Alleviative Effects And Its Mechanism Of Exogenous Spermidine On Tomato Seedlings Under Salinity-alkalinity Mixed Stress

Saline-alkaline stress is one of the major abiotic stresses which causes crop failures, sothe research for improving the saline-alkaline tolerance of crops has been attracted greatattention. As a kind of secondary metabolites in organisms, polyamines （PAs） in plants havemulti-functions, such as regulating basic metabolisms, influencing growth and development,adjusting morphogenesis and slowing the aging process. Putrescine （Put）, spermidine （Spd）and spermine （Spm） are common PAs, and Spd is the most closely associated PA with stresstolerance in plants. Under salt stress conditions, exogenous Spd application has beensuccessfully used for alleviating the growth inhibitory effects, and even enhancing theproductive potential of crops exposed to high-salinity stress. However, the current researchabout the influence mechanisms of exogenous Spd on plant resistance, mostly focus on theneutral NaCl solution, while there is nearly no related report on the alkaline salt tolerance.Tomato （Solanum lycopersicum） is one of the widely cultivated and consumed crop inthe world, and it is also a good model for the plant genetics and solanaceous crops research.Cultivated tomatoes are moderately sensitive to salt stress, and salinity-alkalinity seriouslyreduces its production. In the present study, the effects of exogenous0.25mmol·L^-1Spd（foliar spraying or seed soaking） on the plant growth, photosynthetic fluorescencecharacteristics, main mineral elements contents, antioxidant system, osmotic adjustmentsystem, nitrogen metabolism and PAs metabolism were investigated, in two cultivars oftomato seedlings （‘Jinpengchaoguan’ and ‘Zhongza No.9’, the former being more tolerant tosaline-alkaline stress than the latter）, grown under salinity-alkalinity stress condition （NaCl:Na₂SO4: NaHCO₃: Na₂CO₃=1:9:9:1）, to explore the physiological functions of exogenous Spdon improving the saline-alkaline tolerance of tomato. Meanwhile, the differentially expressedproteins in leaves of ‘Zhongza No.9’ tomato seedlings under each treatment were identifiedand classified by MALDI-TOF/MS, in order to preliminarily reveal the protein foundationsinduced by Spd under saline-alkaline stress. Main research results were as follows:1. Under salinity-alkalinity stress, the seedling growth of the two tomato cultivars wasinhibited. The net photosynthetic rate, stomatal conductance, transpiration rate, chlorophyllcontents, photochemical quantum efficiency of PSII, photosynthetic electron transport rate, photochemical quenching coefficient and total P contents in leaves, as well as the total N,total K, Ca²⁺, Mg²⁺contents in leaves and roots, were all significantly reduced undersaline-alkaline stress. However, the leaf non-photochemical quenching coefficient and the Na⁺contents in leaves and roots were all significantly increased. Besides, there was greaterinfluence on ‘Zhongza No.9’. At the fifth day of treatment, the main limiting factor ofphotosynthesis in ‘Zhongza No.9’ tomato seedling was non-stomatal factor undersaline-alkaline stress, while it was stomatal factor for‘Jinpengchaoguan’. Under stressconditions, exogenous foliar spraying Spd could significantly improve the shoots growth, theleaf chlorophyll contents and PSII photochemical activities of both tomato cultivars.Moreover, the inhibition of leaf photosynthetic electron transportation, as well as the stomataland non-stomatal limitation, caused by salinity-alkalinity stress, could be effectively reducedby exogenous Spd, meanwhile, the main mineral elements contents were maintained in aproper balance, and the alleviative effect of Spd treatment on saline-alkaline damages wasmore obvious in ‘Zhongza No.9’ cultivar.2. Exogenous Spd may help reduce salinity-alkalinity stress-induced increases in freeamino acids, ammonium （NH₄⁺） contents, and glutamate dehydrogenase （GDH） activities;depress stress-induced decreases in nitrate contents, and nitrate reductase, nitrite reductase,glutamine synthetase （GS）, glutamate synthase （GOGAT）, glutamate oxaloacetatetransaminase （GOT）, and glutamate pyruvate transaminase （GPT） activities, especially for‘Zhongza No.9’. The results showed that exogenous Spd promotes the assimilation of excesstoxic NH₄⁺, by coordinating and strengthening the synergistic action of GDH, GS/GOGAT,and transamination pathways, all during saline-alkaline stress. Subsequently, NH₄⁺and itsrelated enzymes （GDH, GS, GOGAT, GOT and GPT）, in vivo, are maintained in a proper andbalanced state, to enable mitigation of stress-resulted nitrogen metabolic disturbances, andeventually promote plant growth.3. Under saline-alkaline stress, the Oˉ2.production rate and H2O2contents in leaves androots of both tomato cultivars increased at different degrees, and the higher levels of reactiveoxygen species （ROS） aggravated the membrane lipid peroxidation, which facilitated theproduction of malondialdehyde. Moreover, the activities of superoxide dismutase （SOD）,peroxidase （POD） and catalase （CAT）, as well as the accumulation of proline and solublesugar, could be induced by salinity-alkalinity stress to some extent. Exogenous Spd, on onehand, may help to alleviate the membrane lipid peroxidative injury, by further increasing thestress-induced SOD, POD and CAT activities to reduce the excessive ROS level. On the otherhand, Spd treatment can enhance the saline-alkaline tolerance of tomato seedlings, throughinducing the further biosynthesis and accumulation of osmotic adjustment materials. 4. PAs content, in any form, increased significantly during salinity-alkalinity stress. Theactivities of S-adenosylmethionine decarboxylase （SAMDC） and diamine oxidase （DAO）,contents of free Spd, soluble conjugated Spd and Spm, and insoluble bound Spd in roots wereincreased to a greater extent in ‘Jinpengchaoguan’ tomato seedlings exposed to saline-alkalinestress. Exogenous Spd markedly suppressed the accumulation of free Put, but promoted anincrease in free Spd and Spm contents, as well as soluble conjugated Spd and insoluble boundPut in both cultivars. From these data, we deduced that exogenous Spd promotes theconversion of free Put into free Spd and Spm, and soluble conjugated forms and insolublebound forms PAs under salinity-alkalinity stress. Furthermore, under saline-alkaline stress,exogenous Spd enhanced the activities of ornithine decarboxylase （ODC）, SAMDC and DAO,and reduced the activities of arginine decarboxylase （ADC） and polyamine oxidase （PAO） in‘Zhongza No.9’ roots. In addition, exogenous Spd reduced the activities of ADC and ODC,and increased the activities of DAO and SAMDC in ‘Jinpengchaoguan’ roots undersaline-alkaline stress. These results suggest that Spd treatment can regulate the metabolicstatus of PAs caused by saline-alkaline stress, and the effects of exogenous Spd on differentcultivars with differential salinity-alkalinity tolerance exist certain differences.5. Two dimensional gel electrophoresis analysis results showed that the leaf protein spotsin control, single Spd, single salinity-alkalinity, and salinity-alkalinity plus Spd treated plantswere1084,1243,954and1172, respectively. Eighty-two differentially expressed proteinspots were identified by MALDI-TOF/MS and related databases, in which72protein spotsgot valid results. The identified proteins were associated with the regulation of photosynthesis,photo respiration, energy pathway, signal transduction, amino acid metabolism, proteinfolding, post-transcriptional regulation, and so on. This provides further evidence thatsaline-alkaline stress and exogenous Spd treatment could induce the synergy of numerousmetabolic processes in plants to adapt to the changing environment.