The Research Of Mechanism Of Calcium Effect On SA-Induced Resistance To Botrytis Cinerea In Tomato

Tomao （Solanum lycopersicum Mill） is an important kind in horticulture production inthe world and China. However, tomato is also a frequently-occurring crop. Currently, theproducer mainly depended on the chemicals to prevent disease，which lead to environmentaland products pollution. The induced resistance is an action mode that comes from plantsthemselves with the characteristic of activity. And which was worth to be researching andapplication. We should carry on depth research on the mechanism of induced resistance if wewant to exploit the plant induce resistance in order to enhance plant defence.Ca²⁺and Salicylic acid are considered that the important resistance substance （Sugimotoet al.,2005; Volpin and Elad,1991; Yoon CS et al.,2010; White,1979; Malamy et al.,1990;Gaffney et al.,1993; Durrant2004; Mauch2001）. A growing number of detail reports indicatethat Ca²⁺have strength role on SA induced effects, such as ROS, sub-high temperatureresistance, drought resistance, on-off of stomata and pos-harvest physiology of peach （Pei etal.,2000；Liu et al.,2005; Wang et al.,2003; Liu et al.,2003; Ao,2003）. There is not detailreport on the mechanism of Ca²⁺effect on SA induced resistance on Botrytis cinerea infectedtomato, and research of the mechanism is lack.The cultivated tomato ‘L402’ which was sensitive to Botrytis cinerea, was employed inthis experiment, the treatments of with or without Ca²⁺was designed, base on the level ofphysiology and molecule analysised the Ca²⁺effect on SA induced resistance on Botrytiscinerea inoculation. It is helpful to study the mechanism to achieve the finally objective toenhanced the SA resistance efficiency and the yield and qulity both in scientific and practicalfields. The main research results are as follows:1. It was clear that Ca²⁺have the ablity to enhanced plants resistance through anaylisis thedisease index of different Ca²⁺concentration of nutrient solution and foliar application Ca²⁺.Compare with control, the disease index declined63.61%in the treatment of supplementingCa²⁺to nutrient solution; and the disease index reduced6.04%in treatment of foliarapplication Ca²⁺compare to control; which illustrated that it was efficiency to apply Ca²⁺tothizosphere. It was found that Magnesium (Mg²⁺) did negative effect on tomato resistance onBotrytis cinerea, in alone Mg²⁺treatment the disease index was78.25，which was increase4.54%compares with control.2. Aimed to analysis the detail reasons of effectively enhancing the tomato resistanceunder thizosphere Ca²⁺supplementation. It was found that the effect of Ca²⁺treatment on SAaccumulation and SA synthesis in tomato leaves. It was showed that the expression level ofCM、PAL and activity of BA2H increased significantly Ca²⁺treated, however, the expressionlevel of PAD4and ICS1were not significantly. So SA accumulation in Ca²⁺concentrationtreated, which was the results of PAL pathway related genes expression levels’ increase.3. It was clear that the disease index reduced significantly by foliar appling SA; andMg²⁺did negative effect on SA induced resistance on Botrytis cinerea. The efficiency was difference that Ca²⁺and SA in different order were applied to test the resistance on Botrytiscinerea. The Ca+SA treatment have the best effect on defense, the disease index decrease17.93%compare with alone SA treatment, and13.34%compare with SA+Ca, there was notdifference between SA+Ca and SA treated. The Mg²⁺declined SA-induced resistance on graymold; the results of application of SA after Ca²⁺indicated that Ca²⁺enhanced SA-inducedresistance on gray mold in tomato.4. It was found that Ca²⁺, EGTA, SA alone and applied complex led to ROS, enzymesactivity and genes expression increase under induced treatments or inoculation with graymold after induced treatments. Among the different treatments, the results showed SA caninduce ROS and Ca²⁺enhanced the SA-induced ROS. In contrast，pretreatment with Ca²⁺chelator EGTA, weakened the ROS and weakened the SA-induced ROS. The similar resultswere found in PAL, chintase, β-1,3-glucancse activity and genes expressions （PR1、 PR2aand PR3b）. It indicated that SA enhanced tomato resistance by regulation of ROS andpathogensis related protein activity and expression, and Ca²⁺promoted SA-induced resistanceto tomato gray mold by further increase these index.5. It was clear that the function of proteins expression of Ca²⁺regulating SA-inducedresistance to gray mold. The tomato leaves protein was extracted with TCA/acetone under theCK、SA、Ca+SA treatments inoculation with gray mold after48h. There were more than500protein spots in gel image through2-DE technical and the software of PD-Quest. We totallyfound out65differentially expressed protein spots. In detail, there were19spots wup-regulated,6spots down-regulated,9new spots in SA and Ca+SA treatemts; in addition,2spots up-regulated,3spots down-regulated,2new spots and2spots disappeared only in SAtreatment; and8spots up-regulated,1spots down-regulated,10new spots and3spotsdisappeared only in Ca+SA treatemts.6.50differentially expressed protein spots with stable expression and higher than1.5-fold spot density were analysed by MODI-TOF-TOF. The42protein spots were obtainedby NCBI database searehing and intereomparison preliminary identifieation. These proteinswere associated with a variety of functions, including photosynthesis, energy metabolism,metabolism, ROS, protein synthesis and degradation, cell growth/division, signal transduction,and transcriptional. The key protein and enzyme of Ca²⁺enhanced SA-induced resistance ontomato Botrytis cinerea were pathogenesis-related protein5, PR STH-2, acidic endochitinase,SOD and APX.8. It was found that the disease index of treatments of Ca²⁺concentration of nutrientsolution with SA application reduced significantly in ‘NahG’ and WT, which proved that Ca²⁺enhanced tomato resistance at the condition of SA presence. Effects of Ca²⁺on SA signalingtransduction in tomato was observed in this paper. The SA-dependent signaling related keygene （NPR1、PR1、TGA1a、TGA2.2） and two mitogen-activated protein kinase （MPK2、MPK4）expression level were analysised. An obvious increase of NPR1、PR1、TGA1a、TGA2.2、MPK2transcript was observed in8mM Ca²⁺treated tomato seedlings. In contrary, the MPK4 expression level was up-regulation only in0mM Ca²⁺treated, and the resistance declinedsignificantly in the treatment of0mM Ca²⁺. Looking From the rule of changes, Ca²⁺concentration of nutrient solution with SA application enhanced tomato resistance, which wasresults of up-regulation expression of SA signaling pathway related genes and MPK2, not theMPK4.9. The gene expression of Ca²⁺related receptor had a great change after treatment ofCa²⁺concentration of nutrient solution with SA application. The transcription of CDPKenhanced obviously in8mM Ca²⁺comparation with3mM Ca²⁺, which expression level waslower in0mM Ca²⁺than3mM Ca²⁺treatment. From the analysis of relationship betweenCa²⁺concentration and CaM expression level, it was indicated that the CaM transcription waspositively correlated to SA simulation. The most probably of CBL transcription situation wasCa²⁺and SA co-regulation. So we speculated that Ca²⁺related receptor partly participated inthe process of Ca²⁺concentration with SA application treatment to enhance tomato resistance.7. It was clear that Ca²⁺have the ablity to enhanced plants resistance through treated‘Moneymaker’ and’NahG’ with different Ca²⁺concentration of nutrient solution andanaylisis the disease index. The disease was lower in ‘Moneymaker’comparation to ‘NahG’.Further more, the disease index was lower in ‘Moneymaker’ and’NahG’ treated with2mMSA after appling with different Ca²⁺concentration of nutrient solution comparation totreatments of different Ca²⁺concentration of nutrient solution, it showed that Ca²⁺not onlyhad the ablity to reduce tomato disease index combined with SA, but also had the function toinclined tomato disease index by the mode of SA-independent. The detail mechanism need tofurther research.