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Characterization of SIP470, A Plant Lipid Transfer Protein, and its Role in Plant Defense SignalingAndrews, Shantaya Biunca, Audam, Timothy, Kumar, Dhirendra, Dr. 04 April 2018 (has links)
Plants are resilient organisms that are continually evolving and continue to withstand an adverse and dynamic world. SABP2-interacting protein (SIP)-470 is a non-specific lipid transfer protein (nsLTP) that was identified in tobacco. SIP470 was discovered during a yeast two-hybrid screening with SABP2, which is an important methyl esterase enzyme which catalyzes the conversion of immobile MeSA into active salicylic acid (SA) during pathogenic challenge. SA activation and mobility allows for immunity to be carried to other, non-infected parts of the plant. This induced responses is called systemic acquired resistance (SAR) and it is a broad spectrum defense. Like many nsLTPs, SIP470 is small and has a predicted characteristic hydrophobic cavity. nsLTPs are found in higher plants and have repeatedly demonstrated protection in biotic stress including disease resistance, and greater resistance to both bacterial and fungal pathogens in overexpressed transgenic lines. This diverse class is also significantly involved in plant adaptation to environmental changes, namely drought, salinity, and freezing, but also in osmotic stress and wounding. Furthermore, nsLTPs are involved in wax metabolism and seed development. Subcellular localization of nsLTPs varies considerably during in vitro and in recent in vivo studies. SIP470 was originally identified in tobacco plants, and therefore, it is important to study its role directly in tobacco plants. SIP470 and eGFP fusion construct has been generated to study the subcellular localization of SIP470 in tobacco cells. SIP470 localization has shown a discontinuous, punctate arrangement around the membrane periphery which is being further verified by subcellular fractionation. Transgenic tobacco lines that are silenced in SIP470 via RNAi have been generated, and these plants are being screened. Overexpressor transgenic lines of SIP470 have been generated and are under the control of an estradiol-inducible promoter. These transgenic lines will be tested for their response in basal resistance and SAR.
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EXPLORING THE ROLE OF DIR1 AND OTHER PHLOEM-MOBILE PROTEINS DURING SARCarella, Philip January 2016 (has links)
Systemic acquired resistance (SAR) is a defense response induced by an initial
localized infection that leads to the generation of long-distance immune signals that
travel to distant leaves to provide enhanced resistance to subsequent infections. The
lipid transfer protein (LTP) DEFECTIVE IN INDUCED RESISTANCE1 (DIR1) travels
via the phloem from induced to distant leaves during SAR and may chaperone
several long-distance signal candidates. In this thesis, the role of DIR1 during SAR is
explored by examining the route of DIR1 movement, investigating the conservation
of DIR1 structure and function, and by identifying DIR1-interacting proteins. I
demonstrate that Arabidopsis plant lines with restricted cell-to-cell movement through
plasmodesmata are negatively impacted in long-distance DIR1 movement,
suggesting that cell-to-cell movement is important for DIR1 to access distant leaves.
To elucidate the molecular function of DIR1, orthology analysis was performed with
putative DIR1 orthologs. Structurally important amino acid residues that contribute to
the hydrophobicity of the LTP cavity were identified, supporting the idea that DIR1
binds a hydrophobic ligand during SAR. RNAi-mediated knockdown of the DIR1
paralog DIR1-like did not impact the SAR response, supporting the idea that DIR1-
like plays a lesser role in SAR. In addition, targeted protein-protein interaction assays
determined that LTP1 and LTP2 interact with DIR1, and SAR phenotypic analysis of
an ltp2-1 mutant supported a role for LTP2 in SAR. Lastly, a comparative proteomics
approach identified several proteins with differential abundance in phloem exudates
collected during the induction of SAR. Of these proteins, m-type thioredoxins, a
major latex protein-like protein, and the UV-B photoreceptor UVR8 were essential for
the manifestation of SAR. Together, these data provide insight into DIR1 function by
identifying the importance of cell-to-cell movement through plasmodesmata, the
DIR1 hydrophobic cavity, and DIR1-interacting proteins for DIR1-mediated SAR. In
addition, this work identifies new phloem-localized proteins that contribute to the
SAR response, providing fundamental knowledge on protein composition within the
phloem during biotic stress. / Dissertation / Doctor of Philosophy (PhD)
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LTP1 and LOX-1 in barley malt and their role in beer production and qualityNieuwoudt, Melanie 04 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Selection of raw materials for a consistent and high quality end product has been a
challenge for brewers globally. Various different factors may influence quality and although a
great number of methods for malt analysis exist today for the prediction of end product quality,
some still do not accurately represent malt performance in beer. This research focussed on
determining parameters in malts to predict two of the major beer quality determining factors
namely, foam- and flavour stability. Specific biochemical markers in barley malt such as lipid
transfer protein 1 (LTP1) lipoxygenase-1 (LOX-1), anti-radical/oxidant potential (AROP), free
amino nitrogen and intact protein were determined and used in beer quality prediction from malt
character. These biochemical quality predictions were then correlated with the end product beer
quality as assessed in sensory analysis trials on micro-brewed beers.
Being such a multi-faceted factor in beer, LTP1 have already become an attractive field
of study. LTP1 is primarily associated with stable beer foam, as a foam protein in its own right,
and acting as a lipid scavenger. This protein is also theorised to play a role in the stability of beer
flavour by possibly acting as anti-oxidant. Lastly LTP1 is known to have anti-yeast activity,
which could negatively impact fermentation. In this study LTP1 and its lipid bound isoform
LTP1b were successfully purified in an economical and easy five step protocol. Both isoforms
showed temperature stability at temperatures >90°C and prefer more neutral and basic pH
environments. Although the reported antioxidant activity was not observed, both purified LTP1
and LTP1b inhibited lipoxygenase-1 (LOX-1) activity, which is responsible for the enzymatic
breakdown of linoleic acid to form 2(E)-nonenal. This is a novel finding that links LTP1 also to
flavour stability. LTP1 exhibited anti-yeast activity whereas LTP1b lost most if not all the
activity. However, since most of the LTP1 is converted to LTP1b and glycosylated isoforms
during the brewing process fermentation will not be greatly influenced, while foam and flavour
stability could still be promoted by the presence of LTP1b.
Flavour deterioration of the final packaged product is partially due to the enzymatic
production of 2(E)-nonenal by LOX-1 and the presence of free oxygen radical species, limited
anti-radical/oxidant potential (AROP) and LTP1. The development of two 96-well micro-assays based on the ferrous oxidation-xylenol orange (FOX) assay for the determination of LOX-1 and
AROP was successfully accomplished and compared well with established assays. The LOXFOX
and AROP-FOX assays were specifically developed for the on-site, high throughput
comparative determination of LOX-1 and AROP in malt and other brewery samples.
The AROP-FOX and LOX-FOX micro-assays and a number of established assays were
used to categorise malts in different predicted quality groups, various biochemical markers were
measured which included LOX activity, LTP1 content, FAN values, intact protein concentration
and AROP. An excellent trend (R2=0.93) was found between FAN/LOX and LTP1/LOX which
also correlated with the novel observation that LOX-1 activity is inhibited by LTP1 at various
concentrations. These trends could assist brewers in optimal blending for not only high quality
end products but also fermentation predictions.
To determine whether these biochemical markers selected for screening in barley malt are
predictive of shelf life potential of the end product, sensory trials were performed. Three barley
malt cultivars were selected for LOX, AROP, LTP1, protein and FAN content and used in
micro-brewery trials at 0 and 3 months and evaluated using sensory analysis. Good correlation
was found between the biochemical predictors and sensory trial for the best quality malt and
beer. These parameters were therefore highly relevant for predicting shelf life potential, although
additional research is required to elucidate the effect of LTP1 and LOX-1 on each other during
the brewing process, since it seems that high LOX-1 concentrations could be leading to LTP1
decreases. With this study it is proposed that if more detailed protein or FAN characterisation is
used together with the screening of LOX-1, LTP1 and AROP, an more accurate shelf life
prediction, based on malt analysis, is possible and with the help of these parameters brewers can
simply blend malts accordingly. / AFRIKAANSE OPSOMMING: Die keuse van roumateriaal om 'n konstante eindproduk van goeie kwaliteit te lewer, was
nog altyd 'n uitdaging vir brouers wêreldwyd aangesien verskeie faktore 'n invloed het op die
kwaliteit van die produk. Alhoewel daar tans verskeie metodes vir moutanalise bestaan wat die
eindproduk–kwaliteit voorspel, is daar min wat werklik die eindproduk kwaliteit soos voorspel
deur moutanalise verteenwoordig. Hierdie navorsing fokus op die bepaling van mout-eienskappe
om twee van die belangrikste bierkwaliteitvereistes, naamlik skuim- en geurstabiliteit te
voorspel. Spesifieke biochemiese eienskappe in garsmout soos lipiedtransportproteien-1 (LTP1),
lipoksigenase-1 (LOX-1), antioksidant-antiradikaal potensiaal (AROP), vry aminostikstof (FAN)
is geïdentifiseer en gebruik in voorspelling van bierkwaliteit vanaf moutkarakter. Hierdie
biochemiese kwaliteit voorspellings is dan gekorreleer met die eindproduk soos ge-evalueer
d.m.v sensoriese analise op mikro-gebroude bier.
Omdat LTP1 soveel fasette in bier beïnvloed, het dit reeds 'n aanloklike studiefokus
geword. LTP1 word hoofsaaklik geassosieer met stabiele skuimkwaliteit in bier en tree op as 'n
lipiedmop (“lipid scavenger”). Die proteien speel teoreties ook 'n rol in die stabiliteit van bier
geur deur moontlik as 'n anti-oksidant op te tree. Laastens is LTP1 bekend vir sy antigis
aktiwiteit wat moontlik 'n negatiewe uitwerking op fermentasies het. Gedurende hierdie
navorsing is LTP1 en sy lipiedbinding isoform LTP1b suksesvol gesuiwer met 'n ekonomies en
eenvoudige 5-stap protokol. Beide isoforme het stabiliteit by temperature >90°C en meer
neutrale en basiese pH omgewings getoon. Alhoewel die voorheen gerapporteerde anti-oksidant
aktiwiteit vir LTP1 nie bevestig kon word nie, is daar wel gevind dat beide LTP1 en LTP1b,
LOX-1, wat verantwoordelik is vir die ensimatiese afbraak van linoleensuur na 2(E)-nonenal, se
aktiwiteit inhibeer. Dit is 'n unieke bevinding wat LTP1 ook koppel aan geurstabiliteit. LTP1 het
antigis aktiwiteit getoon, maar LTP1b het die meeste, indien nie alle antigis-aktiwiteit verloor.
Omdat die meeste van die LTP1's omgeskakel word na LTP1b's en geglikosileerde isoforme
tydens die brouproses, sal fermentasie nie beduidend beinvloed word nie, maar die skuim- en
geurstabiliteit sal steeds bevorder word deur die blote teenwoordigheid van die LTP1b. Geurverval van die finale verpakte produk is gedeeltelik a.g.v die ensimatiese produksie
van 2(E)-nonenal deur LOX-1 en die teenwoordigheid van vry suurstofradikaal spesies, beperkte
AROP en LTP1. Die ontwikkeling van twee 96-putjie mikroessaïs, gebasseer op die yster
oksidasie-xilenol oranje (FOX) essai vir die bepaling van LOX-1 en AROP, was suksesvol en
het goed vergelyk met reeds gevestigde essaïs. Die LOX-FOX en AROP-FOX mikroessaïs is
spesifiek ontwikkel vir die residente, hoë deurvloei vergelykende bepaling van LOX-1 en AROP
in mout en ander brouery-monsters.
Die AROP-FOX en LOX-FOX mikroessaïs en 'n paar gevestigde essaïs is gebruik om
moute te kategoriseer in die verskillende voorspelde kwaliteitsgroepe. Die biochemiese merkers
wat gemeet is het die volgende ingesluit: LOX aktiwiteit, LTP1 inhoud, FAN waardes, proteïen
konsentrasie en AROP. 'n Merkwaardige korrelasie (R2=0.93) is gevind tussen FAN/LOX en
LTP1/LOX wat ook ooreenstem met die waarneming dat LOX-1 aktiwiteit onderdruk word deur
LTP1 by verskeie konsentrasies. Hierdie korrelasies kan brouers help met optimale versnitting
van moute vir, nie net die hoogste kwaliteit eindproduk nie, maar ook vir fermentasie
voorspellings.
Om te bepaal of hierdie geselekteerde biochemiese merkers in mout die potensieële
raklewe van die eindproduk verteenwoordig, is sensoriese evaluerings uitgevoer. Drie gars-mout
kultivars is geselekteer o.g.v LOX-, AROP-, LTP1-, proteïen- en FAN-inhoud en gebruik in
mikro-brouery proewe en op 0 en 3 maande en is ge-evalueer deur sensoriese analise. Goeie
korrelasie is gevind tussen die biochemiese voorspellers en sensoriese evaluering vir die beste
kwaliteit mout en bier. Hierdie maatstawwe is daarom uiters relevant vir voorspelling van die
potensiele rakleeftyd, alhoewel addisionele navorsing nodig is om die effek van LTP1 en LOX-1
op mekaar gedurende die brouproses te bepaal. Dit blyk dat 'n hoë LOX-1 konsentrasies kan lei
tot 'n afname in LTP1. Met hierdie studie word dit voorstel dat, as meer gedetaileerde proteien
of FAN karakterisering saam met LOX-1, LTP1, en AROP analise uitgevoer word, 'n meer
akkurate raklewe voorspelling moontlik is en met behulp van hierdie parameters kan brouers
moute dienooreenkomstig versnit.
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MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF OLEATE- AND GLYCEROL-3-PHOSPHATE-REGULATED SIGNALING IN PLANTSMandal, Mihir Kumar 01 January 2012 (has links)
Oleic acid (18:1), a monounsaturated fatty acid (FA), is synthesized upon desaturation of stearic acid (18:0) and this reaction is catalyzed by the plastidal enzyme stearoyl-acyl carrier protein-desaturase (SACPD). A mutation in the SSI2/FAB2 encoded SACPD lowers 18:1 levels, which correlates with induction of various resistance (R) genes and increased resistance to pathogens. Genetic and molecular studies have identified several suppressors of ssi2 which restore altered defense signaling either by normalizing 18:1 levels or by affecting function(s) of a downstream component. Characterization of one such ssi2 suppressor mutant showed that it is required downstream of low 18:1-mediated constitutive signaling and partially restores altered defense signaling in the ssi2 mutant. Molecular and genetic studies showed that the second site mutation was in the Nitric Oxide Associated (NOA) 1 gene, which is thought to participate in NO biosynthesis. Consistent with this result, ssi2 plants accumulated high levels of NO and showed an altered transcriptional profile of NO-responsive genes. Interestingly, the partial defense phenotypes observed in ssi2 noa1 plants were completely restored by an additional mutation in either of the two nitrate reductases NIA1 or NIA2. This suggested that NOA1 and NIA proteins participated in NO biosynthesis in an additive manner. Biochemical studies showed that 18:1 physically bound NOA1, in turn leading to its degradation in a protease-dependent manner. In concurrence, overexpression of NOA1 did not promote NO-derived defense signaling in wild-type plants unless 18:1 levels were lowered. Subcellular localization showed that NOA1 and the 18:1-synthesizing SSI2 were present in close proximity within the nucleoids of chloroplasts. Indeed, pathogen- or low 18:1- induced accumulation of NO was primarily detected in the chloroplasts and their nucleoids. Together, these data suggested that 18:1 levels regulate NO synthesis and thereby NO-mediated retrograde signaling between the nucleoids and the nucleus. Since cellular pools of glycerol-3-phosphate (G3P) regulate 18:1 levels, I next analyzed the relationship between G3P and 18:1. Interestingly, unlike 18:1, an increased G3P pool was associated with enhanced systemic immunity in Arabidopsis. This was consistent with G3P-mediated transcriptional reprogramming in the distal tissues. To determine mechanism(s) underlying G3P-conferred systemic immunity, I analyzed the interaction between G3P and a lipid transfer protein (LTP), DIR1. In addition, I monitored localization of DIR1 in both Arabidopsis as well as tobacco. Contrary to its predicted apoplastic localization, DIR1 localized to endoplasmic reticulum and plasmodesmata. The symplastic localization of DIR1 was confirmed using several different assays, including co-localization with plasmodesmatal-localizing protein, plasmolysis and protoplast-based assays. Translocation assays showed that G3P increased DIR1 levels and translocated DIR1 to distal tissues. Together, these results showed that G3P and DIR1 are present in the symplast and their coordinated transport into distal tissues is likely essential for systemic immunity.
In conclusion, this work showed that low 18:1-mediated signaling is mediated via NO, synthesis of which is likely initiated in the plastidal nucleoids. In addition, my work shows that G3P functions as an independent signal during systemic signaling by mediating translocation of the lipid transfer protein, DIR1.
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Regulation of non-specific lipid transfer proteins in abiotically stressed Physcomitrella patensJansson, Sandra January 2011 (has links)
Non-specific lipid transfer proteins is a large and diverse protein family found in plants, with roles in biological systems ranging from long distance signaling to plant pathogen defense. Little is known about the roles of nsLTPs, but recent studies have cast some light on the issue, among other things proposing that they may be involved in the cutice formation on land-living liverworts, mosses and non-seedbearing plants. Increased cuticle formation is thought to be a part of a plants defense system against stress. In this experiment, the expression of nsLTPs type G in the moss Physcomitrella patens was examined by qRT-PCR on cDNA synthesized from already existing mRNA samples from moss under different abiotic stresses. The different stresses were UV-light, salt (ion toxicity), heavy metal, cold drought, plant hormone and osmosis. House-keeping gene P. patens beta-tubuline 1 was used as reference and relative expression analysis was performed. The study showed a general down-regulation of PpLTPg's in the abiotically stressed samples, and the possible coupled regulatory response of PpLTPg3 and PpLTPg5. The results imply that the PpLTPg's in Physcomitrella patens could be connected to biological processes that cease during stress, or that they worl through negative feedback to support plant defense against stress.
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Arabidopsis LTP12, A Homolog of SIP470, As a Key Player in Biotic and Abiotic Stress Response Signaling PathwayGiri, Bikram, Mr., Kumar, Dhirendra, Dr. 25 April 2023 (has links) (PDF)
Lipid transfer proteins (LTPs) belong to the pathogenesis-related protein family (PR-14) and are thought to participate in plant defense mechanisms. In this study, we characterize the function of an Arabidopsis thaliana mutant ltp12 (AT3G51590), a homologous lipid transfer protein to SIP470 from Nicotiana tabacum for its role in abiotic and biotic stress. SIP470, a lipid transfer protein, was found to interact with SABP2 in a yeast-two hybrid screen. SABP2 in tobacco is required for inducing a robust SAR response. The objective of this research is to understand the role of LTP12 in mediating abiotic stress as salicylic acid plays an important role in both abiotic and biotic stress in plants. For this research, stressor chemicals, NaCl (salinity), mannitol (osmotic stress), and drought (no water or PEG) will be used. Seedlings were initially germinated and grown on artificial plant growth MS media. The similar-sized young seedlings were transferred to MS media plates supplemented with or without stressor chemicals. Oxidative stress analysis of various antioxidant enzymes, such as catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) will be performed. The Na+ homeostasis for salinity stress will be studied using CoroNaTM dye and confocal microscopy. Our lab has T-DNA insertion knockout mutants of LTP12 that we will be used in the proposed studies. Here, we hypothesize that mutant ltp12 plants will be hypersensitive to abiotic stressors like NaCl, mannitol, and drought, while wildtype Col-0 will be markedly more tolerant. Reports also suggest that knockout lines of other lipid transfer proteins show a defective growth phenotype and lower expression of systemic acquired resistance (SAR). Moreover, to gain a better understanding of both lines' responses to abiotic stress, we need to carry out further studies on the soil as well. The study will also discuss the subcellular localization of ltp12 in Arabidopsis, which will provide an idea of its functional mechanism. Understanding the role of lipid transfer proteins can lead to the development of transgenic plants that are more tolerant to abiotic stresses and climate change.
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Role of Arabidopsis LTP12 in Environmental Stress ResponseGiri, Bikram 01 May 2024 (has links) (PDF)
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.
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Biochemical Characterization of SBIP-470 and its role in SA-mediated Signaling in PlantsChapagai, Danda P 01 December 2014 (has links)
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.
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Expressão diferencial de genes normalizadores e da família LTPs1, em genótipos de arroz sob estresse salino / Differential expression of normalizing genes and the family LTPs1, in genotypes of rice under saline stressMoraes, Gabriela Peres 16 August 2013 (has links)
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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.
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Localization of SIP470, a Plant Lipid Transfer Protein in Nicotiana tabacumAndrews, Shantaya 01 December 2018 (has links) (PDF)
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.
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