Claudia Stange - Academia.edu (original) (raw)
Papers by Claudia Stange
Plants
Plant carotenoids are synthesized and accumulated in plastids through a highly regulated pathway.... more Plant carotenoids are synthesized and accumulated in plastids through a highly regulated pathway. Lycopene β-cyclase (LCYB) is a key enzyme involved directly in the synthesis of α-carotene and β-carotene through the cyclization of trans-lycopene. Daucus carota harbors two LCYB genes, of which DcLCYB2 (annotated as CCS-Like) is mostly expressed in mature storage roots, an organ that accumulates high α-carotene and β-carotene content. In this work, we determined that DcLCYB2 of the orange Nantes variety presents plastid localization and encodes for a functional LCYB enzyme determined by means of heterologous complementation in Escherichia coli. Also, ectopic expression of DcLCYB2 in tobacco (Nicotiana tabacum) and kiwi (Actinidia deliciosa) plants increases total carotenoid content showing its functional role in plants. In addition, transgenic tobacco T2 homozygous plants showed better performance under chronic salt treatment, while kiwi transgenic calli also presented a higher surviv...
Daucus carota DcPSY2 and DcLCYB1 as Tools for Carotenoid Metabolic Engineering to Improve the Nutritional Value of Fruits
Frontiers in Plant Science
Carotenoids are pigments with important nutritional value in the human diet. As antioxidant molec... more Carotenoids are pigments with important nutritional value in the human diet. As antioxidant molecules, they act as scavengers of free radicals enhancing immunity and preventing cancer and cardiovascular diseases. Moreover, α-carotene and β-carotene, the main carotenoids of carrots (Daucus carota) are precursors of vitamin A, whose deficiency in the diet can trigger night blindness and macular degeneration. With the aim of increasing the carotenoid content in fruit flesh, three key genes of the carotenoid pathway, phytoene synthase (DcPSY2) and lycopene cyclase (DcLCYB1) from carrots, and carotene desaturase (XdCrtI) from the yeast Xanthophyllomyces dendrorhous, were optimized for expression in apple and cloned under the Solanum chilense (tomatillo) polygalacturonase (PG) fruit specific promoter. A biotechnological platform was generated and functionally tested by subcellular localization, and single, double and triple combinations were both stably transformed in tomatoes (Solanum ly...
Agrobacterium tumefaciens-Mediated Stable Transformation of Daucus carota
Methods in Molecular Biology
International Journal of Molecular Sciences
Light is an important cue that stimulates both plastid development and biosynthesis of carotenoid... more Light is an important cue that stimulates both plastid development and biosynthesis of carotenoids in plants. During photomorphogenesis or de-etiolation, photoreceptors are activated and molecular factors for carotenoid and chlorophyll biosynthesis are induced thereof. In fruits, light is absorbed by chloroplasts in the early stages of ripening, which allows a gradual synthesis of carotenoids in the peel and pulp with the onset of chromoplasts’ development. In roots, only a fraction of light reaches this tissue, which is not required for carotenoid synthesis, but it is essential for root development. When exposed to light, roots start greening due to chloroplast development. However, the colored taproot of carrot grown underground presents a high carotenoid accumulation together with chromoplast development, similar to citrus fruits during ripening. Interestingly, total carotenoid levels decrease in carrots roots when illuminated and develop chloroplasts, similar to normal roots exp...
Plants
Reductions in crop yields brought about by abiotic stress are expected to increase as climate cha... more Reductions in crop yields brought about by abiotic stress are expected to increase as climate change, and other factors, generate harsher environmental conditions in regions traditionally used for cultivation. Although breeding and genetically modified and edited organisms have generated many varieties with greater abiotic stress tolerance, their practical use depends on lengthy processes, such as biological cycles and legal aspects. On the other hand, a non-genetic approach to improve crop yield in stress conditions involves the exogenous application of natural compounds, including plant metabolites. In this review, we examine the recent literature related to the application of different natural primary (proline, l-tryptophan, glutathione, and citric acid) and secondary (polyols, ascorbic acid, lipoic acid, glycine betaine, α-tocopherol, and melatonin) plant metabolites in improving tolerance to abiotic stress. We focus on drought, saline, heavy metal, and temperature as environmen...
A de novo transcriptome analysis revealed that photomorphogenic genes are required for carotenoid synthesis in the dark-grown carrot taproot
Molecular Genetics and Genomics
Boosting carotenoid content in Malus domestica var. Fuji by expressing AtDXR through an Agrobacterium ‐mediated transformation method
Biotechnology and Bioengineering
Differential role of the two ζ-carotene desaturase paralogs in carrot (Daucus carota): ZDS1 is a functional gene essential for plant development and carotenoid synthesis
Plant Science
AtA6PR1 and AtA6PR2 encode putative aldose 6-phosphate reductases that are cytosolically localized and respond differentially to cold and salt stress in Arabidopsis thaliana
Journal of Plant Biochemistry and Biotechnology
Ciencia e investigación agraria
C. Stange. 2006. Plant-virus interactions during the infective process. Cien. Inv. Agr. 33(1): 3-... more C. Stange. 2006. Plant-virus interactions during the infective process. Cien. Inv. Agr. 33(1): 3-21. Viruses that infect plants are generally single-stranded (ss) positive-sense RNA viruses. The accumulation of the virus progeny inside the plant cells involves translation, replication, cell-to-cell and long-distance movement of viral sequences. Over the past 30 years high progress has been made in understanding the interactions between the virus and the host plant during these processes. Reports of host factors implicated in promoting viral cycle and the characterization of plant virus receptors (R) and their resistance mechanisms in Solanaceae, Cucurbitaceae, Leguminoseae and in Arabidopsis thaliana have contributed extensively to understanding this complex interaction. Almost all of the R genes cloned share structural similarity, harbouring LRR, NBS, TIR and LZ domains, suggesting a convergence in the signal transduction machinery in plant defence. Plant viruses evolve very rapidly. This is possible because of their very short replication cycles, large numbers of genomes within each cell and across many cells per host, and many hosts infected. Therefore, viruses readily produce new avirulence factors and resistancebreaking viral genotypes. To overcome the appearance of new viral races, plants generate R gene variants through recombination processes and develop specialized defence mechanisms such as post-transcriptional gene silencing. However, viruses such as Potyvirus X can overcome this type of plant resistance. Recent insights into virus-host interactions have been compiled in this review, focusing on the interaction between Tobacco mosaic virus and the N receptor in Nicotiana tabacum, to describe the possible transduction mechanisms that trigger a cascade of downstream events leading to viral defence in plants.
PLOS ONE
Increasing β-carotene (a vitamin A precursor) content in Triticum turgidum L. ssp. durum (durum w... more Increasing β-carotene (a vitamin A precursor) content in Triticum turgidum L. ssp. durum (durum wheat) grains is important to improve pasta nutritional quality. Studies in other species show that altering the expression of LCYE genes increases the flux towards the β-β branch, accumulating higher β-carotene levels. Durum wheat is a tetraploid species that has two LCYE genes (LCYE-A and LCYE-B) associated to the A and B genomes. The objective of this work was to produce durum wheat LCYE mutants through EMS to potentially increase β-carotene content. The LCYE point mutations created with EMS were identified using a Kronos TILLING (Targeting Induced Local Lesion IN Genomes) mutant population. Specific primers that amplified exons 3 through 10 of the LCYE genes were designed and validated. To simplify the TILLING procedure, fragments were digested with CJE (Celery Juice Extract) and visualized on 2% agarose gels. 6X mutant pools were identified, which showed cleavage products and then made into 2X pools to identify mutant individuals. LCYE mutants were then sequenced and evaluated with BLOSUM62, SIFT and PSSM algorithms. Mutants with substitutions W437*, P334L and G368R in LCYE-A and P405L, G352R and T393I in LCYE-B predicted to affect protein function were selected. Substitution W437* increased β-carotene in 75% and overall total carotenoids content in leaves of the mutant 2426 (A1 mutant line), but no significant differences relative to the control were found in grains through HPLC. Finally, the increased levels of β-carotene on leaves have potential applications to improving plant resistance under contaminated environmental conditions.
Unraveling the induction of phytoene synthase 2 expression by salt stress and abscisic acid in Daucus carota
Journal of experimental botany, Jan 18, 2018
Phytoene synthase (PSY) is the first committed enzyme of the carotenoid biosynthesis pathway and ... more Phytoene synthase (PSY) is the first committed enzyme of the carotenoid biosynthesis pathway and the most important point of regulation. Carotenoids are precursors of abscisic acid (ABA), which mediates abiotic stress tolerance responses in plants. ABA activates the synthesis of its own precursors through induction of PSY expression. Carrot, a species that accumulates very high amounts of carotenoids in its reserve root, has two PSY paralog genes that are expressed differentially in the root. Here, we determined that DcPSY2 expression is induced by salt stress and ABA. A DcPSY2 promoter fragment was obtained and characterized. Bioinformatic analysis showed the presence of three ABA responsive elements (ABREs). Through overexpressing pPSY2:GFP in Nicotiana tabacum we determined that all three ABREs are necessary for the ABA response. In the carrot transcriptome, we identified three ABRE binding protein (DcAREB) transcription factor candidates that localized in the nucleus, but only o...
Illuminating colors: regulation of carotenoid biosynthesis and accumulation by light
Current opinion in plant biology, Jun 1, 2017
Light stimulates the biosynthesis of carotenoids and regulates the development of plastid structu... more Light stimulates the biosynthesis of carotenoids and regulates the development of plastid structures to accommodate these photoprotective pigments. Work with Arabidopsis revealed molecular factors coordinating carotenoid biosynthesis and storage with photosynthetic development during deetiolation, when underground seedlings emerge to the light. Some of these factors also adjust carotenoid biosynthesis in response to plant proximity (i.e., shade), a mechanism that was readapted in tomato to monitor fruit ripening progression. While light positively impacts carotenoid production and accumulation in most cases, total carotenoid levels decrease in roots of colored carrot cultivars when illuminated. The recent discovery that such cultivars might be photomorphogenic mutants provides an explanation for this striking phenotype.
Frontiers in Plant Science, 2016
Carotenoids and chlorophylls are photosynthetic pigments synthesized in plastids from metabolic p... more Carotenoids and chlorophylls are photosynthetic pigments synthesized in plastids from metabolic precursors provided by the methylerythritol 4-phosphate (MEP) pathway. The first two steps in the MEP pathway are catalyzed by the deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR) enzymes. While DXS has been recently shown to be the main flux-controlling step of the MEP pathway, both DXS and DXR enzymes have been proven to be able to promote an increase in MEP-derived products when overproduced in diverse plant systems. Carrot (Daucus carota) produces photosynthetic pigments (carotenoids and chlorophylls) in leaves and in light-exposed roots, whereas only carotenoids (mainly αand β-carotene) accumulate in the storage root in darkness. To evaluate whether DXS and DXR activities influence the production of carotenoids and chlorophylls in carrot leaves and roots, the corresponding Arabidopsis thaliana genes were constitutively expressed in transgenic carrot plants. Our results suggest that DXS is limiting for the production of both carotenoids and chlorophylls in roots and leaves, whereas the regulatory role of DXR appeared to be minor. Interestingly, increased levels of DXS (but not of DXR) resulted in higher transcript abundance of endogenous carrot genes encoding phytoene synthase, the main rate-determining enzyme of the carotenoid pathway. These results support a central role for DXS on modulating the production of MEP-derived precursors to synthesize carotenoids and chlorophylls in carrot, confirming the pivotal relevance of this enzyme to engineer healthier, carotenoid-enriched products.
Carotenoid Biosynthesis in Daucus carota
Subcellular Biochemistry, 2016
Carrot (Daucus carota) is one of the most important vegetable cultivated worldwide and the main s... more Carrot (Daucus carota) is one of the most important vegetable cultivated worldwide and the main source of dietary provitamin A. Contrary to other plants, almost all carrot varieties accumulate massive amounts of carotenoids in the root, resulting in a wide variety of colors, including those with purple, yellow, white, red and orange roots. During the first weeks of development the root, grown in darkness, is thin and pale and devoid of carotenoids. At the second month, the thickening of the root and the accumulation of carotenoids begins, and it reaches its highest level at 3 months of development. This normal root thickening and carotenoid accumulation can be completely altered when roots are grown in light, in which chromoplasts differentiation is redirected to chloroplasts development in accordance with an altered carotenoid profile. Here we discuss the current evidence on the biosynthesis of carotenoid in carrot roots in response to environmental cues that has contributed to our understanding of the mechanism that regulates the accumulation of carotenoids, as well as the carotenogenic gene expression and root development in D. carota.
Biosynthesis of Carotenoids in Plants: Enzymes and Color
Subcellular Biochemistry, 2016
Apocarotenoids: A New Carotenoid-Derived Pathway
Subcellular Biochemistry, 2016
Carotenoids are precursors of carotenoid derived molecules termed apocarotenoids, which include i... more Carotenoids are precursors of carotenoid derived molecules termed apocarotenoids, which include isoprenoids with important functions in plant-environment interactions such as the attraction of pollinators and the defense against pathogens and herbivores. Apocarotenoids also include volatile aromatic compounds that act as repellents, chemoattractants, growth simulators and inhibitors, as well as the phytohormones abscisic acid and strigolactones. In plants, apocarotenoids can be found in several types of plastids (etioplast, leucoplast and chromoplast) and among different plant tissues such as flowers and roots. The structural similarity of some flower and spice isoprenoid volatile organic compounds (β-ionone and safranal) to carotenoids has led to the recent discovery of carotenoid-specific cleavage oxygenases, including carotenoid cleavage dioxygenases and 9-cis-epoxydioxygenases, which tailor and transform carotenoids into apocarotenoids. The great diversity of apocarotenoids is a consequence of the huge amount of carotenoid precursors, the variations in specific cleavage sites and the modifications after cleavage. Lycopene, β-carotene and zeaxanthin are the precursors of the main apocarotenoids described to date, which include bixin, crocin, picrocrocin, abscisic acid, strigolactone and mycorradicin.The current chapter will give rise to an overview of the biosynthesis and function of the most important apocarotenoids in plants, as well as the current knowledge about the carotenoid cleavage oxygenase enzymes involved in these biosynthetic pathways.
Molecules and Cells, Jul 1, 2005
When inoculated into sensitive tobacco Xanthi-nn plants, the crucifer and garlic-infecting Tobacc... more When inoculated into sensitive tobacco Xanthi-nn plants, the crucifer and garlic-infecting Tobacco mosaic virus (TMV-Cg) induces local necrotic lesions that resemble those seen in the hypersensitive response (HR) of resistant tobacco plants. However, unlike these, tobacco Xanthi-nn plants do not become resistant to infection and the virus spreads systemically causing a severe disease characterized by necrotic lesions throughout the plant. To identify the viral protein that elicits this necrotic response, we used a set of hybrid viruses constructed by combination of TMV-Cg and the tobacco mosaic virus strain U1 (TMV-U1). In this study we present evidence that the coat protein of TMV-Cg (CPCg) is the elicitor of the necrotic response in tobacco Xanthi-nn plants. Local and systemic necrotic lesions induced by TMV-Cg and by the hybrid U1-CPCg-that carries CPCg in a TMV-U1 context-are characterized by cell death and by the presence of autoflorescent phenolic compounds and H 2 O 2 , just like the HR lesions. In addition, defense-related genes and detoxifying genes are induced in tobacco Xanthi-nn plants after TMV-Cg and U1-CPCg inoculation. We postulate that in our system, CPCg is recognized by sensitive tobacco plants that mount an incomplete defense response. We call this an HR-like since it is not enough to induce plant resistance.
C. Stange. 2006. Interacción planta-virus durante el proceso infectivo. Cien. Inv. Agr. 33(1):3-2... more C. Stange. 2006. Interacción planta-virus durante el proceso infectivo. Cien. Inv. Agr. 33(1):3-21. Los virus que infectan plantas son generalmente de tipo DNA o RNA de cadena simple y positiva. El ciclo viral se inicia al penetrar el virus en la célula hospedera. Este comienza con el desensamblaje, replicación del RNA, traducción de proteínas, ensamble, liberación, movimiento de célula a célula y a larga distancia. El conocimiento de los mecanismos de interacción entre la planta hospedera y el virus, ha progresado considerablemente en los últimos treinta años. Por ejemplo, se ha determinado la participación de componentes del citoesqueleto y de proteínas del hospedero en movimiento local (célula a célula) y a larga distancia (movimiento sistémico) de los virus en las plantas. Además, se han caracterizado numerosos receptores virales codificados por genes de resistencia (R) y se ha determinado el mecanismo de defensa en Arabidopsis thaliana y en especies de las familias Solanaceae, ...
Molecules and cells, Jan 30, 2005
When inoculated into sensitive tobacco Xanthi-nn plants, the crucifer and garlic-infecting Tobacc... more When inoculated into sensitive tobacco Xanthi-nn plants, the crucifer and garlic-infecting Tobacco mosaic virus (TMV-Cg) induces local necrotic lesions that resemble those seen in the hypersensitive response (HR) of resistant tobacco plants. However, unlike these, tobacco Xanthi-nn plants do not become resistant to infection and the virus spreads systemically causing a severe disease characterized by necrotic lesions throughout the plant. To identify the viral protein that elicits this necrotic response, we used a set of hybrid viruses constructed by combination of TMV-Cg and the tobacco mosaic virus strain U1 (TMV-U1). In this study we present evidence that the coat protein of TMV-Cg (CPCg) is the elicitor of the necrotic response in tobacco Xanthi-nn plants. Local and systemic necrotic lesions induced by TMV-Cg and by the hybrid U1-CPCg -that carries CPCg in a TMV-U1 context- are characterized by cell death and by the presence of autoflorescent phenolic compounds and H2O2, just li...
Plants
Plant carotenoids are synthesized and accumulated in plastids through a highly regulated pathway.... more Plant carotenoids are synthesized and accumulated in plastids through a highly regulated pathway. Lycopene β-cyclase (LCYB) is a key enzyme involved directly in the synthesis of α-carotene and β-carotene through the cyclization of trans-lycopene. Daucus carota harbors two LCYB genes, of which DcLCYB2 (annotated as CCS-Like) is mostly expressed in mature storage roots, an organ that accumulates high α-carotene and β-carotene content. In this work, we determined that DcLCYB2 of the orange Nantes variety presents plastid localization and encodes for a functional LCYB enzyme determined by means of heterologous complementation in Escherichia coli. Also, ectopic expression of DcLCYB2 in tobacco (Nicotiana tabacum) and kiwi (Actinidia deliciosa) plants increases total carotenoid content showing its functional role in plants. In addition, transgenic tobacco T2 homozygous plants showed better performance under chronic salt treatment, while kiwi transgenic calli also presented a higher surviv...
Daucus carota DcPSY2 and DcLCYB1 as Tools for Carotenoid Metabolic Engineering to Improve the Nutritional Value of Fruits
Frontiers in Plant Science
Carotenoids are pigments with important nutritional value in the human diet. As antioxidant molec... more Carotenoids are pigments with important nutritional value in the human diet. As antioxidant molecules, they act as scavengers of free radicals enhancing immunity and preventing cancer and cardiovascular diseases. Moreover, α-carotene and β-carotene, the main carotenoids of carrots (Daucus carota) are precursors of vitamin A, whose deficiency in the diet can trigger night blindness and macular degeneration. With the aim of increasing the carotenoid content in fruit flesh, three key genes of the carotenoid pathway, phytoene synthase (DcPSY2) and lycopene cyclase (DcLCYB1) from carrots, and carotene desaturase (XdCrtI) from the yeast Xanthophyllomyces dendrorhous, were optimized for expression in apple and cloned under the Solanum chilense (tomatillo) polygalacturonase (PG) fruit specific promoter. A biotechnological platform was generated and functionally tested by subcellular localization, and single, double and triple combinations were both stably transformed in tomatoes (Solanum ly...
Agrobacterium tumefaciens-Mediated Stable Transformation of Daucus carota
Methods in Molecular Biology
International Journal of Molecular Sciences
Light is an important cue that stimulates both plastid development and biosynthesis of carotenoid... more Light is an important cue that stimulates both plastid development and biosynthesis of carotenoids in plants. During photomorphogenesis or de-etiolation, photoreceptors are activated and molecular factors for carotenoid and chlorophyll biosynthesis are induced thereof. In fruits, light is absorbed by chloroplasts in the early stages of ripening, which allows a gradual synthesis of carotenoids in the peel and pulp with the onset of chromoplasts’ development. In roots, only a fraction of light reaches this tissue, which is not required for carotenoid synthesis, but it is essential for root development. When exposed to light, roots start greening due to chloroplast development. However, the colored taproot of carrot grown underground presents a high carotenoid accumulation together with chromoplast development, similar to citrus fruits during ripening. Interestingly, total carotenoid levels decrease in carrots roots when illuminated and develop chloroplasts, similar to normal roots exp...
Plants
Reductions in crop yields brought about by abiotic stress are expected to increase as climate cha... more Reductions in crop yields brought about by abiotic stress are expected to increase as climate change, and other factors, generate harsher environmental conditions in regions traditionally used for cultivation. Although breeding and genetically modified and edited organisms have generated many varieties with greater abiotic stress tolerance, their practical use depends on lengthy processes, such as biological cycles and legal aspects. On the other hand, a non-genetic approach to improve crop yield in stress conditions involves the exogenous application of natural compounds, including plant metabolites. In this review, we examine the recent literature related to the application of different natural primary (proline, l-tryptophan, glutathione, and citric acid) and secondary (polyols, ascorbic acid, lipoic acid, glycine betaine, α-tocopherol, and melatonin) plant metabolites in improving tolerance to abiotic stress. We focus on drought, saline, heavy metal, and temperature as environmen...
A de novo transcriptome analysis revealed that photomorphogenic genes are required for carotenoid synthesis in the dark-grown carrot taproot
Molecular Genetics and Genomics
Boosting carotenoid content in Malus domestica var. Fuji by expressing AtDXR through an Agrobacterium ‐mediated transformation method
Biotechnology and Bioengineering
Differential role of the two ζ-carotene desaturase paralogs in carrot (Daucus carota): ZDS1 is a functional gene essential for plant development and carotenoid synthesis
Plant Science
AtA6PR1 and AtA6PR2 encode putative aldose 6-phosphate reductases that are cytosolically localized and respond differentially to cold and salt stress in Arabidopsis thaliana
Journal of Plant Biochemistry and Biotechnology
Ciencia e investigación agraria
C. Stange. 2006. Plant-virus interactions during the infective process. Cien. Inv. Agr. 33(1): 3-... more C. Stange. 2006. Plant-virus interactions during the infective process. Cien. Inv. Agr. 33(1): 3-21. Viruses that infect plants are generally single-stranded (ss) positive-sense RNA viruses. The accumulation of the virus progeny inside the plant cells involves translation, replication, cell-to-cell and long-distance movement of viral sequences. Over the past 30 years high progress has been made in understanding the interactions between the virus and the host plant during these processes. Reports of host factors implicated in promoting viral cycle and the characterization of plant virus receptors (R) and their resistance mechanisms in Solanaceae, Cucurbitaceae, Leguminoseae and in Arabidopsis thaliana have contributed extensively to understanding this complex interaction. Almost all of the R genes cloned share structural similarity, harbouring LRR, NBS, TIR and LZ domains, suggesting a convergence in the signal transduction machinery in plant defence. Plant viruses evolve very rapidly. This is possible because of their very short replication cycles, large numbers of genomes within each cell and across many cells per host, and many hosts infected. Therefore, viruses readily produce new avirulence factors and resistancebreaking viral genotypes. To overcome the appearance of new viral races, plants generate R gene variants through recombination processes and develop specialized defence mechanisms such as post-transcriptional gene silencing. However, viruses such as Potyvirus X can overcome this type of plant resistance. Recent insights into virus-host interactions have been compiled in this review, focusing on the interaction between Tobacco mosaic virus and the N receptor in Nicotiana tabacum, to describe the possible transduction mechanisms that trigger a cascade of downstream events leading to viral defence in plants.
PLOS ONE
Increasing β-carotene (a vitamin A precursor) content in Triticum turgidum L. ssp. durum (durum w... more Increasing β-carotene (a vitamin A precursor) content in Triticum turgidum L. ssp. durum (durum wheat) grains is important to improve pasta nutritional quality. Studies in other species show that altering the expression of LCYE genes increases the flux towards the β-β branch, accumulating higher β-carotene levels. Durum wheat is a tetraploid species that has two LCYE genes (LCYE-A and LCYE-B) associated to the A and B genomes. The objective of this work was to produce durum wheat LCYE mutants through EMS to potentially increase β-carotene content. The LCYE point mutations created with EMS were identified using a Kronos TILLING (Targeting Induced Local Lesion IN Genomes) mutant population. Specific primers that amplified exons 3 through 10 of the LCYE genes were designed and validated. To simplify the TILLING procedure, fragments were digested with CJE (Celery Juice Extract) and visualized on 2% agarose gels. 6X mutant pools were identified, which showed cleavage products and then made into 2X pools to identify mutant individuals. LCYE mutants were then sequenced and evaluated with BLOSUM62, SIFT and PSSM algorithms. Mutants with substitutions W437*, P334L and G368R in LCYE-A and P405L, G352R and T393I in LCYE-B predicted to affect protein function were selected. Substitution W437* increased β-carotene in 75% and overall total carotenoids content in leaves of the mutant 2426 (A1 mutant line), but no significant differences relative to the control were found in grains through HPLC. Finally, the increased levels of β-carotene on leaves have potential applications to improving plant resistance under contaminated environmental conditions.
Unraveling the induction of phytoene synthase 2 expression by salt stress and abscisic acid in Daucus carota
Journal of experimental botany, Jan 18, 2018
Phytoene synthase (PSY) is the first committed enzyme of the carotenoid biosynthesis pathway and ... more Phytoene synthase (PSY) is the first committed enzyme of the carotenoid biosynthesis pathway and the most important point of regulation. Carotenoids are precursors of abscisic acid (ABA), which mediates abiotic stress tolerance responses in plants. ABA activates the synthesis of its own precursors through induction of PSY expression. Carrot, a species that accumulates very high amounts of carotenoids in its reserve root, has two PSY paralog genes that are expressed differentially in the root. Here, we determined that DcPSY2 expression is induced by salt stress and ABA. A DcPSY2 promoter fragment was obtained and characterized. Bioinformatic analysis showed the presence of three ABA responsive elements (ABREs). Through overexpressing pPSY2:GFP in Nicotiana tabacum we determined that all three ABREs are necessary for the ABA response. In the carrot transcriptome, we identified three ABRE binding protein (DcAREB) transcription factor candidates that localized in the nucleus, but only o...
Illuminating colors: regulation of carotenoid biosynthesis and accumulation by light
Current opinion in plant biology, Jun 1, 2017
Light stimulates the biosynthesis of carotenoids and regulates the development of plastid structu... more Light stimulates the biosynthesis of carotenoids and regulates the development of plastid structures to accommodate these photoprotective pigments. Work with Arabidopsis revealed molecular factors coordinating carotenoid biosynthesis and storage with photosynthetic development during deetiolation, when underground seedlings emerge to the light. Some of these factors also adjust carotenoid biosynthesis in response to plant proximity (i.e., shade), a mechanism that was readapted in tomato to monitor fruit ripening progression. While light positively impacts carotenoid production and accumulation in most cases, total carotenoid levels decrease in roots of colored carrot cultivars when illuminated. The recent discovery that such cultivars might be photomorphogenic mutants provides an explanation for this striking phenotype.
Frontiers in Plant Science, 2016
Carotenoids and chlorophylls are photosynthetic pigments synthesized in plastids from metabolic p... more Carotenoids and chlorophylls are photosynthetic pigments synthesized in plastids from metabolic precursors provided by the methylerythritol 4-phosphate (MEP) pathway. The first two steps in the MEP pathway are catalyzed by the deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR) enzymes. While DXS has been recently shown to be the main flux-controlling step of the MEP pathway, both DXS and DXR enzymes have been proven to be able to promote an increase in MEP-derived products when overproduced in diverse plant systems. Carrot (Daucus carota) produces photosynthetic pigments (carotenoids and chlorophylls) in leaves and in light-exposed roots, whereas only carotenoids (mainly αand β-carotene) accumulate in the storage root in darkness. To evaluate whether DXS and DXR activities influence the production of carotenoids and chlorophylls in carrot leaves and roots, the corresponding Arabidopsis thaliana genes were constitutively expressed in transgenic carrot plants. Our results suggest that DXS is limiting for the production of both carotenoids and chlorophylls in roots and leaves, whereas the regulatory role of DXR appeared to be minor. Interestingly, increased levels of DXS (but not of DXR) resulted in higher transcript abundance of endogenous carrot genes encoding phytoene synthase, the main rate-determining enzyme of the carotenoid pathway. These results support a central role for DXS on modulating the production of MEP-derived precursors to synthesize carotenoids and chlorophylls in carrot, confirming the pivotal relevance of this enzyme to engineer healthier, carotenoid-enriched products.
Carotenoid Biosynthesis in Daucus carota
Subcellular Biochemistry, 2016
Carrot (Daucus carota) is one of the most important vegetable cultivated worldwide and the main s... more Carrot (Daucus carota) is one of the most important vegetable cultivated worldwide and the main source of dietary provitamin A. Contrary to other plants, almost all carrot varieties accumulate massive amounts of carotenoids in the root, resulting in a wide variety of colors, including those with purple, yellow, white, red and orange roots. During the first weeks of development the root, grown in darkness, is thin and pale and devoid of carotenoids. At the second month, the thickening of the root and the accumulation of carotenoids begins, and it reaches its highest level at 3 months of development. This normal root thickening and carotenoid accumulation can be completely altered when roots are grown in light, in which chromoplasts differentiation is redirected to chloroplasts development in accordance with an altered carotenoid profile. Here we discuss the current evidence on the biosynthesis of carotenoid in carrot roots in response to environmental cues that has contributed to our understanding of the mechanism that regulates the accumulation of carotenoids, as well as the carotenogenic gene expression and root development in D. carota.
Biosynthesis of Carotenoids in Plants: Enzymes and Color
Subcellular Biochemistry, 2016
Apocarotenoids: A New Carotenoid-Derived Pathway
Subcellular Biochemistry, 2016
Carotenoids are precursors of carotenoid derived molecules termed apocarotenoids, which include i... more Carotenoids are precursors of carotenoid derived molecules termed apocarotenoids, which include isoprenoids with important functions in plant-environment interactions such as the attraction of pollinators and the defense against pathogens and herbivores. Apocarotenoids also include volatile aromatic compounds that act as repellents, chemoattractants, growth simulators and inhibitors, as well as the phytohormones abscisic acid and strigolactones. In plants, apocarotenoids can be found in several types of plastids (etioplast, leucoplast and chromoplast) and among different plant tissues such as flowers and roots. The structural similarity of some flower and spice isoprenoid volatile organic compounds (β-ionone and safranal) to carotenoids has led to the recent discovery of carotenoid-specific cleavage oxygenases, including carotenoid cleavage dioxygenases and 9-cis-epoxydioxygenases, which tailor and transform carotenoids into apocarotenoids. The great diversity of apocarotenoids is a consequence of the huge amount of carotenoid precursors, the variations in specific cleavage sites and the modifications after cleavage. Lycopene, β-carotene and zeaxanthin are the precursors of the main apocarotenoids described to date, which include bixin, crocin, picrocrocin, abscisic acid, strigolactone and mycorradicin.The current chapter will give rise to an overview of the biosynthesis and function of the most important apocarotenoids in plants, as well as the current knowledge about the carotenoid cleavage oxygenase enzymes involved in these biosynthetic pathways.
Molecules and Cells, Jul 1, 2005
When inoculated into sensitive tobacco Xanthi-nn plants, the crucifer and garlic-infecting Tobacc... more When inoculated into sensitive tobacco Xanthi-nn plants, the crucifer and garlic-infecting Tobacco mosaic virus (TMV-Cg) induces local necrotic lesions that resemble those seen in the hypersensitive response (HR) of resistant tobacco plants. However, unlike these, tobacco Xanthi-nn plants do not become resistant to infection and the virus spreads systemically causing a severe disease characterized by necrotic lesions throughout the plant. To identify the viral protein that elicits this necrotic response, we used a set of hybrid viruses constructed by combination of TMV-Cg and the tobacco mosaic virus strain U1 (TMV-U1). In this study we present evidence that the coat protein of TMV-Cg (CPCg) is the elicitor of the necrotic response in tobacco Xanthi-nn plants. Local and systemic necrotic lesions induced by TMV-Cg and by the hybrid U1-CPCg-that carries CPCg in a TMV-U1 context-are characterized by cell death and by the presence of autoflorescent phenolic compounds and H 2 O 2 , just like the HR lesions. In addition, defense-related genes and detoxifying genes are induced in tobacco Xanthi-nn plants after TMV-Cg and U1-CPCg inoculation. We postulate that in our system, CPCg is recognized by sensitive tobacco plants that mount an incomplete defense response. We call this an HR-like since it is not enough to induce plant resistance.
C. Stange. 2006. Interacción planta-virus durante el proceso infectivo. Cien. Inv. Agr. 33(1):3-2... more C. Stange. 2006. Interacción planta-virus durante el proceso infectivo. Cien. Inv. Agr. 33(1):3-21. Los virus que infectan plantas son generalmente de tipo DNA o RNA de cadena simple y positiva. El ciclo viral se inicia al penetrar el virus en la célula hospedera. Este comienza con el desensamblaje, replicación del RNA, traducción de proteínas, ensamble, liberación, movimiento de célula a célula y a larga distancia. El conocimiento de los mecanismos de interacción entre la planta hospedera y el virus, ha progresado considerablemente en los últimos treinta años. Por ejemplo, se ha determinado la participación de componentes del citoesqueleto y de proteínas del hospedero en movimiento local (célula a célula) y a larga distancia (movimiento sistémico) de los virus en las plantas. Además, se han caracterizado numerosos receptores virales codificados por genes de resistencia (R) y se ha determinado el mecanismo de defensa en Arabidopsis thaliana y en especies de las familias Solanaceae, ...
Molecules and cells, Jan 30, 2005
When inoculated into sensitive tobacco Xanthi-nn plants, the crucifer and garlic-infecting Tobacc... more When inoculated into sensitive tobacco Xanthi-nn plants, the crucifer and garlic-infecting Tobacco mosaic virus (TMV-Cg) induces local necrotic lesions that resemble those seen in the hypersensitive response (HR) of resistant tobacco plants. However, unlike these, tobacco Xanthi-nn plants do not become resistant to infection and the virus spreads systemically causing a severe disease characterized by necrotic lesions throughout the plant. To identify the viral protein that elicits this necrotic response, we used a set of hybrid viruses constructed by combination of TMV-Cg and the tobacco mosaic virus strain U1 (TMV-U1). In this study we present evidence that the coat protein of TMV-Cg (CPCg) is the elicitor of the necrotic response in tobacco Xanthi-nn plants. Local and systemic necrotic lesions induced by TMV-Cg and by the hybrid U1-CPCg -that carries CPCg in a TMV-U1 context- are characterized by cell death and by the presence of autoflorescent phenolic compounds and H2O2, just li...