Proteome And Physiological Mechanisms Of Improved Ca（NO₃）₂ Stress Tolerance Of Cucumber Seedlings By Exogenous Spermidine

The over overuse of fertilizers and mismanagement in protected horticulture have created a serious problem of secondary salinization that limits vegetable sustaninable development. It is reported that Ca2+ accounts for over 60% of total cations and NO3- accounts for 67-76% of the total anions in secondary salinization of soil. The high level of Ca(NO3)2 accumulation was one of the main reasons for soil salinization. Polyamines are a class of organic polycations present ubiquitously in living organisms, which involved in helping plants deal with a wide range of abiotic stresses. The application of exogenous polyamines provides a convenient and effective approach to enhance plant tolerance under abiotic stress, and Spd showed the most closely relationshiop with stress resistance. Cucumber (Cucumis sativus L.)is an economically important horticultural crop and highly sensitive to saliny. The present investigation was carried out to study the effects of exogenously sprayed of 1 mM spermidine(Spd) on the proteome and physiology mechanisms of salt-sensitive cucumber cultivar(Jinyou No. 4) exposed to Ca(NO3)2 stress (80 mM). The main results are as follows:1. Exogenous Spd alleviate the inhibition of plant growth in response Ca(NO3)2 stress. A 2-DE gel electrophoresis and MALDI-TOF/TOF-MS were performed. A total of 71 differential protein spots were confidently identified in response to Ca(NO3)2 stress or Spd.The resulting proteins involved in protein metabolism, carbohydrate and energy metabolism,ROS homeostasis and stress defense,cell wall related,transcription and others. Most proteins belong to protein metabolism, carbohydrate and energy metabolism, ROS homeostasis and stress defense and were visibly increased by exogenous Spd. Consistently, the Spd-responsive protein interaction network revealed 13 key proteins, which all involved in protein metabolism, carbohydrate and energy metabolism, ROS homeostasis and stress defense, indicated their important role in Spd-induced Ca(NO3)2 stress resistance.2. Ca(NO3)2 stress caused a notable reduction of PN,which is mainly due to stomatal limitations. Exogenous Spd increased the chlorophyll content, Gs and Ci, thus promoted the net photosynthetic rate. Exogenous Spd elevated non-photochemical quenching (qN),enhanced the electron transport capacity (ΦPSⅡ), photochemical quenching (qP) and the maximal quantum yield of PSII photochemistry (Fv/Fm), thus avoiding the photoinhibitio in cucumber seedlings leaves.3. Under Ca(NO3)2 stress, Exogenous Spd significantly increased the activities of antioxidant enzymes (SOD, POD, CAT, APX, GR, MDAR and DHAR) and the the content of non-enzymatic antioxidants (AsA and GSH), resulting the decrease of the reactive oxygen species (ROS, including O2·- and H2O2) and MDA content, ultimately alleviate the oxidative damage induced by Ca(NO3)2 stress, and avoided the lipid peroxidation.4. Under Ca(NO3)2 stress,exogenous Spd enhanced the inactivated enzymes activities involved in the nitrogen metabolism, including NR, NiR, GS, GOGAT and GDH, thus reducing the accumulation of NO3- and NH4+, which enhanced the capacity of nitrate reduction and ammonia assimilation. In addition, Spd treatment remarkably increased the accumulation of soluble carbohydrate (including sucrose, fructose and glucose), thus alleviate the osmotic stress caused by Ca(NO3)2 stress,protected enzyme activities related in N metabolism, and promoted the conversion of NO3- and NH4+ to aminos acids and soluble protein. In the presence of Spd, total C content and the C/N radio increased significantly,while total N decreased in response to Ca(NO3)2 stress. Based on our results, we suggest that exogenous Spd could effectively accelerate nitrate transformation into amino acids and improve the accumulation of carbon assimilation production, thereby enhancing the ability of the plants to maintain their C-N balance in response to Ca(NO3)2 stress.5. Under Ca(NO3)2 stress, exogenous Spd significantly induced the expressions of poly amine synthesis gene (SAMDC, ADC and SPMS), auxin synthesis gene (TAR, YUCCA and NIT), polar auxin transport genes (AUX and PIN) and ethylene synthetic gene (SAMS and ACS), and decreased ABA biosynthetic genes (ZEP, NCED and AAO) expression, thus increased the contents of free Put, free Spd, free Spm and IAA and the release rate of ethylene,and reduced ABA content, ultimately increased the length of lateral to some extent, and recover the root apical externalmorphology in cucumber.