Introdução: A obesidade e a dislipidemia são grandes contribuintes dos agravos cardiovasculares. O consumo de vegetais, principalmente de suas proteínas, atua de forma protetora na magnitude destes agravos. Há grandes indícios de que a proteína do amaranto possui efeito hipocolesterolemizante pela ação de peptídeos, originários de sua digestão incompleta. Objetivo: Verificar a ação, in vitro, do hidrolisado proteico do amaranto, submetido a diferentes processamentos, na solubilização micelar do colesterol e inibição da atividade enzimática HMGR. Métodos: As farinhas processadas e crua foram analisadas quanto seu teor de aminoácidos. Os isolados proteicos das farinhas do grão de amaranto tostado, extrusado e cru, foram submetidos à hidrólise enzimática e em seguida, foi elaborada uma solução de sais biliares e colesterol para avaliar a capacidade dos hidrolisados proteicos em diminuir a solubilização micelar de colesterol. Utilizaram-se os ultra filtrados (PM menor que 3 kDa) em concentração de 3 mg/mL em equivalentes de albumina, e para os de peso moleculares maiores foram utilizados 10 mg/mL. Com o intuito de verificar o mecanismo de inibição da síntese endógena de colesterol, somente, foram utilizados os hidrolisados ultra filtrados. Nos ensaios de inibição enzimática da HMGR foram utilizadas concentrações de hidrolisados (0,1, 0,5 e 1 mg/mL) para avaliar a inibição e comparar a pravastatina (inibidor conhecido). Resultados: A composição de aminoácidos demonstrou-se adequada, quando comparada a recomendação de aminoácidos essenciais para crianças de 2 a 5 anos. Os aminoácidos hidrofóbicos constituem 30 por cento do total de aminoácidos. Ao avaliar o efeito do hidrolisado na solubilização micelar do colesterol, foi observado que houve diferença (p < 0,004) devido ao processamento. O hidrolisado proteico da farinha crua (IPHc), com peptídeos de peso molecular maior que 3 kDa, reduziu a solubilização micelar do colesterol em 44,09 ± 1,5 por cento , enquanto que os hidrolisados de farinha tostada (IPHt) e extrusada (IPHe) reduziram em 31,24 ± 5,9 por cento e 24,97 ± 4,1 por cento . Já os hidrolisados com peso molecular menor que 3 kDa apresentaram pouca diferença (p < 0,03) em relação ao processamento. A redução da solubilidade micelar observada pelos IPHc e IPHe foi semelhante: 37,21 ± 1,65 por cento e 35,45 ± 0,4 por cento , respectivamente. O IPHt apresentou a menor redução de 22,47 ± 4,6 por cento . Os hidrolisados da farinha de amaranto também foram capazes de inibir a atividade da enzima HMGR em diversas concentrações. O controle da atividade normal da enzima apresentou 0,65 ± 0,05 µmol de NAPH oxidada min/mg equivalente de albumina. O IPHc, em concentrações de 0,1 e 0,5 mg/mL, apresentou efeito similar ao da pravastatina, diferindo do controle (p < 0,05), produzindo: 0,24 ± 0,03 e 0,29 ± 0,13 de µmol de NAPH oxidada min/mg equivalente de albumina. Por outro lado o IPHt apresentou efeito similar ao da pravastatina em concentração superior ao cru; em 1 mg/mL produziu 0,20 ± 0,09 de µmol de NAPH oxidada min/mg equivalente de albumina. O IPHe apresentou efeito inibidor da enzima em concentração de 0,1 mg/mL, porém menor do que o observado para a pravastatina. Conclusões: A proteína do grão de amaranto hidrolisada possui indícios de atividade hipocolesterolêmica. Sendo capaz de atuar tanto na via exógena quanto na via endógena, inibindo a absorção do colesterol e sua síntese de forma indireta. O processamento térmico diminuiu esta capacidade, mas ainda demonstra resultados significativos. Dentre os processamentos, a extrusão mostrou ter diminuído este efeito, embora os seus resultados possam ter sido influenciados pela quantidade de componentes no isolado proteico, como lipídeos e compostos fenólicos. / Introduction: Obesity and dyslipidemia are major contributors of cardiovascular diseases. The consumption of vegetables, especially their protein, acts protectively on the magnitude of these injuries. There is evidence that amaranth protein has a cholesterol-lowering effect by the action of peptides originating from its incomplete digestion. Objective: To assess the effect, in vitro, of the hydrolyzed protein of amaranth, submitted to different processes, on the reduction of the micellar solubilization of cholesterol and on the inhibition of HMGR enzyme activity. Methods: The raw and processed flours were analyzed for their content of amino acids. The isolated protein from amaranth grain flour toasted, extruded and raw, were subjected to enzymatic hydrolysis. Subsequently, it was prepared a solution of bile salts and cholesterol to assess the ability of the hydrolyzed protein to decrease the micellar solubilization of cholesterol. It was used the ultra-filtered peptides (MW up to 3 kDa) at concentration of 3 mg/mL equivalent albumin; and for higher molecular weights, it was used 10 mg/mL. In order to verify the mechanism of inhibition of the cholesterol endogenous synthesis, only it was used the hydrolyzed ultra-filtered peptides with MW < 3 KDa. In the assays of HMGR inhibition, several concentrations of peptides were used (0.1, 0.5 and 1 mg/mL) to compare the inhibition to pravastatin (a known inhibitor). Results: The amino acid composition showed to be adequate when compared to the recommendation of essential amino acids for children 2-5 years. Hydrophobic amino acids compose 30 per cent in total amino acids. When evaluating the effect of the hydrolyzate micellar solubilization of cholesterol has been observed that significant difference (p <0.004) from the processing. The peptides from raw flour hydrolyzed protein (IPHc) with a molecular weight greater than 3 kDa reduced micellar solubilization of cholesterol by 44.09 ± 1.5 per cent , while those from the roasted flour (IPHt) and extruded flour (IPHe) reduced by 31.24 ± 5.9 per cent and 24.97 ± 4.1 per cent . Peptides with a molecular weight up to 3 kDa showed little difference (p < 0.03) due to processing. The reduction of the observed micellar solubility of IPHc and IPHe were similar: 37.21 ± 0.4 per cent and 35.45 ± 1.65 per cent , respectively. The IPHt showed the smallest decrease of 22.47 ± 4.6 per cent . The peptides from amaranth flour were also able to inhibit the activity of the enzyme HMGR in various concentrations. The control of normal enzyme activity showed 0.65 ± 0.05 mol of NAPH oxidized min/mg equivalent of albumin. The IPHc at concentrations of 0.1 and 0.5 mg/mL had an effect similar to that of pravastatin, different from control (p < 0.05), yielding: 0.24 ± 0.03 and 0.29 ± 0.13 mol of the NAPH oxidized min/mg equivalent of albumin. On the other hand, IPHt showed a similar effect to higher concentration of pravastatin in the raw, and in 1 mg/mL produced from 0.20 ± 0.09 mol of oxidized NAPH min/mg equivalent of albumin. The IPHe showed the inhibitory effect on the enzyme concentration as lower as 0.1 mg/mL, but less pronounced than pravastatin. Conclusions: The peptides from hydrolysis of amaranth grain has evidence of hypocholesterolemic activity. They are able to act both in exogenously and endogenous pathways, inhibiting the absorption of cholesterol and its synthesis. The thermal processing reduces this capacity, but still shows significant results. Among the processing, extrusion deserves less attention than other one, although their results may have been influenced by the amount of the isolated protein components, such as lipids and phenolic compounds.

Introdução: A obesidade e a dislipidemia são grandes contribuintes dos agravos cardiovasculares. O consumo de vegetais, principalmente de suas proteínas, atua de forma protetora na magnitude destes agravos. Há grandes indícios de que a proteína do amaranto possui efeito hipocolesterolemizante pela ação de peptídeos, originários de sua digestão incompleta. Objetivo: Verificar a ação, in vitro, do hidrolisado proteico do amaranto, submetido a diferentes processamentos, na solubilização micelar do colesterol e inibição da atividade enzimática HMGR. Métodos: As farinhas processadas e crua foram analisadas quanto seu teor de aminoácidos. Os isolados proteicos das farinhas do grão de amaranto tostado, extrusado e cru, foram submetidos à hidrólise enzimática e em seguida, foi elaborada uma solução de sais biliares e colesterol para avaliar a capacidade dos hidrolisados proteicos em diminuir a solubilização micelar de colesterol. Utilizaram-se os ultra filtrados (PM menor que 3 kDa) em concentração de 3 mg/mL em equivalentes de albumina, e para os de peso moleculares maiores foram utilizados 10 mg/mL. Com o intuito de verificar o mecanismo de inibição da síntese endógena de colesterol, somente, foram utilizados os hidrolisados ultra filtrados. Nos ensaios de inibição enzimática da HMGR foram utilizadas concentrações de hidrolisados (0,1, 0,5 e 1 mg/mL) para avaliar a inibição e comparar a pravastatina (inibidor conhecido). Resultados: A composição de aminoácidos demonstrou-se adequada, quando comparada a recomendação de aminoácidos essenciais para crianças de 2 a 5 anos. Os aminoácidos hidrofóbicos constituem 30 por cento do total de aminoácidos. Ao avaliar o efeito do hidrolisado na solubilização micelar do colesterol, foi observado que houve diferença (p < 0,004) devido ao processamento. O hidrolisado proteico da farinha crua (IPHc), com peptídeos de peso molecular maior que 3 kDa, reduziu a solubilização micelar do colesterol em 44,09 ± 1,5 por cento , enquanto que os hidrolisados de farinha tostada (IPHt) e extrusada (IPHe) reduziram em 31,24 ± 5,9 por cento e 24,97 ± 4,1 por cento . Já os hidrolisados com peso molecular menor que 3 kDa apresentaram pouca diferença (p < 0,03) em relação ao processamento. A redução da solubilidade micelar observada pelos IPHc e IPHe foi semelhante: 37,21 ± 1,65 por cento e 35,45 ± 0,4 por cento , respectivamente. O IPHt apresentou a menor redução de 22,47 ± 4,6 por cento . Os hidrolisados da farinha de amaranto também foram capazes de inibir a atividade da enzima HMGR em diversas concentrações. O controle da atividade normal da enzima apresentou 0,65 ± 0,05 µmol de NAPH oxidada min/mg equivalente de albumina. O IPHc, em concentrações de 0,1 e 0,5 mg/mL, apresentou efeito similar ao da pravastatina, diferindo do controle (p < 0,05), produzindo: 0,24 ± 0,03 e 0,29 ± 0,13 de µmol de NAPH oxidada min/mg equivalente de albumina. Por outro lado o IPHt apresentou efeito similar ao da pravastatina em concentração superior ao cru; em 1 mg/mL produziu 0,20 ± 0,09 de µmol de NAPH oxidada min/mg equivalente de albumina. O IPHe apresentou efeito inibidor da enzima em concentração de 0,1 mg/mL, porém menor do que o observado para a pravastatina. Conclusões: A proteína do grão de amaranto hidrolisada possui indícios de atividade hipocolesterolêmica. Sendo capaz de atuar tanto na via exógena quanto na via endógena, inibindo a absorção do colesterol e sua síntese de forma indireta. O processamento térmico diminuiu esta capacidade, mas ainda demonstra resultados significativos. Dentre os processamentos, a extrusão mostrou ter diminuído este efeito, embora os seus resultados possam ter sido influenciados pela quantidade de componentes no isolado proteico, como lipídeos e compostos fenólicos. / Introduction: Obesity and dyslipidemia are major contributors of cardiovascular diseases. The consumption of vegetables, especially their protein, acts protectively on the magnitude of these injuries. There is evidence that amaranth protein has a cholesterol-lowering effect by the action of peptides originating from its incomplete digestion. Objective: To assess the effect, in vitro, of the hydrolyzed protein of amaranth, submitted to different processes, on the reduction of the micellar solubilization of cholesterol and on the inhibition of HMGR enzyme activity. Methods: The raw and processed flours were analyzed for their content of amino acids. The isolated protein from amaranth grain flour toasted, extruded and raw, were subjected to enzymatic hydrolysis. Subsequently, it was prepared a solution of bile salts and cholesterol to assess the ability of the hydrolyzed protein to decrease the micellar solubilization of cholesterol. It was used the ultra-filtered peptides (MW up to 3 kDa) at concentration of 3 mg/mL equivalent albumin; and for higher molecular weights, it was used 10 mg/mL. In order to verify the mechanism of inhibition of the cholesterol endogenous synthesis, only it was used the hydrolyzed ultra-filtered peptides with MW < 3 KDa. In the assays of HMGR inhibition, several concentrations of peptides were used (0.1, 0.5 and 1 mg/mL) to compare the inhibition to pravastatin (a known inhibitor). Results: The amino acid composition showed to be adequate when compared to the recommendation of essential amino acids for children 2-5 years. Hydrophobic amino acids compose 30 per cent in total amino acids. When evaluating the effect of the hydrolyzate micellar solubilization of cholesterol has been observed that significant difference (p <0.004) from the processing. The peptides from raw flour hydrolyzed protein (IPHc) with a molecular weight greater than 3 kDa reduced micellar solubilization of cholesterol by 44.09 ± 1.5 per cent , while those from the roasted flour (IPHt) and extruded flour (IPHe) reduced by 31.24 ± 5.9 per cent and 24.97 ± 4.1 per cent . Peptides with a molecular weight up to 3 kDa showed little difference (p < 0.03) due to processing. The reduction of the observed micellar solubility of IPHc and IPHe were similar: 37.21 ± 0.4 per cent and 35.45 ± 1.65 per cent , respectively. The IPHt showed the smallest decrease of 22.47 ± 4.6 per cent . The peptides from amaranth flour were also able to inhibit the activity of the enzyme HMGR in various concentrations. The control of normal enzyme activity showed 0.65 ± 0.05 mol of NAPH oxidized min/mg equivalent of albumin. The IPHc at concentrations of 0.1 and 0.5 mg/mL had an effect similar to that of pravastatin, different from control (p < 0.05), yielding: 0.24 ± 0.03 and 0.29 ± 0.13 mol of the NAPH oxidized min/mg equivalent of albumin. On the other hand, IPHt showed a similar effect to higher concentration of pravastatin in the raw, and in 1 mg/mL produced from 0.20 ± 0.09 mol of oxidized NAPH min/mg equivalent of albumin. The IPHe showed the inhibitory effect on the enzyme concentration as lower as 0.1 mg/mL, but less pronounced than pravastatin. Conclusions: The peptides from hydrolysis of amaranth grain has evidence of hypocholesterolemic activity. They are able to act both in exogenously and endogenous pathways, inhibiting the absorption of cholesterol and its synthesis. The thermal processing reduces this capacity, but still shows significant results. Among the processing, extrusion deserves less attention than other one, although their results may have been influenced by the amount of the isolated protein components, such as lipids and phenolic compounds.

L'enzim HMG-CoA reductasa (HMGR) catalitza la primera etapa limitant en la síntesi d'isoprenoides citosòlics. En plantes, és un enzim de membrana i la seva primera destinació subcel·lular és el reticle endoplasmàtic. Estructuralment, està formada per un domini amino-terminal (que inclou una regió amino-terminal citosòlica i dos fragments transmembrana) i un domini catalític altament conservat en tota l'escala evolutiva. En totes les espècies de plantes conegudes fins al moment, existeixen isoformes de l'HMGR codificades per diferents gens. Concretament, en Arabidopsis thaliana el gen hmg1 (expressat de forma majoritària) codifica per a les isoformes HMGR1S i HMGR1L, i el gen hmg2 (expressat a arrels, plàntules i inflorescències) codifica per a la isoforma HMGR2. A nivell d'estructura primària l'HMGR1L difereix de l'HMGR1S per la presència d'una regió extra de 50 residus aminoacídics a l'extrem amino-terminal. El domini amino-terminal confereix diferents destinacions de localització subcel·lular a la proteïna. En un treball anterior, es van identificar tres proteïnes que interaccionen amb les isoformes derivades del gen hmg1: AtB"&#945;, AtB"&#946; i AtKLC-1. Les dues primeres interaccionen específicament amb la regió amino-terminal de les isoformes HMGR1S i HMGR1L. Les proteïnes AtB"&#945; i AtB"&#946; són isoformes de la subunitat B" reguladora del complex proteïna fosfatasa 2A (PP2A). En el genoma d'A. thaliana hi ha cinc seqüències que codifiquen per a isoformes de la subunitat B" que comparteixen una gran homologia. En l'estructura primària de la subunitat B" s'han identificat motius EF-Hand (implicats en la unió a calci). S'ha demostrat que tant AtB"&#945; com AtB"&#946; uneixen calci. La tercera proteïna identificada, AtKLC-1, interacciona específicament amb la regió amino-terminal de l'HMGR1L. S'ha determinat per assaigs de doble híbrid en llevat i, posteriorment confirmats in vitro, que la PR65, proteïna estructural del complex PP2A, interacciona específicament amb l'AtKLC-1. La regió de la PR65 suficient per a la interacció amb la subunitat B" reguladora, AtB"&#945;, i amb l'AtKLC-1 comprèn la mateixa seqüència aminoacídica. Per tant, l'AtB"&#945; i l'AtKLC-1 podrien competir per a l'associació amb la PR65. Així, en la cèl·lula, la PP2A podria regular d'una forma particular les isoformes HMGR1S i HMGR1L. Assaigs in vivo i in vitro demostren que la PP2A és un regulador negatiu de l'enzim HMGR. Els ions calci inhibeixen també l'activitat HMGR. Per a què es dugui a terme la repressió de l'activitat HMGR tant per calci com per PP2A, es requereix el domini amino-terminal de la proteïna. Per tant, els resultats són consistents amb què les subunitats AtB" i/o AtKLC-1 duen a terme un paper mediador en la modulació de l'HMGR per la PP2A. El domini amino-terminal de l'HMGR1S està implicat en la morfogènesi de vesícules derivades del reticle endoplasmàtic. La subunitat AtB"&#945; i la resta del complex PP2A participen en aquest procés. El domini amino-terminal de l'HMGR1L dirigeix la proteïna a la trama de reticle endoplasmàtic. La PP2A participa també en la localització subcel·lular d'aquesta isoforma.S'ha caracteritzat una línia mutant en el gen hmg1 d'A. thaliana que mostra absència del transcrit hmg1 i una reduïda activitat HMGR. Les plantes d'aquest mutant creixen normalment en un medi de cultiu estèril i manifesten severes alteracions del desenvolupament quan són cultivades en terra. L'addició de mevalonat, producte de la reacció catalitzada per l'HMGR, no reverteix el fenotip. Les dades indiquen que el gen hmg1 duu a terme una funció no metabòlica relacionada amb l'adaptació al medi. En aquestes condicions de creixement, les estructures vesiculars induïdes per l'HMGR1S podrien tenir alguna funció essencial relacionada amb la resposta a estrès. / "ROLE OF THE PROTEINS AtKLC-1 AND AtB" IN THE REGULATION OF HMG-CoA REDUCTASE IN ARABIDOPSIS THALIANA".TEXT: The enzyme HMG-CoA reductase (HMGR) catalyses the formation of mevalonate in the first rate-limiting step of the mevalonate pathway for isoprenoid biosynthesis. All known plant HMGR isoforms are primarily targeted to the endoplasmic reticulum (ER).These proteins contain two domains: an N-terminal domain (which includes an N-terminal cytosolic region and two membrane-spanning sequences) and a conserved catalytic domain. In all plant species, there are a variety of HMGR isoforms encoded by a multigene family. In Arabidopsis thaliana, two genes (hmg1 and hmg2) encode three HMGR isoforms (HMGR1S, HMGR1L and HMGR2). Isoforms HMGR1S and HMGR1L are identical in sequence but HMGR1L is extended 50 amino acid residues at the N-terminal end. In a previous study, three proteins had been identified which specifically interact with the HMGR1L N-terminal end. Two of these proteins, designed AtB"&#61537; and AtB"&#61538;, are regulatory B" subunits of protein phosphatase 2A (PP2A). They bind the N-terminal region of HMGR1S and HMGR1L. The third one, AtKLC-1, exclusively interact with the N-terminal region of HMGR1L. In vitro assays have revealed that PR65, PP2A scaffolding subunit, specifically interact with AtKLC-1. Genetic and pharmacological approaches demonstrate that PP2A exerts an inhibitory control over HMGR. These results indicate that AtB" or AtKLC-1 could have a mediating role in the HMGR regulation carried out by PP2A. The N-terminal domain of the HMGR1S isoform is involved in the biogenesis of vesicle structures derived from the ER membranes. The PP2A complex is concerned in this process and the subunit AtB"&#61537; acts as an interceding factor. Furthermore, we have characterized an Arabidopsis thaliana insertion mutant for hmg1. This mutant exhibits no visible phenotype under sterile growth conditions but shows dwarfing and sterility when it is grown in a soil substrate. The addition of mevalonate doesn't rescue this phenotype. These data suggest that hmg1 performs a no-metabolic function related with environment adaptation. In this context, ER derived vesicles induced by HMGR1S could play an essential role related with stress response.

Malaria är en farlig tropiksjukdom orsakad av parasiten Plasmodium som vållar många dödsfall varje år. Sedan några år tillbaka rekommenderar Världshälsoorganisationen (WHO) användandet av artemisinin och dess derivat för behandlandet av malaria. Artemisinin syntetiseras normalt i växten Artemisia annua i lågt utbyte. På grund av det låga utbytet är läkemedlet väldigt dyrt. Då parasiten blivit resistent mot de flesta malarialäkemedel är artemisinin ett viktigt preparat i kampen mot malaria. Forskning pågår för att hitta nya eller effektivare metoder för framställning av substansen då en oro finns att produktionen från A. annua inte kommer kunna möta kraven från läkemedelsindustrin. En av teorierna är ifall andra växter inom Artemisia-släktet kan syntetisera artemisinin då flera växter uppvisat helande effekter vid andra sjukdomar. I denna studie undersöktes det ifall växterna A. vulgaris och A. absinthium från Artemisia-släktet skulle kunna syntetisera artemisinin. Med hjälp av molekylärbiologiska tekniker isolerades genetiskt material ifrån växterna. Materialet granskades efter ribonukleinsyra (RNA)- och deoxiribonukleinsyra (DNA) -sekvenser för funktionella enzym som katalyserar reaktioner i artemisinins biosyntes. Ifall generna uttrycks för dessa enzym kan eventuellt artemisinin bildas. Växterna hämtades från Revsudden, Sverige och genetiskt material isolerades. Förekomsten av genuttryck för fem viktiga enzym i artemisinins biosyntes undersöktes med Polymerase Chain Reaction (PCR). Resultatet blev att växterna hade genuttryck för två respektive tre av de fem enzymen. Detta pekar mot att varken A. vulgaris eller A. absinthium kan syntetisera artemisinin då de saknade några viktiga nyckelenzym i syntesen. Trots att en tidigare studie indikerar närvaro av artemisinin i dessa växter kan slutsatsen dras att A. vulgaris och A. absinthium inte kan bilda artemisinin. / Malaria is a tropical disease that accounts for the death of many people annually and is caused by a parasite called Plasmodium. The World Health Organization (WHO) recommends artemisinin and its derivates for malaria treatment. Artemisinin is synthesized generally in Artemisia annua in small amounts. The artemisinin-treatment is very expensive due to the small amounts produced in the plant. Since the parasite has developed resistance towards many antimalarial drugs, artemisinin is an important drug against malaria. Research to find alternative methods for artemisinin-production has begun because there is a great concern that artemisinin-production at current rate will not meet the demand from the pharmaceutical industry. Some speculate if artemisinin can be synthesized in other plants from the Artemisia-genus since many plants have shown healing properties towards other diseases. In this study, we investigated if A. vulgaris and A. absinthium could produce artemisinin. Using molecular biology techniques, genetic material was isolated from the plants. Ribonucleotide (RNA)- and deoxyribonucleotide (DNA)- sequences which encode important enzymes in the artemisinin biosynthesis were examined. In case all the genes were expressed, artemisinin may be synthesized. The plants were picked on Revsudden, Sweden and genetic material was isolated. The presence of gene expression of five important enzymes in the artemisinin biosynthesis was investigated by Polymerase Chain Reaction (PCR). The results showed that the plants had gene expression of two respectively three of the five enzymes. Due to the fact that the plants need all five enzymes to synthesize artemisinin, even though a recent study has shown presence of artemisinin in these plants, this study concludes that artemisinin cannot be synthesized in A. vulgaris and A. absinthium.

3-Hydroxy-3-methylglutaryl CoA reductase (HMGR, EC 1.1.1.34) catalyzes the reaction from hydroxymethylglutaryl CoA to mevalonate in the isoprenoid pathway. In solanaceous plants, one class of endproducts of the pathway are sesquiterpenoid phytoalexins, antibiotic compounds produced by plants in response to pathogens. We are interested in the role of the defense-inducible isoforms of HMGR in phytoalexin production and disease resistance. Transgenic tobacco, constitutively expressing the defense-inducible tomato hmgr isogene, hmg2, showed fewer and smaller lesions following tobacco mosaic virus (TMV) inoculation. There is little evidence of phytoalexins acting directly against viruses, but they may reduce the spread of viruses as part of the hypersensitive response resulting in death of the host cell. Transmission electron microscopy of leaf cells of the transgenic plants revealed a larger volume of cytosol and accumulation of electron-dense inclusion bodies within the vacuoles. No structures resembling crystalloid ER or karmellae, caused by overexpression of hmgr in mammalian or yeast cells, respectively, were observed. Similar inclusion bodies were found in the vacuoles of wild-type tobacco leaf cells adjacent to necrotic cells in a TMV lesion. Tobacco expressing a truncated (membrane domain) form of hmg2 did not show enhanced resistance to TMV or any ultrastructural changes, indicating the importance of catalytically active HMG2 in mediating these changes. Sesquiterpene cyclase (a key branch point enzyme controlling sesquiterpene phytoalexin biosynthesis) was not induced and the amount of capsidiol, the tobacco phytoalexin, was not elevated by expression of hmg2. After TMV-inoculation, HMGR activity and the amount of capsidiol were higher in the wild-type than in the transgenic plants. Consequently, the enhanced resistance to TMV was not due to constitutive capsidiol production. The transgenic plants may have been able to produce sesquiterpenoid phytoalexins faster due to constitutive hmg2- expression and restricted the spread of the virus earlier, so that only a few cells were sacrificed. The subcellular localization of the defense-specific HMG2 isoform was determined by tagging tomato hmg2 with a c-myc epitope, and constitutively expressing the construct in transgenic tobacco plants. In non-induced leaves, MYC-HMG2 was found localized in small clusters associated with the ER. In TMV-inoculated leaves MYC-HMG2 co-localized with sesquiterpene cyclase to the vacuolar inclusion bodies suggesting that they may contain a defense-induced, membrane-associated multienzyme complex dedicated to sesquiterpene production. Our results support the hypothesis of the multibranched plant isoprenoid pathway being partly regulated by pathway partitioning. / Ph. D.

The rate limiting step in isoprenoid biosynthesis is catalyzed by 3-hydroxy-3-methylglutaryl CoA reductase (HMGR, EC 1.1.1.34). In plants, HMGR is encoded by small gene families whose members are differentially expressed. In tomato, hmg2 was previously isolated and sequenced. We report the isolation and sequence analysis of a clone (pCD4) encompassing exon I of tomato hmg1 which encodes the putative membrane domain. Sequence comparisons of plant HMGR proteins reveal two hydrophobic stretches within the amino terminus which are highly conserved among species. Using in vitro transcription and translation systems, the membrane domain structure of two tomato HMGR isoforms, HMG1 and HMG2, were analyzed. Results from these experiments reveal that tomato HMGRs are targeted to microsomal membranes in a cotranslational fashion that does not involve cleavage of an N-terminal targeting peptide. Membrane topography of HMGR was revealed by protease protection studies, indicating that both tomato HMGRs span the membrane two times such that both the C- and N-termini are located within the cytosol. HMG2 but not HMG1 was glycosylated in the in vitro system. Deletion of the hmg1 5' untranslated regions and sequences encoding the first six highly charged amino acids resulted in inefficient translation in vitro. However, targeting to microsomes was unchanged. HMG1 membrane domain was tagged with a FLAG epitope to facilitate in vivo studies. Agrobacterium-mediated transformation was used to introduce the tagged hmg1 gene into two Nicotiana tabacum cell lines, BY-2 and KY-14. The slow growth kinetics of KY-14 prevented effective recovery of transformed lines, however, Northern analyses of BY-2 showed that the hmg1 transgene was expressed. Comparisons of BY-2 and KY-14 revealed differences in defense responses to elicitor treatment. BY-2 cells showed minimal defense capabilities, whereas KY-14 cells were rapidly induced as indicated by increased HMGR enzyme activity and browning of the cells. HMGR enzyme activity was decreased in both KY-14 and BY-2 cells following sterol treatment, but the reduction was more pronounced in KY-14 cells. Thus transgenic BY-2 cells may be useful in future in vivo immunolocalization studies, but analyses of HMGR transcriptional regulation and regulated degradation will require use of the more responsive KY-14 cells.. / Ph. D.

3-Hydroxy-3-methylglutaryl CoA reductase (HMGR, EC 1.1.1.34) mediates the first regulatory step (HMG-CoA reduction to mevalonate) in isoprenoid biosynthesis. The tomato genome contains at least four differentially regulated hmg isogenes encoding HMGR. Functions of tomato hmg2 in defense responses were studied by promoter analyses of hmg2:GUS gene fusions, overexpression of hmg2 cDNA, and antisense inhibition of hmg1 and hmg2 in transgenic plants. Activity of the hmg2 promoter is developmentally regulated showing expression in seedling cotyledons and hypocotyls, in trichomes, and in reproductive tissues including pollen, stigmas, ovules, petals and mature seeds. hmg2:GUS activity is rapidly induced by wounding or in response to pathogenic viruses or bacteria. hmg2:GUS expression is localized to tissue surrounding lesions generated through interactions with either TMV or the bacterial pathogen, Erwinia carotovora subsp. carotovora (Ecc). Tomato hmg2 cDNA was cloned by PCR, expressed in E. coli to confirm its HMGR activity, inserted behind the double enhanced CaMV 35S promoter, and engineered into tobacco. Southern and northern analyses confirmed transformation and message expression. Enzyme activity was enhanced compared to nontransformed plants. Selected transgenic plants were significantly reduced for Ecc tissue maceration. The size of necrotic lesions induced by TMV was also significantly reduced compared to the nontransformed or vector controls. Thus, genetic manipulation of the rate-limiting step in a major defense pathway provides a novel strategy for enhancing disease resistance. We also generated transgenic tobacco and tomato containing antisense constructs for tomato hmg1 and hmg2 to study their effect on disease resistance. Full-length hmg2 and 5' regions of hmg1 or hmg2 were inserted in the antisense orientation behind a 35S promoter. Tomato expressing the full-length hmg2 antisense showed lower HMGR enzyme activity and were more susceptible to soft rot by Ecc than control plants. In contrast, expression of either antisense hmg/ or antisense hmg2 in the heterologous tobacco system resulted in plants with enhanced resistance to Ecc and reduced TMV lesion sizes. These results may indicate that antisense inhibition is non-specifically exerted on isogenes other than the defense-specific HMGR gene. / Ph. D.