Alleviative Effects And Its Mechanism Of Exogenous5-aminolevulinic Acid On Tomato Seedlings Subjected To Salt Stress

With the development of protected agricultural specialization and large-scaleproduction in our country, the soil secondary salinization problem in greenhouse isincreasingly outstanding. Soil salinization has become the main factors bringing down thevegetable production, which dramaticaly affects the sustainable development of protectedagriculture.5-aminolevulinic acid (ALA), an critical precursor in the biosynthesis of organiccyclic tetrapyrrole chemical compound, is ubiquitous in higher plants and plays an importantrole in the chlorophyll synthesis, photosynthesis and plant growth physiological processes.Previous studies indicated that, ALA could improve the ability of resistance to abiotic stress inmany different kinds of plants, while what is the best applied concentration of ALA and thebest pattern of application, there is still no consensus conclusion. Whether exogenous ALAcan make up for the mineral nutrition imbalances caused by abiotic stress, or exists regulationeffects for chlorophyll synthesis metabolism, or coordinates the absorption and distribution oflight energy and the expression of aquaporin genes, these have not yet been clearly studied.Thus, there is a big gap on the level of theory and practice according to the current researchsituation.Tomato (Solanum lycopersicum), a moderate salt resistance plant, is one of themajor crops in greenhouse cultivation. However, the productivity and quality of tomato arealways seriously affected by the soil secondary salinization. In the present study, salt-sensitivetomato cultivar (Zhongza No.9) and salt-tolerance one (Jinpeng No.1) were used. It wasconducted hydroponics to investigate the effects of foliage spraying or root applied ALA inthe two different salt tolerance of cultivars exposed to NaCl stress, respectively. Plant growth,physiological, biochemical metabolism and photosynthetic characteristics were measured. Wediscussed effect of the different concentration of exogenous ALA in salt-stressed tomatoplants. Optimum applied concentration of ALA was found as well as best method ofapplication, indicating that exogenous ALA accelerated tomato seedlings photosynthesis andphotosynthetic electron transport. By analyzing mineral element absorption, chlorophyllsynthesis and metabolism, photosynthetic electron flow distribution and homeostaticregulation of ROS, we explicated the physiological mechanisms of ALA-regulated stress tolerance. We also investigated the expression of aquaporins and LeNHX1genes regulated byexogenous ALA in tomato seedlings under salt stress, illustrated its mechanism of increasedsalt tolerance. The main results are as follows:1. The results of seed soaking experiment indicated that, tomato seed germinationrate (GR), germination energy (GE), germination index (GI) and vitality index (VI) weremarkedly enhanced under relatively high (100mmol·L-1) NaCl stress by treated withappropriate concentrations (0.1-0.5mg·L-1) of ALA. Seed soaking with ALA helped improveautioxidant enzyme activities, reduce lipid peroxidation degree, promote the seed germinationand seedlings growth. However, high concentration (10.0mg·L-1) of ALA inhibited seedgermination. Foliage spraying or root soaking with different concentration of ALA effectivelyrelieved the inhibition effect on plant growth of tomato sedlings subjected to NaCl stress,induced the activities of autioxidant system (POD, CAT, SOD and APX) and osmoticregulation system in roots and leaves, reduced the degree of membrane lipid peroxidation,significantly improved the content of chlorophyll and carotenoid. Foliage spraying with50mg·L-1ALA and root soaking with10mg·L-1ALA has the best-mitigated effect, and highconcentrations beyond the optimum concentration have reverse impact.2. With the increase of treat time, net photosynthetic rate and PSII photochemicalefficiency of both cultivar tomato seedlings diminished notably by salt stress, while stomatalimitation and the proportion of excitation energy for antenna heat dissipation significantlyincreased. Zhongza No.9was injured by salt stress more seriously. Nine days after NaCltreatment, Zhongza No.9exerted typical limitation of non-stomata, and yet, the decrease ofphotosynthesis in Jinpeng No.1was inhibited mainly by stomata factor. Foliage spraying orroot soaking with ALA remarkably increased the maximum net photosynthetic rate, lightsaturation point, stomatal conductance and transpiration rate of both cultivar tomato seedlings,alleviated restriction of stomata and non-stomata limitation on photosynthetic carbonassimilation caused by salt stress. The integrity of thylakoid membrane was preserved byexogenous ALA application through increasing carotenoid content. Meanwhile, application ofALA decreased the light energy used for antenna heat dissipation, improved thephotochemistry electron efficiency of PSII reaction center and the ability of light utilization,especially under high light, thus, relieved photoinhibition, which foliage spraying had bettereffect than root application.3. The Rubisco carboxylation rate (Vc) and oxygenation rate (Vo), the maximumpotential rate of electron transport contributing to RuBP regeneration (Jmax) andphotosynthetic carbon reduction electron flow Je(PCR) of both cultivar tomato seedlings subjected to NaCl stress were significantly induced by exogenous ALA application, whichreduced the feedback inhibition of PSII photochemical reaction. Simultaneously, applicationof ALA augmented the photosynthetic carbon oxidation electron flow Je(PCO) and the totalalternate electron flow on the O2-dependent and O2-independent (Ja), which enhanced thedissipation of excess light energy and avoided the generation of reactive oxygen species. Theactivities of monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase(DHAR) and glutathion reductase (GR) in the roots substantially decreased by NaClstress. Meanwhile, the content of ascorbic acid (AsA) and reduced glutathione (GSH)reduced largely in salt-stress plants, which led to the increase of O2·ˉproductive rate andaggravation of lipid peroxidation, thus remained high content of MDA as well. ExogenousALA reinforced the scavenging capacity of reactive oxygen species by improving thewater-water cycle related enzyme activities, in the meantime, dissipated excess light energy,alleviated photoinhibition and augmented the electron transfer for photosynthetic carbon cycle,finally, mediated the balance of the photosynthetic electron flow distribution.4. NaCl stress significantly reduced the content of chlorophyll at different leafpositions in tomato seedlings. At the beginning of salt stress, the content of ALA, PBG andUroIII significantly increased, while the content of ProtoIX, Mg-ProtoIX, Pchl, Chl a and Chlb decreased dramatically, that indicated that during the initial stress, downstream metabolismof chlorophyll precursor substances was blocked which is contributed to the main factor ofchlorophyll reduction and the blocked site was concluded in the process of transformation ofUroIII into ProtoIX. Ten days after salt treatment, the contents of ALA, PBG and UroIIIreduced gradually, which was the same as ProtoIX and its downstream products, thatsuggested that the inhibition of ALA synthesis was the main factor of chlorophyll reduction,and the synthesis was inhibited more significantly in Zhongza No.9than that of Jinpeng No.1.On the contrast, exogenous ALA alleviated the inhibtion effects, improved the transform ofPBG to UroIII and the whole downstream metabolism, and enhanced the synthesis rate ofendogenous ALA at the same time. Thus, ALA improved the synthesis of chlorophyll from thescratch and the transformation of intermediate metabolites (PBG and UroIII) to thedownstream, which contributed together to the increase of chlorophyll content.5. Under salt stress conditions, Na+accumulation significant increased in the roots,stem and leaves of the two tomato cultivars, K+accumulation increased markedly in roots,while declined in leaves, the content of Ca2+and Mg2+significantly decreased in stem andleaves. Simultaneously, the transporting selective ratio of K+, Ca2+and Mg2+from root to stem(SK.Na, SCa.Na, SMg.Na) dropped dramatically. ALA treatment increased the content of K+, Ca2+, Mg2+and the transporting selective ratio of K+and Mg2+(SK.Na, SMg.Na) from stem to leaves inthe salt-tolerance cultivar under salt stress. In salt-sensitive cultivar, the transporting selectiveratio of K+(SK.Na) from stem to leaves, and the transporting selective ratio of Ca2+and Mg2+(SCa.Na, SMg.Na) from root to stem and stem to leaves increased significantly after theapplication of ALA. This indicated that the exogenous ALA was effective in regulating theselectivity of absorption and transportation of K+, Ca2+and Mg2+, improving the transportingselective ratio of K+, Ca2+and Mg2+from stem to leaves, maintaining a balance offundamental mineral elements, thus reduced the injury of salt stress.6. Real-time PCR was used to investigate the expression of aquaporins and LeNHX1gene regulated by exogenous ALA in tomato seedlings under salt stress. Results showed thatthe expression of aquaporins and LeNHX1genes in tomato leaves under salt stress werenotably up-regulated or brought forward by exogenous ALA application. Meanwhile, ALAtreatment reduced the expression of LePIP1, LePIP2and LeAQP2in tomato roots, improvedthe water transport in cell-cell pathway in leaves and lessened the root water loss.Alternatively, in the leaves, expression of LeTIP and LeNHX1genes that located in tonoplastmembrane increased much larger than that of LePIP1, LePIP2and LeAQP2genes that locatedin plasma membrane. That indicated that, according to segregate Na+to vacuole and improvethe vacuole membrane permeability, make full use of the osmotic regulation of vacuole tokeep the water balance and a relatively high physiological activity in leaves, ALA treatmentimproved the tolerance of tomato seedlings to salt stress.