The Effect Of 24-epibrassinolide(24-epiBL)in Salinity Tolerance Of Robinia Pseudoacacia L.and Cinnamomum Camphora L.seedlings

Nowadays,the question of the soil salinity is aggravated and the area of saline soil in China has been the third one globally.Jiangsu province,most densely populated provinces in China,located at economically developed region of southern China,lower reach of Yangtze River and along the coast of the Yellow Sea,has the question of soil salinity and the anthropic and industrial activities are frequent here.As the global warming are lasting,it cause the weaken soil nutrient and the severer evaporation of soil moisture.Evaporation off the soil surface causes salt to accumulate on these sites which increase the saline soil area in this region to limit the regional economic development and disturb the local residents live.The treatment and restoration of saline land are extremely urgent in this district.As one of the control measures of saline land,afforestation has a lasting positive effect on the improvement of saline land and can provide materials and other forest by-products to develop regional economy.Black locust?Robinia pseudoacacia L.?as the major tree species has been widely planted here because it is significantly increase soil nitrate concentrations along the coast and reduce the impact of hurricanes on such stands.Camphor?Cinnamomum camphora L.?as the popularly economic species locally has effected the region economy.As the increasing extent and degree of saline land here,the above two kinds of trees are facing the growth limitation.As a novel plant growth hormone,24-epibrassinolide?24-epiBL?has been widely used to enhance the procreation,growth,resistance of plants to multiple stresses,including salinity,but the research of 24-epiBL in salt tolerance of woody plant was less.In the present study,we analyzed the effects of salt stress and different treatments of 24-eipBL on salinity tolerance in black locust seedlings and camphor seedlings.Plants were pretreated with three levels of 24-epiBL?0.21,0.62,or 1.04?M?by either soaking seeds during the germination phase?Sew?,foliar spraying?Spw?,or root dipping?Diw?.This research picked up the appropriate 24-epibl application methods and concentrations and proclaimed the inhibition mechanism of NaCl and function mechanism of 24-epiBL in regulating the salinity tolerance based on plant growth,photosynthesis and antioxidative ability.The mainly research results as follow:?1?24-epiBL could reduced the salinity index,modify the growth inhibition of black locust and camphor seedlings under salinity stress.It obviously improved the height and diameter growth,maintained the leaf area and accumulation of biomass companying with the increased leaf relative water content and membrane steady index The higher concentration of 24-epiBL the more effective was in 0.21-1.04?M;The positive effects of alleviating effect in R.pseudoacacia seedlings in terms of treatment application was Diw?Sew>Spw and in C.camphora seedlings was Sew>Spw.?2?Regarding the R.pseudoacacia and C.camphora seedlings,increased salinity resulted in a decrease in net photosynthesis rate?P_n?,stomatal conductance?G_s?,intercellular:ambient CO₂concentration ratio?C_i/C_a?,water-use efficiency?WUE_i?;the reduction of maximum quantum yield of PSII?F_v/F_m?was as 5%of black locust and as 28%of camphor,non-photochemical quenching?NPQ?was decreased as47%of camphor but it was increased 2.4-2.8 times in black locust,thermal dissipation?H_d?was increased by 1.2-2.2 times in both plants.All the above varieties accompanied the reduction in pigment concentration?Chl a,Chl b,and Chl?especially in higher salinity?P<0.05?.The membrane lipid peroxidation role?malondialdehyde,MDA?and superoxide dismutase?SOD?was increased were increased 1.2-7.4 times and 12-52%in both plants,the peroxidase?POD?was increased 1.3-2.8 times in black locust but decreased 15.8-28 times on camphor.The mechanism of photosynthetic inhibition caused by salinity based on analysis of leaf photosynthetic indexes was different because of the species difference of this two seedlings.The photo-inhibition of black locust seedlings was the effect in mesophyll conductance?G_m?,stomatal conductance?G_s?,osmotic stress and oxidative stress,but the reason for camphor seedlings was osmotic stress,oxidative stress and mesophyll disturbance not for limitation on G_s.?3?Exogenous 24-epiBL could improve the P_n and WUE_i of black locust and camphor leaves.For black locust,the effect of G_s was not obvious?P>0.05?accompanying with the lower C_i/C_a in SS-S1,the increase of G_s was significant only with weaken increase?¹.37%?in C_i/C_a by Diw treatment compared to SS-S2;for camphor seedlings 24-epiBL treatments had no effect on G_s,Sew-0.62/1.04S1 and Spw-1.04S1 had the obvious decrease?11.9-12.5%?on C_i/C_a in SS-S1 but in SS-S2 only Sew-0.62/1.04S2 had the significant reduction?7.8-12.6%?on C_i/C_a.All 24-epiBL could make increase in F_v/F_m and decrease in H_d obviously in both seedlings?P<0.05?;in SS-S1group,the regulation of NPQ in camphor seedlings was improved by 24-epiBL but the effect in black locust was decreased and only obvious reduction appeared in Diw treatment,in SS-S2 group,only Sew-1.04S2 and Diw-1.04S2 had the 0.6%and 9.5%reductions separately but without significance compared to SS-S2,the NPQ of camphor leaves was significantly improved by all 24-epiBL treatments in SS-S2.All 24-epiBL treatments could improve the photo pigments content in two kinds of seedlings and reduce the MDA content significantly in dependence on the obvious increased POD and SOD activities,the higher concentration of 24-epiBL the better effect was.?4?The salinity caused impair in photosynthetic organ including physiological and structural level in both two kinds of plant in this research.Salinity induced the increased accumulation of Na⁺(26.1-116.7?m?mg^-1 prot in black locust and 60.3-77.4?m?mg^-1 in camphor)and decreased accumulation of K⁺(1.6-3.5?m?mg^-1 prot in black locust,23.3-28.8?m?mg^-1 prot in camphor),Ca²⁺(7.0-7.8?m?mg^-1 prot in black locust,62.9-71.1?m?mg^-1 prot in camphor)in chloroplasts which broke the ion balance in organ to result in the osmotic stress and ion poison.The above physiological disturbance caused the harm on structure of chloroplast including elliptoid chloroplasts,large or less starch granules,irregular thylakoids,many swollen plastoglobuli and loosen thylakoid.The destroyed structure of chloroplast induced the defect in photosynthetic pigments synthesize?Chl a,Chl b,and Chl?and photosynthetic electron transport in chloroplast.Osmotic stress and ion poison induced the destroy the antioxidant defense system as such significant increase in hydrogen peroxide?H₂O₂?as 4.3-15.2 times of CK and MDA as 1.6-3.5times of CK).The ability of antioxidant defense in black locust chloroplast was better than it in camphor?s which showed the activities of ascorbic acid peroxidase?APX?and SOD in black locust chloroplast were obvious higher than 1.6-3.5 times of blank control?CK?beside APX in SS-S2and APX,SOD in camphor chloroplast had no difference in CK and salt stress control?SS?.?5?Exogenous 24-epiBL could reduce the H₂O₂ as 56-70%and MDA as 70-73%in chloroplast significantly,improve the activities of antioxidant enzymes?APX as 1.5-3.1 fold of SS and SOD as 1.3-2.1 fold of SS?,weaken the oxidative harm caused by salinity stress,induced K⁺increase as 1.5-2.2 times of SS and Ca²⁺increase as 3.5-4.0 times of SS and decrease in Na⁺,which was benefit to pigment synthesis.Different application methods of 24-epiBL could maintain regular chloroplast and thylakoid membrane structures especially in higher NaCl stress.In black locust chloroplast,Sew and Spw had no obvious distinguish between each other,but Diw kept the better structure of chloroplast and increased the number of plastoglobuli in SS-S1;In SS-S2,Diw was less better than Sew and Spw,it could maintain the normal structure of chloroplast and the much number of plastoglobuli.For the camphor chloroplast,the Sew was obviously better than Spw especially in SS-S1,both pretreatments of 24-epiBL got the atrophic chloroplast and starch granules specially showed in Spw and Sew could keep the better normal structure of chloroplast,Sew and Spw could keep the better regular thylakoids particular in SS-S1 which no difference compared to CK.Although the higher salinity caused the less loosen thylakoids by 24-epiBL treatments but better than stress control.?6?Our study revealed 24-epiBL improved the salt tolerance of C.camphor by modifying the photosynthetic limitation based on physiological analysis in leaves and chloroplast.Camphor seeds and seedlings were pretreated with 1.04?M 24-epiBL either by soaking during the germination phase or by foliar spraying and then salinity treatment with 103.45mM NaCl for 50 days.Base on the iTRAQ analysis,there were 240 DEPs;22.1%DEPs were involved in energy metabolism pathways including photosynthesis?23 DEPs?,photosynthesis-antenna?10 DEPs?,and carbon fixation in photosynthetic organisms pathways?20 DEPs?caused by NaCl.The total of 106 DEPs were enriched in 17 functions related to oxidative effects.The effects of 24-epiBL were better via the seed soaking method?S+B/S?than via the foliar spraying method?S+E/S?.Both two kinds applications of 24-epiBL could alter the expression of proteins that reflect oxidative stress?eg.,18.2 kDa class I heat shock family protein and superoxide dismutase family protein?.By the analysis of protein-protein interactions?PPIs?in response to S+B/S,sixteen DEPs strongly regulated the inhibition of photosynthesis including two verified proteins named superoxide dismutase family protein?gi|566168071?and O2-evolving complex 33 kD family protein?gi|566168071?,which two protein was down-expressed protein in salt stress?the separate expressions were:0.1±0.02 and 0.23±0.17?and 24-epiBL induced them up-regulated?the separate expressions were:17.2±1.38 and 4.79±2.41?as 172-fold and 20.82-fold compared salt stress control?S/C?.To sum up,salinity could inhibit the plant growth in black locust and camphor seedlings especially obvious in photosynthesis with different mechanism.Exogenous 24-epiBL could modify the negative effect caused by salinity and the Diw>Sew>Spw appeared in R.pseudoacacia seedlings,Sew>Spw was in C.camphora seedlings,the best concentration of 24-epiBL was 1.04?m.24-epiBL could improve the expression of anti-oxidative proteins and modify the injure caused by oxidative stress by salinity to protect chloroplast normal structure and keep the basic function steady,improve the antioxidant capacity,regulate the ion poison situation which were better in photosynthetic normal running to release the photo-inhibition and improve the salinity tolerance of R.pseudoacacia and C.camphora seedlings.