Lipid transfer proteins (LTPs) constitute a pervasive class of small proteins implicated in many biological and physiological processes, including seed development, germination, cuticle formation, and abiotic stress responses. In this study, we explored the role of Arabidopsis LTP12 protein in mitigating environmental stresses. To address this, we analyzed the T-DNA knockout mutant ltp12, focusing on its responses to salinity and osmotic stress. Utilizing antioxidant enzyme assays, phenotypic analyses (including water loss, chlorophyll content, seed germination rates, root length, and relative water content), and lipid profile analysis via Thin Layer Chromatography (TLC), we found that ltp12 mutants showed reduced catalase and peroxidase activities and poorer hydration, chlorophyll content, germination, and growth under stress, compared to wild-type Col-0 plants. Mutants delayed vegetative-to-floral phase transition compared to wild-type. Additionally, lipid analysis indicated that the wild-type plants had increased phospholipids under high osmotic stress, suggesting LTP12's involvement in lipid reorganization during stress responses.

Salicylic acid binding protein 2 (SABP2) is known to play a key role in Salicylic acid mediated defense pathway. SBIP-470 is SABP2 interacting protein that might be putatively involved in transfer of lipids. SBIP-470 was cloned without the signal peptide and expressed in E. coli. In vitro lipid binding assay using recombinant SBIP-470 failed to detect lipid binding. In vitro lipid transfer assay showed recombinant SBIP-470 does not transfer phospholipid. Study has shown that SBIP-470 is highly inducible upon infection with viral as well as bacterial pathogens. Induction of SBIP-470 expression upon the TMV infection most likely depends upon the SABP2 while its expression upon non-host bacterial pathogens is most probably inhibited by the SABP2. A study of Arabidopsis knockout mutants (ltp12 mutant and ltp2 mutant) lacking the SBIP-470 homolog genes showed defects in growth phenotype, and they were found susceptible to bacterial pathogens.

SIP470, a putative tobacco lipid transfer protein, was identified in a yeast two-hybrid screen to interact with SABP2. SABP2 is a critical role in SA-mediated signaling in tobacco and other plants. In vitro studies using purified recombinant SIP470 confirmed that it is a lipid binding protein. In an attempt to determine its role in mediating stress responses, Arabidopsis T-DNA insertion knockout lines lacking SIP470 homolog were used for the analysis. These mutant plants were defective in basal resistance against microbial pathogens. Expression of defense gene PR-1 was also delayed in these mutant plants. Interestingly, these mutant plants were not defective in inducing systemic acquired resistance. Besides biotic stress, these mutant plants also showed increased susceptibility to abiotic stresses. To directly study the role of SIP470 in tobacco plants, transgenic tobacco lines, with reduced levels of SIP470 expression, were generated using RNAi and transgenic lines overexpressing SIP470 were also generated.

All plants have a protection against the surrounding environment called a cuticle coating. When this cuticle coating is constructed it is believed that the family of protein called lipid transfer proteins (LTPs) is involved. The LTPs are small and cysteine rich. In Marchantia polymorpha the groups of LTPs called LTPd and LTPg can be found. 8 and 4 in each group respectively. In the genes of LTPd there is an intron placed downstream of the start codon. Firstly, a sequence database search was performed and LTPd2 and LTPd3 were chosen for further experiments in this study. Secondly, a control that the intron was present in the samples were done by preforming a PCR reaction of cDNA from isolated RNA taken from untreated Marchantia polymorpha. A gel electrophoresis of the product was also performed. Lastly, the amount of alternative splicing in LTPd2 and LTPd3 from Marchantia polymorpha after treated with cold and dehydration were studied using quantitative PCR. For the qPCR MpACT and the exon of respective gene were used as references. The ΔCt values and the expression fold (2ΔΔCt) calculated from the qPCR results showed that most of the transcript with introns preserved were upregulated after subjected to stress. Only the intron in MpLTPd2 and MpLTPd3 with MpACT as reference showed a small downregulation after the cold treatment. The intron in MpLTPd3 with MpLTPd3s exon as reference didn’t show any difference. None of the intron transcript in any of the genes on the other hand showed any significant difference in the alternative splicing. This could be because of small sample groups when the test was performed. In conclusion, there were no significant difference in intron expression between treated and control samples. Therefore, nothing can be said about the change in alternative splicing in MpLTPds after cold and dehydration treatments.

Made available in DSpace on 2014-08-20T13:59:06Z (GMT). No. of bitstreams: 1 dissertacao_gabriela_peres_moraes.pdf: 2721394 bytes, checksum: 37931b48d7259651d911f5ce19c45303 (MD5) Previous issue date: 2013-08-16 / On the production of rice, the salinization of the soil and the water of irrigation, during the establishment phases its closely related to the variation in the levels of production. Among the main damages caused by saline stress at cellular level, are the disturbances in the plasmatic membrane, being its effects manifested by alterations on the permeability, lipid composition, electrical potential and activity of proteins linked to it. The proteins LTPs ("Lipid Transfer Proteins") are related to the transfer and connection of fatty acids and phospholipids between membranes, in addition to being involved in the modification of the lipid composition and their biogenesis. On this note, the objective of this work was to analyze the expression of ten candidate genes to normalizing for studies of genetic expression and verify the differential expression of eleven genes LTPs in rice seedlings. The genotypes BRS Bojuru (tolerant) and BRS Ligeirinho (sensitive) were exposed to 150mM of NaCl in times 0, 24, 48, 72 and 96 hours. The normalizing gene UBQ10 was the most stable for these experimental conditions tested, while the least stable were IF-4a, Tip41-Like and Cyclophilin, being, therefore not in, the least indicated. Among the analyzed LTPs genes, LTP10 presented a high expression value (70 times more) in the 96 hour of stress, when compared to the control in sensitive genotype. For the BRS Bojuru this same gene kept the expression standards while exposed in different times to the stress. The gene LTP14 showed similar patterns of expression between the genotypes studied. In the beginning of the stress, the level of expression practically unaltered, followed by increase and successive decrease after 72 hrs of salt exposure. After the correlation analysis, it was observed that LTP7 and 14 showed a positive correlation, while LTPs 10,26 and 23 showed negative correlation among genotypes. The phylogenetic tree showed a grouping tendency similar to the nucleotides and amino acids sequences analyzed. Based on the results of this study, it was concluded that the gene UBQ10 is the best normalizing for the experimental conditions tested and the genes LTP10, 23 and 26 can be used as a marker for the assisted selection of rice plants for the saline stress for showing contrasting response of expression between genotypes. / Na orizicultura, a salinização do solo e da água de irrigação, durante as fases de estabelecimento e reprodutiva está intimamente relacionada com a variação nos níveis de produção. Dentre os principais danos causados pelo estresse salino em nível celular, estão os distúrbios na membrana plasmática, sendo seus efeitos manifestados por alterações na permeabilidade, na composição lipídica, potencial elétrico e atividade de enzimas e proteínas ligadas a ela. As proteínas LTPs ( Lipid Transfer Proteins ) estão relacionadas com a transferência e ligação de ácidos graxos e fosfolipídios entre as membranas, além de estarem envolvidas na modificação da composição lipídica e biogênese destas. Desta forma, o objetivo deste trabalho foi analisar a expressão de dez genes candidatos a normalizadores para estudos de expressão gênica e verificar a expressão diferencial de 11 genes LTPs em plântulas de arroz. Os genótipos BRS Bojuru (tolerante) e BRS Ligeirinho (sensível) foram expostos a 150 mM de NaCl nos tempos 0, 24, 48, 72 e 96 horas. O gene normalizador UBQ10 foi o mais estável para estas condições experimentais testadas, enquanto que os menos estáveis foram IF-4α, TIP41-LIKE e Cyclophilin, sendo, portanto, os menos indicados. Dentre os genes LTPs avaliados, LTP10 apresentou alto valor de expressão (70 vezes mais) nas 96 horas de estresse, quando comparado ao controle no genótipo sensível. Para BRS Bojuru esse mesmo gene manteve o padrão de expressão durante os tempos de exposição ao estresse. O gene LTP14 apresentou padrão de expressão semelhante entre os genótipos estudados. No início do estresse, o nível de expressão manteve-se praticamente inalterado, seguido de aumento e sucessiva diminuição a partir das 72 h de exposição ao sal. A partir da análise de correlação observou-se que LTP7 e 14 apresentaram correlação positiva, enquanto que LTPs 10, 26 e 23 apresentaram correlação negativa entre os genótipos. As árvores filogenéticas mostraram uma tendência de agrupamento semelhante entre as sequências de nucleotídeos e aminoácidos analisadas. Com base nos resultados obtidos, conclui-se que o gene UBQ10 é o melhor normalizador para as condições experimentais testadas e os genes LTP10, 23 e 26 poderão ser utilizados como possíveis marcadores para a seleção assistida de plantas de arroz para o estresse salino por apresentarem resposta de expressão contrastante entre os genótipos.

SABP2-interacting protein 470 (SIP470), a non-specific lipid transfer protein (nsLTP), was discovered in a yeast two-hybrid screening using SABP2 as bait and tobacco leaf proteins as prey. SABP2 is an important enzyme in systemic acquired resistance that converts salicylic acid to methyl salicylate. Localization studies are an important aspect to understanding the biological function of proteins. nsLTPs are generally considered apoplastic proteins and has been localized intracellularly and extracellularly. Transient expression shows highest expression of SIP470-eGFP at 2 days post infiltration into Nicotiana benthamiana. Confocal microscopy showed localization near the periphery of the cell. Subcellular localization using differential centrifugation showed that SIP470 is localized in the mitochondria. Mitochondria membranes are rich in lipids and have shown lipid exchange with the endoplasmic reticulum in mammalian systems. Co-localization of SIP470-eGFP+mCherry did not express complete co-localization in the targeted organelles. Co-localization pattern suggests possible localization in the endoplasmic reticulum.

<p>  </p> <p>Plants synthesize and release a variety of volatile organic compounds (VOCs) that are important for their reproduction, defense, and communication. These low-molecular-weight, lipophilic molecules also serve as practical products in industries such as food additives, fragrances, colorants, nutraceuticals, and pharmaceuticals. In addition, they have agricultural applications such as sustainable methods for pest control. Therefore, identifying the biological mechanisms involved in volatile emission could help researchers develop new ways to control the timing and release of volatiles, defend against pests, and engineer the production of these valuable chemicals.</p> <p>While progress has been made in understanding plant volatile biosynthesis, their release from the cell remains incomplete. For plant VOCs to be emitted into the environment, they must move from their site of biosynthesis through the cytosol, transverse the plasma membrane, hydrophilic cell wall, and sometimes cuticle to exit the cell. It was previously shown by mathematical modeling that to achieve observed emission rates solely by diffusion, VOCs would accumulate in the cellular membranes to levels that are likely detrimental to the membrane integrity and function. Hence, it was proposed that there are biological mechanisms involved to lower VOC concentrations in membranes. In this work, we focus on the aqueous cell wall, the thickest layer among the three subcellular barriers that should act as a barrier for the diffusion of VOCs. We hypothesize that the transport of VOCs across the cell wall is facilitated by lipid transfer proteins (LTPs) which enhance the solubility of hydrophobic volatiles in the aqueous environment, prevent their back partition into the plasma membrane after entering the cell wall, and hence enhance their net diffusion. To investigate if the presence of LTPs has influence on the total VOC efflux, we use reverse-genetic, biochemical, and mathematical modeling approaches. Out of three highly expressed LTPs identified in the petunia petal, only downregulation of <em>PhLTP3</em> expression led to a decrease in VOC emission in the corresponding transgenic plants. A facilitated diffusion model was built to quantify the VOC flux difference with the presence of LTPs in the cell wall. Modeling of the steady state system revealed the facilitation of VOC flux by LTPs is greatest when the VOC concentration gradient across the cell wall is shallow, which is a physiologically relevant condition. In addition, there exists an optimal protein dissociation constant value for maximal facilitated flux, indicating the balance between the binding and the unloading of VOC is critical. With the in vitro displacement assay, the binding constants of candidate PhLTPs with VOCs were obtained and were all found to be in the µM range, which is close to our model predicted optimal value. The results revealed that LTPs, specifically PhLTP3, play a role in the export of VOCs from the plasma membrane, across the cell wall, to the cuticle.</p> <p>In our earlier mathematical model, the emission of VOCs from the petunia flowers was modeled assuming negligible mass transfer resistance on the surface of the cuticle because of their high volatility. However, the resistance imposed by the surface boundary layer was not considered. To examine if surface convection influences VOC emission, a model system which utilized a model cuticular wax film containing 2-phenylethanol (2-PE) was built to imitate the VOC emission from plant cuticle. The convection mass transfer coefficient of 2-PE emission from a model cuticular wax film was obtained by experimental data fitting and calculated from the correlation that involves Sherwood number. The obtained values that were smaller than unity indicates that the surface boundary layer imposes a higher mass transfer resistance than the model cuticle for the emission of 2-PE in the range of wind velocities investigated. The examination of petunia flowers under air flow showed increases in total emission but no significant differences in total internal pools, which indicates an increase in biosynthesis. The emission changes of individual compounds were different and does not clearly correlate to any molecular properties of the compounds.  </p>

Une distribution lipidique hétérogène est essentielle à l’identité et fonction des organelles, mais l’échange par trafic vésiculaire tend à annuler cette distribution. Il existe donc des mécanismes qui assurent l’homéostasie des lipides. Les protéines Osh (S. cerevisiae) et les OSBP-Related Proteins (ORP, H. sapiens), sont des transporteurs de lipides. Osh4 est capable d’échanger de l’ergostérol contre le phosphatidylinositol-4-phosphate (PI4P), présent sur l’appareil de Golgi. Utilisant des outils fluorescents mesurant avec une précision inégalée le transport de stérol et de PI4P, nous démontrons qu’Osh4 transporte du stérol contre son gradient de concentration en utilisant l’énergie d’un gradient de PI4P. Un couplage au métabolisme du PI4P permettrait à Osh4 d’alimenter le Golgi avec du stérol, ainsi créant le gradient de stérol entre ces organelles. La protéine OSBP participe, via sa capacité à connecter la membrane du RE à celle du trans-Golgi, à la création de jonctions entre ces organelles. Nous avons montré qu’OSBP, par échange stérol/PI4P, utilise le PI4P pour transférer du cholestérol au Golgi, mais également pour autoréguler sa capacité à former les jonctions. Osh6 lie la phosphatidylsérine, nous permettant d’étudier un nouveau mécanisme d’échange. Nous avons résolu la structure cristallographique d’un complexe Osh6/PI4P et avons pu observer l’échange de ces deux ligands par Osh6 entre deux membranes. Cette étude nous permet de suggérer que l’échange de PI4P avec divers lipides, via les protéines Osh/ORP, serait un mécanisme général permettant aux cellules de maintenir le gradient lipidique entre le RE et les membranes tardives de la voie sécrétoire. / An uneven lipid distribution is essential for the function of eukaryotic organelles. However, exchange of material by vesicular trafficking has a tendency to perturb this distribution; mechanisms must though exist to ensure lipid homeostasis. Osh proteins (S. cerevisiae) and OSBP-Related Proteins (ORPs, H. sapiens), are lipid transfer proteins (LTPs). Osh4 is capable of exchanging ergosterol for phosphatidylinositol 4-phosphate (PI4P), found on the Golgi. Using novel fluorescent tools to measure with unprecedented precision the transport of sterol and PI4P, we find that Osh4 can transport sterol against its concentration gradient using the energy of a PI4P gradient. Coupled to phosphoinositide metabolism, this allows Osh4 to transport sterol to the trans-Golgi and create the sterol gradients observed between these organelles. OSBP participates in the creation of membrane contact sites (MCSs) via its capacity to connect ER membranes to those of the trans-Golgi. We have shown that it uses PI4P for transporting cholesterol from the ER to the trans-Golgi by sterol/PI4P counterexchange, hence also autoregulating its tethering activity. Finally, the identification of phosphatidylserine as a ligand for Osh6 allowed us to analyze the possible extrapolation of the PI4P counterexchange mechanism. We have solved the crystal structure of Osh6 in complex with PI4P and have been able to follow counterexchange of PI(4)P and PS in vitro. Concluding, our studies allow us to suggest a general mechanism for ORP/Osh-mediated counterexchange of PI4P for other lipids to maintain lipid gradients between the ER and late membranes of the secretory pathway.

Due to global warming, our planet will experience more extreme weather conditions. Plants can protect themselves against these abiotic stress conditions with their stress response, which includes alternative splicing of certain genes. Alternative splicing is a post-transcriptional process where a single gene gives rise to different mRNAs, which in turn produces different proteins. In plants, this is usually done by intron retention. One type of protein that may be involved in this stress response are the non-specific lipid transfer proteins (LTPs). Indeed, evidence of intron retention has been found in the LTP genes in the liverwort Marchantia polymorpha, called MpLTPd. To investigate whether this alternative splicing is caused by abiotic stress or not, I subjected the moss to two different types of stress trials, drought and cold, and compared the general expression of the intron in MpLTPd2 and MpLTPd3 from the stressed samples to samples from a moss grown under normal conditions. I found that the expression of the intron did change in the stressed moss, but none of the differences were significant. This suggests that alterative splicing in MpLTPd2 and MpLTPd3 is not caused by cold and drought and that the intron-containing protein plays no role in the protection of M. polymorpha against abiotic stress.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / This work reports the biochemical characterization, cytotoxic and allergenic effects of a lipid transfer protein isolated from M. citrifolia seeds (McLTP1), with trypsin and alpha-amylase inhibition properties. McLTP1 was purified with a procedure involving trichloroacetic acid precipitation and gel filtration chromatography. This protein showed significant inhibitory activities against trypsin (767,10  8,36 TIU/mgP), chymotrypsin (25,36  0,86 IU/mgP), papain (65,419  0,152 IU/mgP) and alpha-amylase (24,40%). Atomic force microscopy displayed that McLTP1 oligomerized in tetramers showing a central channel. Fluorescence and CD assays revealed that the McLTP1 structure is highly stable, regardless of pH and temperature levels. In vitro, McLTP1 presented a selective cytotoxic effect to human ovarian cancer cells (OVCAR-8; IC50 of 16,6 &#956;g/mL) and demonstrated hemolytic effect against fresh rabbit red blood cels. Similarly to other non-specific lipid transfer protein reported, McLTP1 showed allergenic properties in mice, being considered as a true food allergen since it was able to sensitize the animals via the gastrointestinal tract. / Morinda citrifolia L. à uma espÃcie nativa do Sudeste da Ãsia intensamente investigada em funÃÃo de suas propriedades terapÃuticas reportadas hà mais de 2.000 anos. Recentemente, uma proteÃna transferidora de lipÃdeos denominada McLTP1 (UniProt Accession Number: C0HJH5) foi isolada de sementes de noni pelo nosso grupo de pesquisa. McLTP1 à uma proteÃna termoestÃvel de massa molecular 9,4 kDa, resistente à proteÃlise e dotada de atividades moduladoras da inflamaÃÃo e da dor pela via oral, promissoras e inÃditas para esse grupo de molÃculas. Este trabalho objetivou caracterizar bioquimicamente McLTP1, bem como avaliar o seu potencial alergÃnico em camundongos, como etapas bÃsicas para o seu uso racional e seguro do ponto de vista farmacolÃgico. Em adiÃÃo, as propriedades terapÃuticas de McLTP1 foram tambÃm ampliadas, atravÃs da investigaÃÃo de seu efeito citotÃxico em diferentes linhagens de cÃlulas tumorais. A proteÃna em estudo foi isolada utilizando o protocolo jà estabelecido, envolvendo as etapas de precipitaÃÃo seletiva de proteÃnas do extrato total das sementes de noni com Ãcido tricloroacÃtico 2,5% e cromatografia de exclusÃo molecular. O ensaio de alergenicidade in vivo foi conduzido apÃs prÃvia aprovaÃÃo pelo Comità de Ãtica para Uso de Animais da Universidade Federal do Cearà e utilizou fÃmeas nulÃparas com massa corporal entre 25 e 30 g. McLTP1 apresentou in vitro atividades inibitÃrias de tripsina (767,10  8,36 UIT/mgP), quimotripsina (25,36  0,86 UI/mgP), papaÃna (65,419  0,152 UI/mgP) e alfa-amilase (24,40%). A atividade inibitÃria de tripsina de McLTP1 foi reduzida significativamente em temperaturas superiores a 37 ÂC, apresentando atividade residual de apenas 5,91% quando aquecida a 100 ÂC por 30 min. Essa atividade foi tambÃm influenciada pelo pH, sendo de apenas 30,13% e 39,05% quando a proteÃna foi incubada em tampÃes de pH 3,0 e 12,0. O padrÃo de oligomerizaÃÃo de McLTP1 demonstrou a formaÃÃo de agregados dimÃricos/tetramÃricos delimitando um canal central de diÃmetro de 4,4 nm. As anÃlises espectroscÃpicas mostraram que McLTP1 apresenta espectro de CD similar Ãquele apresentado por outras proteÃnas transferidoras de lipÃdeos e caracterÃstico de proteÃnas ricas em alfa-hÃlice. Espectro de CD de McLTP1 nÃo mostrou alteraÃÃes significativas em diferentes temperaturas e pHs, corroborando com os dados de estabilidade obtidos anteriormente. Diferentemente, em condiÃÃes redutoras (DTT 1 mM) o espectro de CD mostrou alteraÃÃo na estrutura secundÃria da proteÃna e os mÃnimos e mÃximos de elipticidade molar foram tambÃm alterados na presenÃa de micelas iÃnicas de SDS (10 mM). McLTP1 apresentou atividade citotÃxica seletiva contra cÃlulas de cÃncer de ovÃrio (Ovcar-8; CI50: 16,6 &#956;g/mL), nÃo sendo citotÃxica para as cÃlulas tumorais de cÃlon humano (HCT-116), leucemia humano (HL-60) e glioblastoma humano (SF-295) testadas. McLTP1 foi capaz de promover hemÃlise significativa em hemÃcias de coelho a partir da concentraÃÃo de 0,005 mgP/mL. McLTP1 apresentou potencial efeito alergÃnico in silico e em camundongos imunizados pela via oral, induzindo a sÃntese de anticorpos IgG e IgG1. Tal como descrito na literatura para outras LTPs, anticorpos anti-McLTP1 produzidos em coelho foram tambÃm capazes de reconhecer proteÃnas presentes em extratos de Rosaceae, Cucurbitaceae e na polpa do fruto de noni. Os dados obtidos permitiram caracterizar parcialmente a proteÃna em estudo, bem como avaliar o seu potencial imunogÃnico apÃs administraÃÃo oral. Novos testes serÃo conduzidos objetivando avaliar a importÃncia clÃnica dessas respostas, uma vez que testes de toxicidade demonstraram que McLTP1 nÃo foi capaz de promover reaÃÃes adversas em camundongos, mesmo apÃs administraÃÃo da dose de 8 mg/kg por 28 dias.