| Heat stress due to increased temperature is an agricultural problem in many areas in the world, which seriously affects plant growth and development and may lead to a drastic reduction in economic yield. Cucumber (Cucumis sativus L.) is one of the important vegetable crops worldwide, and also is the main type of vegetables cultivated in protected condition in China. However, in most parts of our protection facilities, the midday temperatures often above40℃, heat injury has become a main limiting factor in summer-autumn cucumber cultivation in open and protected field. Polyamines (PAs) are low molecular weight aliphatic amines with biological activity produced during metabolism, necessary for normal cell growth, morphogenesis, aging and stress response, mainly including putrescine (Put), spermidine (Spd) and spermine (Spm). In recent years, growing interest has focused on the subject that using exogenous PAs to regulate and mediate a variety of stress tolerance of plants. The information between PAs and high temperature stress in plant physiology is very limited; especially proteomics study is still blank.In the present study, the more heat-resistant cucumber cultivar cv.’Jinchun No.4’is used in climate chambers and hydroponics. By using exogenous Spd (1mM) foliar spray, we determined the effects of high temperature stress (42℃/32℃) on the plant growth, photosynthesis and chlorophyll fluorescence, chloroplast ultrastructure, membrane lipid peroxidation, antioxidant enzymes activity and isozymes, ascorbate-glutathione cycle, nitrogen metabolism and so on. We also investigated on changes of soluble protein, proteomics and mRNA transcription level in leaves of cucumber seedlings to explore the molecular mechanism why exogenous Spd increased the heat resistance of cucumber seedlings. The main results are as follows: 1. Effects of exogenous Spd on growth and endogenous poyamines contents of cucumber seedlings under high temperature stress:under high temperature stress, seedling growth was inhibited, plant height, stem diameter, fresh and dry weight, and leaf area are significantly decreased, exogenous Spd could availably alleviate the heat damage on growth, increase plant biomass accumulation. Upon high temperature stress, the content of free, soluble conjugated and insoluble bound Put was significantly decreased, and the the content of free, soluble conjugated and insoluble bound Spd and Spm were significantly increased by high temperature stress. From the species of PAs, the conversion from Put to Spd and Spm was likely a kind of adaptive response to heat stress for cucumber seedlings. Exogenous Spd promoted the contents of Put and Spd, but a slight inhibitory effect on Spm content. That is means higher level of Spd, because Put is the precursor of Spd and Spm is the downstream product of Spd. Exogenous Spd promoted high levels of endogenous Spd may contribute to enhanced heat tolerance of cucumber seedlings.2. Exogenous Spd increased the photosynthetic characteristics of cucumber seedlings under high temperature stress:Compared with normal temperature, pigment contents and net photosynthetic rate were decline, stomatal conductance and transpiration rate were increased under high temperature, PS Ⅱ photochemical efficiency was also decreased significantly; application of exogenous Spd under high temperature was helpful to improve the pigment contents, maintain a higher photosynthetic rate and photochemical efficiency. Without application of Spd, high temperature stress induced the accumulation of swollen starch grains in chloroplasts, the extrusion and reduction of grana lamellae, and the ubiquitous appearance of osmiophilic granules. However, the chloroplast ultrastructure of seedlings treated with spermidine was only very mildly affected by high temperatures. Spd protects the organelle structure and photochemical activity of chloroplasts, which is fairly important in the adaptation of cucumber seedlings against high temperature stress. Exogenous Spd also effectively inhibited the hydrolysis of starch under high temperature stress, so the sugars tend to synthesis and accumulated more dry matter.3-Exogenous Spd improved the antioxidant capacity for ROS cleaning in leaves of cucumber seedlings under high temperature stress:Under high temperature stress, membrane lipid peroxidation was significantly increased in leaves of cucumber seedlings, showing the relative conductivity, MDA content and lipoxygenase (LOX) activity were significantly increased, ROS levels (including O2production rate and H2O2content) were remarkably raised. Exogenous Spd treatment significantly inhibited the oxidative damage of membrane lipid in leaves under high temperature stress. Expose to high temperature, SOD activity decreased, POD and APX activity first increased and then decreased, CAT activity first decreased and then increased, the isozymes of these enzymes also changed accordingly; Exogenous Spd treatment significantly promoted the activities of SOD, and the POD, CAT and APX activities were also enhanced to varying degrees. Apart from Spd had little effect on POD isozymes, the expression of other kinds of protective isozymes were significantly enhanced by Spd. In AsA-GSH cycle, MDAR, DHAR and GR activity, and reduced form contents of AsA and GSH were increased under high temperature stress. Exogenous Spd treatment significantly promoted the activities of MDAR, DHAR and GR, and the contents of AsA and GSH under high temperature stress. In particular, the ratio of AsA/GSH and GSH/GSSG were also further increased. Cucumber seedlings have their own antioxidant system against oxidative stress caused by high temperatures, this resistance gradually weakened with the extension of stress time, but exogenous Spd could significantly reduce the lipid peroxidation caused by heat stress, improve antioxidant enzyme activity and enhance isozymes expression, activate the AsA-GSH cycle, so the total antioxidant capacity of cucumber seedlings for was totally improved.4-Effects of exogenous Spd on nitrogen and other metabolism in leaves of cucumber seedlings under high temperature stress:Under high temperature stress, the nitrate content in roots was increased but transport to leaves was blocked, nitrate reductase (NR) in roots was deactivated, the ammonium content in both roots and leaves was largely increased. The exogenous Spd upon high temperature were helpful to regulate the balance of nitrogen metabolism, promoting nitrate, suppressing ammonium and activating the NR. Spd could availably modulate nitrogen metabolism in cucumber seedlings under high temperature stress. Proline is also a kind of important nitrogen-containing compound, the higher level of proline can enhance the ability of osmotic adjustment to maintain the water balance in plants. Proline content in leaves was increased by high temperature treatment, exogenous Spd promoted the enhancement. High temperature induced plasma membrane protein content decreased and tonoplast membrane protein content increased, proton pumps activity were enhanced; exogenous Spd could further improve activity of H+-ATPase but not H+-PPase to stabilize the membrane structure and establish the transmembrane proton driver△pH, promote the generation of ATP and maintenance of cytoplasmic acid-base balance. 5. Effects of exogenous Spd on proteomics changes and mRNA accumulation patterns in leaves of cucumber seedlings under high temperature stress:At the beginning of heat stress, the soluble protein content in leaves was increased, but with the stress time extended to5days, the soluble protein content started to decline, exogenous Spd increased soluble protein. By using the two-dimensional electrophoresis (2-DE) and mass spectrometry (LC-ESI-MS/MS) technology, compared the2-DE protein profiles of treatments of high temperature or exogenous Spd with control, we found62differentially expressed proteins,60spots were successfully been identified by MS. Functional classification revealed that25spots related to photosynthesis,16spots involved in energy and material metabolism,6spots for the molecular chaperone protein,6spots in defense and stress respond and7spots related to protein or nucleic acid biosynthesis, and another two spots identified by MS with too lower scores or EST function unknown protein. The main difference proteins such as Rubisco large/small subunit, Rubisco activase (RCA), Transketolase (TK), phosphate ribulose kinase (PRK), heat shock protein70(HSP70), chaperonin60(CPN60), Cu/Zn-SOD, carotenoids associated protein, protein translation elongation factor (EF-Ts and EF-G) and so on. These proteins related to photosynthesis, energy and metabolism were most seriously affected by high temperature stress. Exogenous Spd was helpful to inhibit parts of the down-regulated proteins under high temperature conditions, showing a positive protective role at protein level. Photosynthesis-related genes (RbcL, RbcS, RCA, OEC23and OEC3, etc.), antioxidant-related genes (SOD, POD and Car, etc.), molecular chaperones (Hsp70, Cpn60, and Hsp20) were prominently response to high temperature stress at mRNA level; exogenous Spd showed significant regulatory role on the expression of some genes. The mRNA transcriptional accumulation patterns of several important high-abundance proteins under high temperatures and/or treated with exogenous Spd were consistent with the proteomics accumulation patterns. |
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