Effects of flooding stress in tomato plants transformed with different levels of mitochondrial sHSP23 6 / Efeitos do estresse por alagamento em plantas de tomateiro transformada com diferentes níveis de sHSP23 6 mitocondrial

Abstract Soil flooding is an environmental stressor for crops that can affect physiological performance and reduce crop yields. Abiotic stressors cause changes in protein synthesis, modifying the levels of a series of proteins, especially the heat shock proteins (HSP), and these proteins can help protect the plants against abiotic stress. The objective of this study was to verify if tomato plants cv. Micro-Tom from different genotypes with varying expression levels of MT-sHSP23.6 (mitochondrial small heat shock proteins) have different responses physiological to flooding. Plants from three genotypes (untransformed, MT-sHSP23.6 sense expression levels and MT-sHSP23.6 antisense expression levels) were cultivated under controlled conditions. After 50 days, the plants were flooded for 14 days. After this period half of the plants from each genotype were allowed to recover. Chlorophyll fluorescence, gas exchange, chlorophyll index, leaf area and dry matter were evaluated. Flood stress affected the photosynthetic electron transport chain, which is related to inactivation of the oxygen-evolving complex, loss of connectivity among units in photosystem II, oxidation-reduction of the plastoquinone pool and activity of photosystem I. The genotype with MT-sHSP23.6 sense expression levels was less sensitive to stress from flooding.

Resumo O alagamento do solo é um estressor ambiental para as culturas e pode afetar o desempenho fisiológico e reduzir a produtividade das culturas. Estresses abióticos causam mudanças na síntese de proteínas, modificando os níveis de uma série de proteínas, em especial as proteínas de choque térmico (HSP) e essas proteínas são conhecidas por proteger as plantas contra estresses abióticos. O objetivo deste estudo foi verificar se as plantas do tomateiro cv. Micro-Tom de distintos genótipos com diferentes níveis de expressão da MT-sHSP23.6 (proteínas mitocondriais de choque térmico com pequena massa molecular), têm diferentes respostas fisiológicas ao alagamento. As plantas de três genótipos (não-transformado, transformado com orientação antisense e transformado com orientação sense para MT-sHSP23.6) foram cultivadas sob condições controladas. Após 50 dias as plantas foram alagadas durante 14 dias. Após esse período as plantas de cada genótipo foram recuperadas. Foram avaliados fluorescência da clorofila, trocas gasosas, índice de clorofila, área foliar e massa seca. O estresse por alagamento afetou a cadeia de transporte de elétrons da fotossíntese, que está relacionado à inativação do complexo de evolução do oxigênio, perda da conectividade entre as unidades do fotossistema II, de oxidação e redução do pool de plastoquinona e atividade do fotossistema I. O genótipo com orientação sense MT-sHSP23.6 foi menos sensível ao estresse por alagamento.

Screening Molecular Chaperones Similar to Small Heat Shock Proteins in Schizosaccharomyces pombe

To screen molecular chaperones similar to small heat shock proteins (sHsps), but without alpha-crystalline domain, heat-stable proteins from Schizosaccharomyces pombe were analyzed by 2-dimensional electrophoresis and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Sixteen proteins were identified, and four recombinant proteins, including cofilin, NTF2, pyridoxin biosynthesis protein (Snz1) and Wos2 that has an alpha-crystalline domain, were purified. Among these proteins, only Snz1 showed the anti-aggregation activity against thermal denaturation of citrate synthase. However, pre-heating of NTF2 and Wos2 at 70degrees C for 30 min, efficiently prevented thermal aggregation of citrate synthase. These results indicate that Snz1 and NTF2 possess molecular chaperone activity similar to sHsps, even though there is no alpha-crystalline domain in their sequences.

Effect of RNA interference on small heat shock protein Sjp40 of Schistosoma japonicum / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effect of RNA interference (RNAi) on small heat shock protein (sHSP) Sjp40 of Schistosoma japonicum and its synergistic effect on the expression of SjHSP60, SjHSP70, and SjHSP90 mRNA, and observe the mRNA expression levels of Sjp40, SjHSP60, SjHSP70, and SjHSP90 in different stages of S.japonicum.</p><p><b>METHODS</b>Double-stranded RNA (dsRNA) of Sjp40 (dsSjp40) and a control dsRNA of green fluorescent protein (dsGFP) were generated by in vitro transcription and transfected into adult worm by immersing the worm in dsRNA solution. The total RNA and proteins were isolated simultaneously from the adult worms using TRIzol reagent 7 days after transfection. The expression levels of Sjp40, SjHSP60, SjHSP70, and SjHSP90 mRNA and the expression level of Sjp40 protein were determined by quantitative real-time PCR (qPCR) and Western blotting, respectively. The mRNA expression of HSPs of S. japonicum in different stages was evaluated by qPCR.</p><p><b>RESULTS</b>Compared with those in the control worms transfected with dsGFP, Sjp40 mRNA level was decreased by 80% in the worms transfected with dsSjp40, and the level of Sjp40 protein showed also a significant decrease. The mRNA expression levels of SjHSP60, SjHSP70, and SjHSP90 did not show an obvious synergism after Sjp40 RNAi. The expression profiles of Sjp40, SjHSP60, SjHSP70, and SjHSP90 showed significant differences in different stages of S. japonicum, and the expression level of Sjp40 mRNA in the egg stage was much higher than that of other HSP genes.</p><p><b>CONCLUSION</b>dsSjp40-RNAi can induce effective suppression of Sjp40 gene expression at both mRNA and protein levels, but no obvious synergism occurs in the mRNA expressions of SjHSP60, SjHSP70, and SjHSP90.</p>

Genetic transformation of Pinellia ternata with Agrobacterium tumefaciens-mediated sHSP genes / 中国中药杂志

<p><b>OBJECTIVE</b>To establish an efficient genetic transformation system of Pinellia ternata.</p><p><b>METHOD</b>With petioles from test-tube seedlings of P. ternata as explants, Agrobacterium tumefaciens mediation method was adopted to explore the effect of phenolic substances, A. tumefaciens's concentration, infection time, pre-incubation time and co-cultivation time on genetic transformation efficiency of P. ternata.</p><p><b>RESULT AND CONCLUSION</b>The genetic transformation efficiency could be effectively enhanced by infecting in A. tumefaciens culture containing AS 40 mg x L(-1) for 15 min for three days. The petioles were put into the differentiation medium containing 150 mg x L(-1) Kan and 350 mg x L(-1) Carb to screening and cultivation. After around 30 days, microtubers could be observed at both sides of the petioles. Gus staining and PCR verification on the regenerated plants showed that the exogenous gene sHSP had been integrated into genome of P. ternata.</p>

Identification of the regulation elements in heat-inducible Lehsp23.8 promoter / 生物工程学报

The promoter of mitochondria-localized small heat shock protein gene in Lycopersicon esculentum (Lehsp23.8) is characterized as strongly heat-inducible. In this study, to determine how the expression of Lehsp23.8 is regulated, we conducted five expression vectors carrying the gus gene driven by the 5' deletion products of the Lehsp23.8 promoter. The corresponding transgenic tobacco plants were generated via Agrobacterium tumefaciens-mediated transformation. Transgenic plants were identified by PCR and Southern blotting analysis. GUS activities under heat-shock conditions were characterized in transgenic tobacco plants. After heat shock, obvious GUS staining was detected in the leaves, shoots, roots, flowers and fruits of the transgenic tobacco plants. The result of fluorometric GUS assays in leaves showed that the heat-induced GUS activity of the 565 bp promoter was the strongest, while that of the 255 bp promoter was the lowest. Deletion analysis shows that the smallest promoter fragment (-255 bp to -23 bp) is sufficient for heat induction. It also indicates that the sequences between -255 bp and -565 bp serve as enhancers, while the sequences between -565 bp and -871 bp can repress the heat-induced activity of the Lehsp23.8 promoter.

Expression of HSP27 and alphaB-crystallin in Avian Cerebellum during Development / 대한해부학회지

It is well known that small heat shock proteins play a role as molecular chaperone. However, during normal development of the cerebellum, expression and distribution of HSP27 and alphaB-crystallin (alphaBC) which are small heat shock proteins have not been reported. To verify the protective role of HSP27 and alphaBC in neurons and glial cells, we examined the expression and distribution of HSP27 and alphaBC in the developing chick cerebellum using immunoblot, immunohistochemical and double immunofluorescence staining. Expression of both HSP27 and alphaBC was first identified in the cerebellum of the embryonic day 14 (E14) embryo, and was increased at E18. Double immunofluorescence analysis with myelin-basic protein (MBP) demonstrated that alphaBC positive (+) cells were mature myelinating oligodendrocytes. alphaBC+ cells were observed in the white matter of the E14 cerebellum. At E18, there were a number of alphaBC+ cells in the white matter and a few cells in the granular layer of the gray matter. On the other hand, HSP27+ cells were observed in the white matter and the Purkinje cell layer at E14. At E18, HSP27+ signals were observed in Purkinje cells and neurons of cerebellar nucleus as well as oligodendrocytes in the white matter and the granular layer. The results that HSP27 and alphaBC were expressed in specific neurons and glial cells in the developing cerebellum suggest that HSP27 and alphaBC may be involved in the protective mechanism for the apoptosis of neurons and the physiological stress occurred in oligodendrocyts during cell maturation.

Mutation analysis of small heat-shock protein 22 gene in Chinese patients with Charcot-Marie-Tooth disease / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the characteristics of the mutation of small heat-shock protein 22 (HSP22) gene in Chinese patients with Charcot-Marie-Tooth (CMT) disease.</p><p><b>METHODS</b>A CMT2L proband with 423(G--> T) mutation in HSP22 gene had been studied and reported by the present authors. In this study, mutation analysis of HSP22 gene was performed using polymerase chain reaction and DNA direct sequencing in 114 CMT probands.</p><p><b>RESULTS</b>In the 114 CMT probands, a 582(C--> T)(T194T)samesense mutation was found in two unrelated families.</p><p><b>CONCLUSION</b>The rate of HSP22 gene mutation in Chinese patients with CMT is as low as 0.87%(1/115).</p>

Reduction of TNF alpha-induced oxidative DNA damage product, 8-hydroxy-2'-deoxyguanosine, in L929 cells stably transfected with small heat shock protein

Previous studies have demonstrated that oxidative stress involving generation of reactive oxygen species (ROS) is responsible for the cytotoxic action of TNF alpha. Protective effect of small heat shock proteins (small HSP) against diverse oxidative stress conditions has been suggested. Although overexpression of small hsp was shown to provide an enhanced survival of TNF alpha-sensitive cells when challenged with TNF alpha, neither the nature of TNF alpha-induced cytotoxicity nor the protective mechanism of small HSP has not been completely understood. In this study, we have attempted to determine whether TNF alpha induces oxidative DNA damage in TNF alpha-sensitive L929 cells. We chose to measure the level of 8-hydroxy-2'-deoxyguanosine (8 ohdG), which has been increasingly recognized as one of the most sensitive markers of oxidative DNA damage. Our results clearly demonstrated that the level of 8 ohdG increased in L929 cells in a TNF alpha dose-dependent manner. Subsequently, we asked whether small HSP has a protective effect on TNF alpha-induced oxidative DNA damage. To accomplish this goal, we have stably transfected L929 cells with mouse small hsp cDNA (hsp25) since these cells are devoid of endogenous small hsps. We found that TNF alpha-induced 8 ohdG was decreased in cells overexpressing exogenous small hsp. We also found that the cell killing activity of TNF alpha was decreased in these cells as measured by clonogenic survival. Taken together, results from the current study show that cytotoxic mechanism of TNF alpha involves oxidative damage of DNA and that overexpression of the small hsp reduces this oxidative damage. We suggest that the reduction of oxidative DNA damage is one of the most important protective mechanisms of small HSP against TNF alpha.