Studies On Physiological Mechanism Of Spinach To Seawater Stress

Water resources in the ocean correspond to the capacity to be about 1.34×1018 m3 which is equivalent to 96.8% of total water of the earth. However, the freshwater on land has an inclination to be lacking. Now, researchers have attempted to cultivate crops by irrigation using seawater for food supply. Spinach (Spinacia olerancea L.) plants are a middle salt tolerant crop, which critical salt concentration is 2 d-S m-1. Therefore, it is supposed to be possible to cultivate spinach plants by using seawater. The physiological response to seawater stress was investigated using two cultivars plants cultured in the Hoagland's nutrients solution with or without seawater (40%). Main research results were as follows:Spinach cultivar such as Helan No.3 was the greatest tolerant to seawater and cv. Yuanye was greatest sensitivity amony fourteen compared cultivars. The appropriate concentration identified was 10% of seawater. And the integrative quality of spinach plants was not enfluenced by nutrient solution with 40% seawater.Dry weight per plant of cv. Helan No.3 was not obviously affected by seawater, but cv. Yuanye spinach was decreased remarkably. During exposure to stress of seawater, malondialdehyde (MDA) content and membrane permeability were both increased, but photo-pigment of leaves was reduced, and theirs change range of cv. Helan No.3 was little than cv. Yuanye spinach (except MDA content of leaves). Stomata limitation of two spinach cultivars induced decrease of intercellular CO2 concentration (Ci) and net photosynthetic rate (Pn) in short term on stress of seawater, however, in long term on stress of seawater, Pn of cv. Helan No.3 resumed to control level, but Pn of cv. Yuanye spinach continued to decrease as a result of assimilation decreased. The analyse of chlorohyll fluorescence kinetica parameters shows that there was little effect on photochemical quenching (qP) of cv.Helan No.3 but quantum yield of PSⅡphotochemisty rate (ΦPSⅡ) increased obviously under seawater stress, that of Yuanye spinach declined when exposure to stress seawater; Decrease amplitude and increase range of cv. Helan No.3 were greater than that of cv. Yuanye spinach; Maximal photochemical efficiency (Fv/Fm) of two spinach cultivars were declined, but the increase of photo-inhibition degree (1-qP/qN) of cv. Yuanye spinach was higher than cv. Helan No.3 under seawater stress. These results suggest that two spinach cultivars were injured by photo-inhibition and photo-oxidation on seawater stress, that lead lipid to peroxidize and chlorophyll (Chl) to decompose; however, tolerance cultivars such as cv. Helan No.3 applied more light energy to photochemical reaction, and caloric dissipation ability was strong, which was depended on xanthophylls cycle; degree of photo-inhibition was lower, reactive oxygen species (ROS), which was excited by excess light energy, were less destroyed membrane system and photopigment to maintained a higher level of Pn, there for, seawater stress had little effect on growth.Apparent quanta yield (AQY) and Pn on saturation photo intensity were remarkably decreased but the allocation of dissipation energy (D) by antenna and non-fluorescence quenching coefficient (NPQ) were increased by the stress of seawater; daily and dynamic movements of NPQ and xanthophyll de-epoxidation state (DES) were presented trends to increased at first and then decreased with extend of stress time and photosynthetic active radiation (PAR) increased during exposure to seawater stress, furthermore, there were played some multinomial regress relationship between NPQ and DES; when treated with ascorbic acid (AsA), NPQ and DES were remarkably increased but decreased by 1, 4-dithiothreitol (DTT); during exposure to seawater stress, AQY and Pn under saturation photo intensity of Yuanye spinach was decrease more than that of cv. Helan No.3, and the increase of D, NPQ and DES were less than that of cv. Helan No.3. These results suggested that photo-inhibition was produced and non-radiant energy was enhanced, and xanthophyll cycle was major approach to thermal dissipation; cv. Helan No.3 has more high activity of de-epoxidation as a result of dissipating excessive energy to decease injury of photosynthesis organs.The production rate of superoxide radical (O2-), content of hydrogen peroxide (H2O2) and MDA in chloroplasts of two spinach cultivars were remarkably increased by seawater with higher rate in cv. Yuanye; In seawater tolerant cultivar (cv. Helan No.3), the ROS elimination is mainly depended on superoxide dismutase (SOD) and ascorbic acid-reduced glutathione recycle system (AsA-GSH), which could alleviate oxidation injury of ROS to chloroplast membrane, and the conversion of glutathione (GSSG) into reduced glutathione (GSH) was maintained to keep correct oxidation and deoxidation circumstance in chloroplasts of spinach leaves during exposure to seawater; while in seawater sensitive cultivar (cv. Yuanye), it is mainly depended on SOD and peroxidase (POD), which has limited ability to eliminate ROS and lead to accumulate mass ROS to severe oxidation injury of chloroplast membrane, and the ratio of GSH/GSSG in chloroplasts was significant decreased by seawater, leading to disturbing of oxidation and deoxidation circumstance in chloroplasts, as a result of declination of seawater tolerance.Spinach plants with seawater were induced to oxidize metabolic substances and to accumulate ROS in chloroplasts of two cultivars to lower the activity of porphobilinogen deaminase (PBGD). Namely, the conversion of porphobilinogen (PBG) into uroporphyrinogenⅢ(UroⅢ) concerning in chlorophyll biosynthetic processes was inhibited by supplying seawater. In Helan No.3 spinach plants with seawater, higher activity of xanthophyll cycle in the leaves played a role of regulation to suppress more excess light energy in order to lower the levels of ROS in chloroplasts. Therefore, the chlorophyll biosynthesis in Helan No.3 leaves with seawater showed quite a weak inhibition and the activity of chlorophllase (Chlase) was not affected by seawater stress. However, more accumulation of ROS in chloroplasts of Yuanye leaves due to the lower activity of xanthophyll cycle severely inhibited the chlorophyll biosynthetic process and remarkably enhanced the activity of Chlase which aggravates the decomposition of chlorophyll. These results suggest that higher activity of xanthophyll cycle regulates to keep chlorophyll metabolic process by decreasing levels of ROS in chloroplasts of spinach plants cultured in the nutrient solution with seawater (40%).The supplement of methyl viologen (MV), the photo-oxidant, enhanced the production rate of O2-, the content of H2O2 and MDA in chloroplast of two spinach cultivars, while the addition of AsA, the eliminator for ROS, decreased ROS level and alleviated oxidization of plasmolemma. The content of chlorophyll b (Chlb), chlorophyll a (Chla) and precursor of Chl such as protochlorophyll (Pchl), Mg-protoporphyrinⅨ(Mg-ProtoⅨ), protoporphyrinⅨ(ProtoⅨ) and uroorphyrinogenⅢ(UroⅢ) was remarkably decreased, but the content of porphobilinogen (PBG) andδ-aminolevulinic acid (ALA) was increased by the stress of seawater, which lead to inhibition for Chl synthesis, and the inhibition was aggravated by MV and eliminated by AsA. Chlorophyllase (Chlase) activity in the leaves of cv. Yuanye was improved while it was not influenced in the leaves of Helan No.3 under seawater stress. The Chlase activity in the leaves of cv. Yuanye was more greatly affected by MV treatment than that of Helan No.3, however, Chlase activity of two cultivars was not influenced by AsA. These results suggest that ROS is closely related to Chl metabolism, which not only injure plasmolemma but also inhibit the process of transformation of PBG to UroⅢas a result of Chl decomposition. In seawater tolerant cultivar (cv. Helan No.3), the ROS elimination is mainly depended on SOD and AsA-GSH system, which could alleviate oxidation injury of ROS to chloroplast membrane and inhibition of Chl synthesis, and Chlase activity was less influenced by seawater stress; while in seawater sensitive cultivar (cv. Yuanye), it is mainly depended on SOD and POD, which has limited ability to eliminate ROS and lead to accumulate mass ROS to severe oxidation injury of chloroplast membrane and inhibition of Chl synthesis, and Chlase activity was significantly improved by seawater stress, which enhanced Chl decomposition.Ionic homeostasis in spinach plants was disturbed by the stress of seawater. Relative contribution of osmolytes such as soluble sugar, free amino acid and Cl- to osmotic adjustment was greater than that of others. In cv. Helan No.3 (tolerant cultivar), soluble sugar was mass accumulated during exposure to seawater, and the selective transport of K+, Ca2+, Mg2+ and SO42- from roots and petioles to leaves was more than that of Na+ and Cl-. Besides, decomposition of proteins and accumulation of free amino acids in seawater stressed plants of cv. Helan No.3 were lower than that in cv. Yuanye; under seawater stress, betaine and proline played a very important role in osmotic balance of roots and leaves, respectively. Plasma membrane H+ -adenosine-5'-triphosphate (H+-ATPase) and tonoplast H+-ATPase were remarkably enhanced, which effectively propelled excess ions to exclude out of cytoplasm and compartmentalized in vacuole, and the activity of Ca2+ -ATPase and Mg2+ -ATPase in chloroplasts were greater than that in cv. Yuanye under seawater stress, which help to propel excess Ca2+ and Mg2+ in cytoplasm transport into chloroplasts, therefore, the levels of Ca2+ and Mg2+ in cytoplasm was stabilized. However, decomposition of protein in leaves of cv. Yuanye (sensitive cultivar) was enhanced by seawater, and the selective transport of ions was lower, these resulted in accumulation of free amino acids, Na+ and Cl- by seawater. During seawater stress, lower ionic exclusion and compartmentalization of cv. Yuanye resulted in mass ionic accumulation in cytoplasm under seawater stress. Therefore, the damage to cells and chloroplasts of cv. Yuanye was more than that of cv. Helan No.3. In summary, greater ability of absorption to K+, Ca2+, Mg2+ and SO42- in leaves and compartmentalization to Na+ and Cl- in the roots and petioles to maintain higher ratio of K/Na, Mg/Na, Ca/Na and SO42-/Cl- and to protect the leaves from excessive Na+ and Cl- toxicity was strategy of ionic selective transportation and absorption against seawater stress; the higher efficiency of the compatible solute accumulation, lower decomposition of proteins and production of free amino acids can be considered as some factors responsible for its tolerance to seawater stress; and higher abilities of ionic exclusion and compartmentalization were regulatory strategy of intracellular ions cellular level against seawater stress.