Molecular Cloning, Expression Characteriazation And Physiological Functions Of A Heat-inducible FtsH Gene From Tomato

FtsH is an ATP-dependent metalloprotease and chaperone which is involved in variousbiological functions and play important roles in organisms. In prokaryotes, functional studieshave revealed an important role for bacterial FtsH in stress responses. Researches from higherplants show that plant FtsH homologs have closely relationship to light stress, coldacclimation, the hypersensitive reaction and so on. However, the precise mechanisms of FtsHduring stress responses are still unknown. The heat-inducible ftsH has been widelycharacterized in prokaryotes but not in higher plants. Therefore, we constructed a cDNAlibrary with heat-shocked tomato leaves and carried out EST analysis, finally resulting in theidentification of a ftsH cDNA clone, named LeftsH6. The analysis of expressioncharacterization revealed that the expression of LeftsH6 was induced only by hightemperature. Then, the 5'-flanking sequence of LeftsH6 was cloned by Inverse PCR andfurther characterized by EMSA and β-glucuronidase (GUS) expression analysis. In addition,we constructed a series of eukaryotic expression vectors including overexpression of LeftsH6,cytoplasm-located LeftsH6 (truncated LeftsH6 cDNA without pre-sequence), antisenserepression of LeftsH6, and carried out tomato transformation. Three kinds of transformationlines were generated and used for investigating the physiological functions of heat-inducibleFtsH protease in tomato.The main results were shown as follows:1. The full-length ftsH-like cDNA containing 2213 bp was cloned from the heat-shockedtomato cDNA library. According to an open reading frame of 2019 bp, the deduced proteinprecursor was predicted to target chloroplast. The putative AAA domain and the Zn2+-bindingdomain, characteristic of FtsH metalloproteases family, were found in the FtsH-like protein.Because the protein structure was highly homologous to Arabidopsis AtFtsH6, the clonedtomato ftsH-like cDNA was named LeftsH6. By Agrobacterium tumefaciens-mediatedtransformation method, a construct encoding the transit sequence of LeFtsH6 and GFP fusionprotein was transfered into tobacco. The expression assay of GFP fluorescence shows thatGFP locates in chloroplasts of tobacco epidermic cells, indicating that LeFtsH6 is achloroplastic protease.Purified FtsH degraded casein but not BSA in vitro, while FtsH mutant with replacementof Glu472 in the Zn2+-binding domain by Gln lost the protease activity. A single copy ofLeftsH6 was detected in tomato genome by Southern blot analysis. Northern and Western blotanalyses revealed consistently the heat-inducible character of the LeftsH6 gene. No LeftsH6expression was detected after cold, salt, drought or light stress. The results provide the firstexperimental evidence of the existence of heat-inducible ftsH gene in higher plants.2. The 5'-flanking sequence of heat-inducible tomato LeftsH6 has been isolated andcharacterized. The LeftsH6 promoter was 1558 bp in length with several typical sequences,such as TATA box and CAAT box. Several heat shock elements (HSEs) were found in thesequence. EMSA analysis showed that the purified HsfA2 protein bound specifically to HSEsof the LeftsH6 promoter in vitro.Transgenic tobacco plants (pF1558,pF587,pF332) carrying the gus gene driven by theLeftsH6 promoter (1558 bp, 587 bp and 332 bp) were generated. High levels of heat-inducedGUS staining were detected in the leaves, roots and flowers of the pF1558 transgenic plants.The ovaries, stigmas, anthers and sepals in the flowers at various developmental stages, aswell as the pollen grains of mature anthers, showed strong heat-induced GUS staining.Deletion analysis showed that the heat-inducible GUS activity of the pF1558 transgenictobacco was the strongest. The 587 bp promoter was able to mediate the heat-inducibleexpression of the gus gene, however, the GUS activity mediated by 587 bp promoter wasmuch lower than 1558 bp promoter. The heat-inducible expression of the gus gene with 332bp promoter was the lowest.In the leaves of transgenic tobacco, the highest level of fluorometric GUS activity wasdetected at 40℃. This heat-induced GUS expression, controlled by the LeftsH6 promoter, wasconsistent with the heat-inducible accumulation pattern of the LeftsH6 mRNA in tomato.Under the same heat shock conditions, the pF1558 transgenic line showed the highest level ofGUS activity among three transgenic lines, which was much higher than pF587 and pF332transgenic tobacco lines. These results show that LeftsH6 is a typical heat shock gene and itsthermoinducibility is mediated by interactions between HSEs of the LeftsH6 promoter andheat shock factor.3. We introduced the full length LeftsH6 gene under the control of cauliflower mosaicvirus 35S promoter into tomato, using an Agrobacterium-mediated transformation. Thetransgenic tomato plants of overexpressing LeftsH6 were generated. Southern blot analysisshowed that the LeftsH6 gene had already been integrated to tomato genome with one orseveral copies. Northern blot analysis showed that the LeftsH6 gene was constitutivelyexpressed in transgenic tomato plants under normal growth temperatures, and the transcriptslevel of LeftsH6 was different among independent transformation lines.The vegetative growth of overexpressing transformants was normal comparing to that ofuntransformed control plants, however, the reproductive growth of transgenic plants appearedabnormal. Firstly, the pollens of overexpressing transformants aborted seriously and no fruitset appeared. The RT-PCR/Southern blot analysis of overexpressing transformants indicatedthat the expected transgene mRNAs were constitutively present in the anther tissues, while incontrol plants the LeftsH6 mRNAs were expressed only under heat stress conditions. Theheat-inducibility of LeftsH6 in the anthers of transformants was disrupted maybe thefundamental reason of pollen abortion. The anther development in wild-type and transgenicplants was observed using paraffin slices. During meiosis, the overexpressing transgenicplants carried apparently deformed tetrads within anther locules. Following meiosis, thepollen grains within overexpressing transgenic lines aborted. Secondly, cross-pollination withnormal pollens made the transgenic plants fruit-setting, however, no viable and full seedsyielded within these fruits. These results provided strong evidence that LeFtsH6 was involvednot only in pollen-formation phase but also in seed development process.4. The eukaryotic expression vector carrying the truncated LeftsH6 cDNA withoutpre-sequence was constructed and introduced into tomato. The transgenic tomato plantsharbouring the cytoplasm-located LeftsH6 were obtained. Northern blot result revealed theconstitutive expression of the truncated LeftsH6 in cytoplasm-located transformants. Thecytoplasm-located transformants had normal phenotypes and morphologically normal flowers.The normal fruits with mature seeds were obtained. The thermotolerance analysis of T1seedlings of cytoplasm-located transformants and control plants showed that no differenceswere detected. The results showed that the correct localization of the chloroplastic LeFtsH6protease was critical to play roles.5. A recombinant vector harboring partial sequence of the LeftsH6 gene (548-basesequence in the 5'-end of LeftsH6) was constructed to repress the expression of endogenousLeftsH6 gene in tomato. Three antisense LeftsH6 lines (AS1, AS2 and AS3) were generated.Northern blot analysis showed that the endogenous LeftsH6 mRNAs of the antisense plantswere reduced in abundance compared to the untransformed plants under heat shock conditions.Under normal growth conditions, the antisense transformation lines had apparently normalphenotypes and morphologically normal flowers. The results of pollen fertility showed thatmost of the pollens were round, full and stained with deep color. Normal fruits were set andgave mature seeds.High temperature treatments of the T3 seedlings of the antisense lines anduntransformed plants showed that the antisense plants were easy to be damaged than controlplants. The chlorophyll fluorescence measurements showed that the antisense plants had amuch higher initial fluorescence Fo and much lower Fv/Fm (representing the maximalphotochemical efficiency of PSⅡ) after heat shock stress comparing to control plants. Itsuggested that the antisense plants submitted the severely damage than control plants underhigh temperatures and the maximal photochemical efficiency of PSⅡ decreased significantly.The transgenic plants didn't easy to restore the normal growth after heat stress. These resultsindicated that the decrease of the endogenous LeftsH6 expression made the antisense linesmore susceptible to heat stress.The innovations of this thesis were shown as follows:1. It was the first time to clone the heat-inducible ftsH gene from higher plants. The resultsfrom Northern and Western blot analyses proved consistently the heat-inducible characterof the LeftsH6 gene. The 1558 bp promoter sequence of heat-inducible ftsH from tomatohas been isolated. EMSA analysis showed that the purified HsfA2 protein boundspecifically to HSEs of the LeftsH6 promoter in vitro. Among three kinds of transgenictobacco plants (pF1558,pF587,pF332) carrying the gus gene driven by the LeftsH6promoter (1558 bp, 587 bp and 332 bp), the heat-inducible GUS activity of the pF1558transgenic tobacco was the strongest. The leaves, roots and flowers of the pF1558transgenic tobacco showed high levels of GUS activity.2. The transgenic tomato plants of overexpression and antisense repression of LeftsH6 weregenerated. Overexpression of the LeftsH6 gene led to abnormal meiosis of pollens. Thepollens aborted seriously and no fruit set appeared in the overexpressing transformants.Cross-pollination using normal pollens made fruit capsules develope, but no viable andfull seeds yielded within capsules. LeFtsH6 was involved in both pollen-formation phaseand seed development process. However, the antisense lines had apparently normalphenotypes and can produce normal fruits and seeds. The antisense repression of theLeftsH6 gene changed the thermotolerance of tomato plants. The results showed thatLeFtsH6 had relation to plant thermotolerance.