The Repression Of LeHsp110/ClpB Expression Decreases The Acquired Thermotolerance In Tomato

Molecular chaperones are a group of proteins that influence the structure andfunction of many polypeptides. They assist the folding, the assembly, the translocationand the aggregation by controlling, binding and releasing the polytides. Under stressconditions, molecular chaperones stabilize the proteins' structure by hindering theaggregation of denatured proteins. ClpB, a member of Clp family, has the function ofmolecular chaperone. ClpB differs from other Clp proteins by possessing a longermiddle region. ClpA and ClpX function as ATPases to regulate ClpP-mediatedproteolysis, while ClpB possesses the molecular chaperone activities to rescue thestress-damaged proteins from an aggregated state. A cDNA coding for aHSP100/ClpB homolog has been cloned from Lycopersicon esculentum, its molecularweight is 110kD and termed as LeHsp110/ClpB. Previous studies showed thatLeHSP110/ClpB was not detected under normal conditions and induced by hightemperature. In this study, an antisense LeHsp110/ClpB cDNA fragment wasintroduced into tomato by Agrobacterium-mediated transformation;the resultsshowed that LeHSP110/ClpB is involved in thermotolerance. The results were shownas follows:1. The intracellular location of LeHSP110/ClpB.The predictions of on-line program TargetP and ChloroP revealed 76 aa ofchloroplast-targeted transit sequence in LeHSP110/ClpB. Chloroplast-localization wascorroborated based on the microscope images of GFP fusion protein that consisted ofboth lead sequence (transit sequence of LeHSP110/ClpB) and GFP. The cells on leaveepidermis peeled from transgenic tobacco were illuminated with blue light(488nm)that caused GFP to fluoresce. All these results indicated that LeHSP110/ClpB was achloroplast protein.2. The generation of tomatoes with repressed LeHsp110/ClpB .In order to study the function of LeHSP110/ClpB during heat stress, weintroduced an antisense LeHsp110/ClpB cDNA fragment into the tomato byAgrobacterium-mediated transformation. A 330 bp cDNA fragment (the highlyvariable region, containing 5'-UTR and the following coding region for chloroplasttransit sequence of LeHSP110/ClpB) was used for the construction of the antisensevector. We generated seventeen antisense transformants. Northern blotting analysisshowed that LeHsp110/ClpB was severely diminished. Western blotting analysis alsoshowed that LeHSP110/ClpB protein exhibited an extreme repression.3. Thermotolerance analysis of transgenic tomatoes.Transgenic plants were exposed to higher temperature after preconditioned at38 ℃ for 2h, and then were transferred to normal conditions for recovery. Theantisense lines were greatly impaired and withered during the recovery phase,whereas for the moment the untransformed control plants and the vector-transformedplants survived, which indicated that the transgenic tomatoes' viability to hightemperature was reduced.Chlorophyll fluorescence measurements showed that a more significant decreaseof Fv/Fm ratios and an increase of Fo values were observed in transgenic plants. Theseresults indicated that antisense lines were more susceptible to thermal irreversibleinactivation than the untransformed and vector-transformed control plants. Thechlorophyll contents in antisense lines declined more quickly. It is probably becausethat pretreatment helped the nontransgenic plans to develop acquired thermotolerance,whereas loss of ClpB synthesis in antisense lines significantly reduced cell viability.These results lead to a conclusion that LeHSP110/ClpB protein plays an essential rolein thermotolerance in higher plants.