, under-NPK,-NK,-NP,-PK, –N,-P、-K treatments. These results proved that –N, but not-P and –K, can significantly cause the increase of the alternative pathway capacity and MDA content in P. annua callus.3. Exogenously added H2O2 biosynthesis inhibitors diphenyliodonium(DPI), H2O2 scavenger catalase(CAT), ethylene biosynthesis inhibitor amino acid oxygen(AOA), ethylene signaling inhibitors AgNO3, and NO scavenger 2-phenyl-4,4,5,5-methylimidazole-1-oxo-3-oxide(PTIO) did not affect-N induced alternative pathway capacity of P. annua callus,implying that these signaling molecules are not involved in the process of alternative pathway capacity induction by nitrogen deprivation in P. annua callus.4. Under 600 mM NaCl treatment, the H2O2, Na+, pyruvate, and MDA content increased by 301.11%, 315.37%, 241.01% and 148.45%, respectively; K+ decreased by 29.1%,;Na+/ K+ ratio increased by 386.55%; electrolyte leakage increased by 151.63%; PM H+-ATPase, ascorbate oxidase(APX), catalase(CAT), peroxidase(POD), and superoxide dismutase(SOD) activities increased by 126.41%, 81.55%, 198.46%, 81.7% and 64.48%, respectively; and the alternative pathway capacity increased by 14.17% in P. annua callus.5. Co-treatment of nitrogen deprivation further improved the capacity of the alternative pathway in P. annua callus, about increased by 60.22%; the activity of PM H+-ATPase, APX, CAT, POD and SOD increased by 119.89%, 28.02%, 44.53%, 32.02% and 64.08%, respectively; pyruvate content increased by 182.61%; MDA and H2O2 content decreased by 29.83% and 99.12%, respectively; electrolyte leakage decreased by 41.02%; Na+/ K+ ratio decreased by 47.25%. These results proved that nitrogen deprivation induces cross-tolerance of P. annua callus to salt stress.6. The application of salicylhydroxamic acid(SHAM, an inhibitor of alternative pathway) partially diminished the effects of nitrogen deprivation pretreatment and the MDA content increased by 36.22%; H2O2 content increased by 101.13%; Na+/ K+ ratio increased by 54.25%; PM H+-ATPase decreased by 39.99%. The effects were eliminated by exogenously added CAT or DPI. 10 mM exogenous pyruvate also induced the cross-tolerance of P. annua callus to salt stress.Taken together, our results suggested that the pretreatment of nitrogen deprivation enhances the capacity of alternative pathway by increasing the pyruvate content and enhances the PM H +-ATPase activity and the ability to maintain the redox state. The increased activity of PM H+-ATPase will enhance the ability to maintain ion homeostasis in P. annua callus, thereby increasing tolerance to salt stress in P. annua callus.