RESUMO
Biofortification was first proposed in the early 1990s as a low-cost, sustainable strategy to enhance the mineral and vitamin contents of staple food crops to address micronutrient malnutrition. Since then, the concept and remit of biofortification has burgeoned beyond staples and solutions for low- and middle-income economies. Here we discuss what biofortification has achieved in its original manifestation and the main factors limiting the ability of biofortified crops to improve micronutrient status. We highlight the case for biofortified crops with key micronutrients, such as provitamin D3/vitamin D3, vitamin B12 and iron, for recognition of new demographics of need. Finally, we examine where and how biofortification can be integrated into the global food system to help overcome hidden hunger, improve nutrition and achieve sustainable agriculture.
Assuntos
Desnutrição , Oligoelementos , Humanos , Biofortificação , Alimentos Fortificados , Desnutrição/prevenção & controle , Vitaminas , MicronutrientesRESUMO
Higher dietary intakes of flavonoids may have a beneficial role in cardiovascular disease prevention. Additionally, supplementation of branched-chain amino acids (BCAAs) in vegan diets can reduce risks associated to their deficiency, particularly in older adults, which can cause loss of skeletal muscle strength and mass. Most plant-derived foods contain only small amounts of BCAAs, and those plants with high levels of flavonoids are not eaten broadly. Here we describe the generation of metabolically engineered cisgenic tomatoes enriched in both flavonoids and BCAAs. In this approach, coding and regulatory DNA elements, all derived from the tomato genome, were combined to obtain a herbicide-resistant version of an acetolactate synthase (mSlALS) gene expressed broadly and a MYB12-like transcription factor (SlMYB12) expressed in a fruit-specific manner. The mSlALS played a dual role, as a selectable marker as well as being key enzyme in BCAA enrichment. The resulting cisgenic tomatoes were highly enriched in Leucine (21-fold compared to wild-type levels), Valine (ninefold) and Isoleucine (threefold) and concomitantly biofortified in several antioxidant flavonoids including kaempferol (64-fold) and quercetin (45-fold). Comprehensive metabolomic and transcriptomic analysis of the biofortified cisgenic tomatoes revealed marked differences to wild type and could serve to evaluate the safety of these biofortified fruits for human consumption.
Assuntos
Aminoácidos de Cadeia Ramificada , Solanum lycopersicum , Humanos , Aminoácidos de Cadeia Ramificada/metabolismo , Solanum lycopersicum/genética , Flavonoides , Leucina , Frutas/genética , Frutas/metabolismo , Isoleucina/metabolismoRESUMO
The presence of anticancer clerodane diterpenoids is a chemotaxonomic marker for the traditional Chinese medicinal plant Scutellaria barbata, although the molecular mechanisms behind clerodane biosynthesis are unknown. Here, we report a high-quality assembly of the 414.98 Mb genome of S. barbata into 13 pseudochromosomes. Using phylogenomic and biochemical data, we mapped the plastidial metabolism of kaurene (gibberellins), abietane, and clerodane diterpenes in three species of the family Lamiaceae (Scutellaria barbata, Scutellaria baicalensis, and Salvia splendens), facilitating the identification of genes involved in the biosynthesis of the clerodanes, kolavenol, and isokolavenol. We show that clerodane biosynthesis evolved through recruitment and neofunctionalization of genes from gibberellin and abietane metabolism. Despite the assumed monophyletic origin of clerodane biosynthesis, which is widespread in species of the Lamiaceae, our data show distinct evolutionary lineages and suggest polyphyletic origins of clerodane biosynthesis in the family Lamiaceae. Our study not only provides significant insights into the evolution of clerodane biosynthetic pathways in the mint family, Lamiaceae, but also will facilitate the production of anticancer clerodanes through future metabolic engineering efforts.
Assuntos
Diterpenos Clerodânicos , Diterpenos , Plantas Medicinais , Scutellaria , Diterpenos Clerodânicos/química , Diterpenos Clerodânicos/metabolismo , Scutellaria/genética , Scutellaria/química , Scutellaria/metabolismo , Abietanos/metabolismo , Diterpenos/química , Diterpenos/metabolismo , Plantas Medicinais/genética , Plantas Medicinais/metabolismoRESUMO
Poor vitamin D status is a global health problem; insufficiency underpins higher risk of cancer, neurocognitive decline and all-cause mortality. Most foods contain little vitamin D and plants are very poor sources. We have engineered the accumulation of provitamin D3 in tomato by genome editing, modifying a duplicated section of phytosterol biosynthesis in Solanaceous plants, to provide a biofortified food with the added possibility of supplement production from waste material.
Assuntos
Solanum lycopersicum , Alimentos Fortificados/análise , Provitaminas , Vitamina A , Vitamina DRESUMO
Wild relatives of tomato are a valuable source of natural variation in tomato breeding, as many can be hybridized to the cultivated species (Solanum lycopersicum). Several, including Solanum lycopersicoides, have been crossed to S. lycopersicum for the development of ordered introgression lines (ILs), facilitating breeding for desirable traits. Despite the utility of these wild relatives and their associated ILs, few finished genome sequences have been produced to aid genetic and genomic studies. Here we report a chromosome-scale genome assembly for S. lycopersicoides LA2951, which contains 37 938 predicted protein-coding genes. With the aid of this genome assembly, we have precisely delimited the boundaries of the S. lycopersicoides introgressions in a set of S. lycopersicum cv. VF36 × LA2951 ILs. We demonstrate the usefulness of the LA2951 genome by identifying several quantitative trait loci for phenolics and carotenoids, including underlying candidate genes, and by investigating the genome organization and immunity-associated function of the clustered Pto gene family. In addition, syntenic analysis of R2R3MYB genes sheds light on the identity of the Aubergine locus underlying anthocyanin production. The genome sequence and IL map provide valuable resources for studying fruit nutrient/quality traits, pathogen resistance, and environmental stress tolerance. We present a new genome resource for the wild species S. lycopersicoides, which we use to shed light on the Aubergine locus responsible for anthocyanin production. We also provide IL boundary mappings, which facilitated identifying novel carotenoid quantitative trait loci of which one was likely driven by an uncharacterized lycopene ß-cyclase whose function we demonstrate.
Assuntos
Solanum lycopersicum , Solanum , Antocianinas/genética , Cromossomos de Plantas/genética , Solanum lycopersicum/genética , Melhoramento Vegetal , Solanum/genéticaRESUMO
Scutellaria baicalensis, is one of the most traditional medicinal plants in the Lamiaceae family, and has been widely used to treat liver and lung complaints and as a complementary cancer treatment in traditional Chinese medicine. The preparation from its roots, called "Huang Qin," is rich in specialized flavones such as baicalein, wogonin, and their glycosides which lack a 4'-hydroxyl group on the B ring (4'-deoxyflavones), with anti-tumor, antioxidant, and antiviral activities. Baicalein has recently been reported to inhibit the replication of the COVID-19 virus. These 4'-deoxyflavones are found only in the order Lamiales and were discovered in the genus Scutellaria, suggesting that a new metabolic pathway synthesizing 4'-deoxyflavones evolved recently in this genus. In this review, we focus on the class of 4'-deoxyflavones in S. baicalensis and their pharmacological properties. We also describe the apparent evolutionary route taken by the genes encoding enzymes involved in the novel, root-specific, biosynthetic pathway for baicalein and wogonin, which provides insights into the evolution of specific flavone biosynthetic pathways in the mint family.
RESUMO
The medicinal plant Scutellaria baicalensis Georgi is rich in specialized 4'-deoxyflavones, which are reported to have many health-promoting properties. We assayed Scutellaria flavones with different methoxyl groups on human cancer cell lines and found that polymethoxylated 4'-deoxyflavones, like skullcapflavone I and tenaxin I have stronger ability to induce apoptosis compared to unmethylated baicalein, showing that methoxylation enhances bioactivity as well as the physical properties of specialized flavones, while having no side-effects on healthy cells. We investigated the formation of methoxylated flavones and found that two O-methyltransferase (OMT) families are active in the roots of S. baicalensis. The Type II OMTs, SbPFOMT2 and SbPFOMT5, decorate one of two adjacent hydroxyl groups on flavones and are responsible for methylation on the C6, 8 and 3'-hydroxyl positions, to form oroxylin A, tenaxin II and chrysoeriol respectively. The Type I OMTs, SbFOMT3, SbFOMT5 and SbFOMT6 account mainly for C7-methoxylation of flavones, but SbFOMT5 can also methylate baicalein on its C5 and C6-hydroxyl positions. The dimethoxylated flavone, skullcapflavone I (found naturally in roots of S. baicalensis) can be produced in yeast by co-expressing SbPFOMT5 plus SbFOMT6 when the appropriately hydroxylated 4'-deoxyflavone substrates are supplied in the medium. Co-expression of SbPFOMT5 plus SbFOMT5 in yeast produced tenaxin I, also found in Scutellaria roots. This work showed that both type I and type II OMT enzymes are involved in biosynthesis of methoxylated flavones in S. baicalensis.
Assuntos
Plantas Medicinais , Scutellaria baicalensis , Flavonoides/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Raízes de Plantas/metabolismo , Scutellaria baicalensis/química , Scutellaria baicalensis/metabolismoRESUMO
The success of Western Scientific approaches to medicine, over the last 150 years, can be measured by substantial increases in life expectancy, reductions in infant mortality and the virtual elimination of many infectious diseases accompanied by development of effective management practices for noncommunicable diseases. However, major challenges remain in the form of infectious diseases that evolve resistance to pharmaceuticals rapidly, new diseases, particularly those caused by viruses and effective long-term treatments for chronic, noncommunicable diseases. Traditional Chinese Medicine (TCM) can offer complementary treatments based on personalised interventions, informed by knowledge accumulated from empirical observations gathered over centuries of practice, that address the impact of disease on the whole body. We provide examples of both infectious and noncommunicable diseases where the combination of Western Scientific Medicine (WSM) and TCM can benefit patients in terms of the speed and efficacy of recovery or disease management. TCM is a healing skill based on practice, while WSM is scientific, based on experiments. Against this background, an understanding of the mechanisms of action of traditional Chinese medicinal preparations will offer fresh routes to discovery and development of new therapeutics as well as patented medical prescriptions, which will rely heavily on modern scientific methodologies for their adoption and success, particularly those in plant genomics, plant breeding and synthetic biology.
Assuntos
Medicina Tradicional Chinesa , China , HumanosRESUMO
Scutellaria baicalensis Georgi is important in Chinese traditional medicine where preparations of dried roots, "Huang Qin," are used for liver and lung complaints and as complementary cancer treatments. We report a high-quality reference genome sequence for S. baicalensis where 93% of the 408.14-Mb genome has been assembled into nine pseudochromosomes with a super-N50 of 33.2 Mb. Comparison of this sequence with those of closely related species in the order Lamiales, Sesamum indicum and Salvia splendens, revealed that a specialized metabolic pathway for the synthesis of 4'-deoxyflavone bioactives evolved in the genus Scutellaria. We found that the gene encoding a specific cinnamate coenzyme A ligase likely obtained its new function following recent mutations, and that four genes encoding enzymes in the 4'-deoxyflavone pathway are present as tandem repeats in the genome of S. baicalensis. Further analyses revealed that gene duplications, segmental duplication, gene amplification, and point mutations coupled to gene neo- and subfunctionalizations were involved in the evolution of 4'-deoxyflavone synthesis in the genus Scutellaria. Our study not only provides significant insight into the evolution of specific flavone biosynthetic pathways in the mint family, Lamiaceae, but also will facilitate the development of tools for enhancing bioactive productivity by metabolic engineering in microbes or by molecular breeding in plants. The reference genome of S. baicalensis is also useful for improving the genome assemblies for other members of the mint family and offers an important foundation for decoding the synthetic pathways of bioactive compounds in medicinal plants.
Assuntos
Vias Biossintéticas/genética , Flavanonas , Flavonoides/genética , Scutellaria baicalensis/genética , Flavanonas/genética , Flavanonas/metabolismo , Flavonoides/metabolismo , Genoma de Planta , Medicina Tradicional Chinesa , Extratos Vegetais , Raízes de Plantas/metabolismo , Plantas Medicinais/genética , Plantas Medicinais/metabolismo , Scutellaria baicalensis/metabolismo , Sequenciamento Completo do GenomaRESUMO
Wild potato species are useful sources of allelic diversity and loci lacking in the cultivated potato. In these species, the presence of anthocyanins in leaves has been associated with a greater tolerance to cold stress. However, the molecular mechanisms that allow potatoes to withstand cold exposure remain unclear. Here, we show that the expression of AN2, a MYB transcription factor, is induced by low temperatures in wild, cold-tolerant Solanum commersonii, and not in susceptible Solanum tuberosum varieties. We found that AN2 is a paralog of the potato anthocyanin regulator AN1, showing similar interaction ability with basic helix-loop-helix (bHLH) co-partners. Their sequence diversity resulted in a different capacity to promote accumulation of phenolics when tested in tobacco. Indeed, functional studies demonstrated that AN2 is less able to induce anthocyanins than AN1, but nevertheless it has a strong ability to induce accumulation of hydroxycinnamic acid derivatives. We propose that the duplication of R2R3 MYB genes resulted in subsequent subfunctionalization, where AN1 specialized in anthocyanin production and AN2 conserved the ability to respond to cold stress, inducing mainly the synthesis of hydroxycinnamic acid derivatives. These results contribute to understanding the evolutionary significance of gene duplication on phenolic compound regulation.
Assuntos
Antocianinas/metabolismo , Proteínas de Plantas/metabolismo , Solanum/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Temperatura Baixa , Ácidos Cumáricos/metabolismo , Genes Duplicados , Pressão Osmótica , Folhas de Planta/genética , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Solanum/fisiologia , Estresse Fisiológico , Nicotiana/genética , Nicotiana/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Specialized secondary metabolites serve not only to protect plants against abiotic and biotic challenges, but have also been used extensively by humans to combat diseases. Due to the great importance of medicinal plants for health, we need to find new and sustainable ways to improve the production of the specialized metabolites. In addition to direct extraction, recent progress in metabolic engineering of plants offers an alternative supply option. We argue that metabolic engineering for producing the secondary metabolites in plants may have distinct advantages over microbial production platforms, and thus propose new approaches of plant metabolic engineering, which are inspired by an ancient Chinese irrigation system. Metabolic engineering strategies work at three levels: introducing biosynthetic genes, using transcription factors, and improving metabolic flux including increasing the supply of precursors, energy, and reducing power. In addition, recent progress in biotechnology contributes markedly to better engineering, such as the use of specific promoters and the deletion of competing branch pathways. We propose that next-generation plant metabolic engineering will improve current engineering strategies, for the purpose of producing valuable metabolites in plants on industrial scales.
Assuntos
Irrigação Agrícola , Engenharia Metabólica/métodos , ChinaRESUMO
Baicalein, wogonin, and their glycosides are major bioactive compounds found in the medicinal plant Scutellaria baicalensis Georgi. These flavones can induce apoptosis in a variety of cancer cell lines but have no effect on normal cells. Furthermore, they have many additional benefits for human health, such as anti-oxidant, antiviral, and liver-protective properties. Here, we report the isolation and characterization of two CYP450 enzymes, SbCYP82D1.1 and SbCYP82D2, which function as the flavone 6-hydroxylase (F6H) and flavone 8-hydroxylase (F8H), respectively, in S. baicalensis. SbCYP82D1.1 has broad substrate specificity for flavones such as chrysin and apigenin and is responsible for biosynthesis of baicalein and scutellarein in roots and aerial parts of S. baicalensis, respectively. When the expression of SbCYP82D1.1 is knocked down, baicalin and baicalein levels are reduced significantly while chrysin glycosides accumulate in hairy roots. SbCYP82D2 is an F8H with high substrate specificity, accepting only chrysin as its substrate to produce norwogonin, although minor 6-hydroxylation activity can also be detected. Phylogenetic analysis suggested that SbCYP82D2 might have evolved from SbCYP82D1.1 via gene duplication followed by neofunctionalization, whereby the ancestral F6H activity is partially retained in the derived SbCYP82D2.
Assuntos
Flavonas/metabolismo , Raízes de Plantas/metabolismo , Scutellaria baicalensis/metabolismo , Apigenina/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Flavanonas/metabolismo , Flavonoides/metabolismo , Humanos , Filogenia , Saccharomyces cerevisiae/metabolismo , Scutellaria baicalensis/genéticaRESUMO
Scutellaria baicalensis Georgi, or Chinese skullcap, has been widely used as a medicinal plant in China for thousands of years, where the preparation from its roots is called Huang-Qin. It has been applied in the treatment of diarrhea, dysentery, hypertension, hemorrhaging, insomnia, inflammation and respiratory infections. Flavones such as baicalin, wogonoside and their aglycones baicalein wogonin are the major bioactive compounds extracted from the root of S. baicalensis. These flavones have been reported to have various pharmacological functions, including anti-cancer, hepatoprotection, antibacterial and antiviral, antioxidant, anticonvulsant and neuroprotective effects. In this review, we focus on clinical applications and the pharmacological properties of the medicinal plant and the flavones extracted from it. We also describe biotechnological and metabolic methods that have been used to elucidate the biosynthetic pathways of the bioactive compounds in Scutellaria.
RESUMO
Wogonin and baicalein are bioactive flavones in the popular Chinese herbal remedy Huang-Qin (Scutellaria baicalensis Georgi). These specialized flavones lack a 4'-hydroxyl group on the B ring (4'-deoxyflavones) and induce apoptosis in a wide spectrum of human tumor cells in vitro and inhibit tumor growth in vivo in different mouse tumor models. Root-specific flavones (RSFs) from Scutellaria have a variety of reported additional beneficial effects including antioxidant and antiviral properties. We describe the characterization of a new pathway for the synthesis of these compounds, in which pinocembrin (a 4'-deoxyflavanone) serves as a key intermediate. Although two genes encoding flavone synthase II (FNSII) are expressed in the roots of S. baicalensis, FNSII-1 has broad specificity for flavanones as substrates, whereas FNSII-2 is specific for pinocembrin. FNSII-2 is responsible for the synthesis of 4'-deoxyRSFs, such as chrysin and wogonin, wogonoside, baicalein, and baicalin, which are synthesized from chrysin. A gene encoding a cinnamic acid-specific coenzyme A ligase (SbCLL-7), which is highly expressed in roots, is required for the synthesis of RSFs by FNSII-2, as demonstrated by gene silencing. A specific isoform of chalcone synthase (SbCHS-2) that is highly expressed in roots producing RSFs is also required for the synthesis of chrysin. Our studies reveal a recently evolved pathway for biosynthesis of specific, bioactive 4'-deoxyflavones in the roots of S. baicalensis.
Assuntos
Flavanonas/biossíntese , Flavonas/biossíntese , Flavonoides/biossíntese , Aciltransferases/genética , Animais , Antioxidantes/química , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Vias Biossintéticas/genética , Linhagem Celular Tumoral , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Flavanonas/química , Flavonas/química , Flavonoides/química , Regulação da Expressão Gênica de Plantas , Humanos , Camundongos , Extratos Vegetais/química , Plantas Medicinais/química , Scutellaria baicalensis/químicaRESUMO
Chlorogenic acid (CGA) is one of the most abundant phenolic compounds in tomato (Solanum lycopersicum). Hydroxycinnamoyl CoA quinate transferase (HQT) is the key enzyme catalysing CGA biosynthesis in tomato. We have studied the relationship between phenolic accumulation and UV-susceptibility in transgenic tomato plants with altered HQT expression. Overall, increased CGA accumulation was associated with increased UV-protection. However, the genetic manipulation of HQT expression also resulted in more complex alterations in the profiles of phenolics. Levels of rutin were relatively high in both HQT gene-silenced and HQT-overexpressing plants raised in plant growth tunnels. This suggests plasticity in the flux along different branches of phenylpropanoid metabolism and the existence of regulatory mechanisms that direct the flow of phenolic precursors in response to both metabolic parameters and environmental conditions. These changes in composition of the phenolic pool affected the relative levels of UV-tolerance. We conclude that the capability of the phenolic compounds to protect against potentially harmful UV radiation is determined both by the total levels of phenolics that accumulate in leaves as well as by the specific composition of the phenolic profile.
Assuntos
Ácido Clorogênico/metabolismo , Fenóis/metabolismo , Solanum/metabolismo , Solanum/efeitos da radiação , Ácido Clorogênico/química , Clorofila/química , Clorofila/metabolismo , Solanum lycopersicum/química , Solanum lycopersicum/metabolismo , Espectrometria de Massas , Extratos Vegetais/química , Solubilidade , Análise EspectralRESUMO
Many angiosperm species produce cones of anthers which release pollen through pores in response to vibration by pollinating bees ("buzz-pollination"). Anther cones of varying degrees of strength are a defining morphological trait for the genus Solanum. Anthers arranged in a robust ('pepper pot') cone are restricted to a single clade within the genus, and may therefore be assumed to be monophyletic. We show that in some species within this clade, such as tomato (Solanum lycopersicum), the anther cone is held together by interlocking hairs (trichomes) along the edges of the anthers. In other species within the clade, such as woody nightshade (Solanum dulcamara), the expanded anther surfaces are closely appressed to form the tightly bound cone, strengthened by extracellular secretions. Ectopic expression of the MIXTA gene from Antirrhinum majus in S. dulcamara results in the formation of ectopic trichomes on the anthers which cause the cone to disintegrate. Therefore, these two species produce the same macroscopic structure through two mutually exclusive developmental routes and the robust anther cone is derived differently within the clade. This example demonstrates that convergence between closely related species can be easily mistaken for homology, and may thus be underestimated.
Assuntos
Evolução Biológica , Solanum/anatomia & histologia , Adaptação Fisiológica/genética , Extensões da Superfície Celular/genética , Extensões da Superfície Celular/ultraestrutura , Solanum lycopersicum/anatomia & histologia , Solanum lycopersicum/genética , Microscopia Eletrônica de Varredura , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Pólen/anatomia & histologia , Pólen/genética , Pólen/ultraestrutura , Proteínas Proto-Oncogênicas c-myb/genética , Solanum/classificação , Solanum/genética , Especificidade da EspécieRESUMO
Starch granule initiation is not understood, but recent evidence implicates a starch debranching enzyme, isoamylase, in the control of this process. Potato tubers contain isoamylase activity attributable to a heteromultimeric protein containing Stisa1 and Stisa2, the products of two of the three isoamylase genes of potato. To discover whether this enzyme is involved in starch granule initiation, activity was reduced by expression of antisense RNA for Stisa1 or Stisa2. Transgenic tubers accumulated a small amount of a soluble glucan, similar in structure to the phytoglycogen of cereal, Arabidopsis, and Chlamydomonas mutants lacking isoamylase. The major effect, however, was on the number of starch granules. Transgenic tubers accumulated large numbers of tiny granules not seen in normal tubers. These data indicate that the heteromultimeric isoamylase functions during starch synthesis to suppress the initiation of glucan molecules in the plastid stroma that would otherwise crystallize to nucleate new starch granules.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Isoamilase/metabolismo , Raízes de Plantas/enzimologia , Solanum tuberosum/citologia , Solanum tuberosum/metabolismo , Amido/metabolismo , Genes de Plantas/genética , Glicogênio/metabolismo , Isoamilase/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Solanum tuberosum/enzimologia , Solanum tuberosum/genéticaRESUMO
Isoamylases are debranching enzymes that hydrolyze alpha-1,6 linkages in alpha-1,4/alpha-1,6-linked glucan polymers. In plants, they have been shown to be required for the normal synthesis of amylopectin, although the precise manner in which they influence starch synthesis is still debated. cDNA clones encoding three distinct isoamylase isoforms (Stisa1, Stisa2, and Stisa3) have been identified from potato. The expression patterns of the genes are consistent with the possibility that they all play roles in starch synthesis. Analysis of the predicted sequences of the proteins suggested that only Stisa1 and Stisa3 are likely to have hydrolytic activity and that there probably are differences in substrate specificity between these two isoforms. This was confirmed by the expression of each isoamylase in Escherichia coli and characterization of its activity. Partial purification of isoamylase activity from potato tubers showed that Stisa1 and Stisa2 are associated as a multimeric enzyme but that Stisa3 is not associated with this enzyme complex. Our data suggest that Stisa1 and Stisa2 act together to debranch soluble glucan during starch synthesis. The catalytic specificity of Stisa3 is distinct from that of the multimeric enzyme, indicating that it may play a different role in starch metabolism.
Assuntos
Glucanos/metabolismo , Isoamilase/genética , Isoamilase/metabolismo , Solanum tuberosum/enzimologia , Sequência de Aminoácidos , Catálise , Ativação Enzimática/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Caules de Planta/enzimologia , Caules de Planta/genética , Homologia de Sequência de Aminoácidos , Solanum tuberosum/genética , Amido/biossínteseRESUMO
Amyloses with distinct molecular masses are found in the starch of pea embryos compared with the starch of pea leaves. In pea embryos, a granule-bound starch synthase protein (GBSSIa) is required for the synthesis of a significant portion of the amylose. However, this protein seems to be insignificant in the synthesis of amylose in pea leaves. cDNA clones encoding a second isoform of GBSSI, GBSSIb, have been isolated from pea leaves. Comparison of GBSSIa and GBSSIb activities shows them to have distinct properties. These differences have been confirmed by the expression of GBSSIa and GBSSIb in the amylose-free mutant of potato. GBSSIa and GBSSIb make distinct forms of amylose that differ in their molecular mass. These differences in product specificity, coupled with differences in the tissues in which GBSSIa and GBSSIb are most active, explain the distinct forms of amylose found in different tissues of pea. The shorter form of amylose formed by GBSSIa confers less susceptibility to the retrogradation of starch pastes than the amylose formed by GBSSIb. The product specificity of GBSSIa could provide beneficial attributes to starches for food and nonfood uses.
Assuntos
Amilose/biossíntese , Pisum sativum/enzimologia , Sintase do Amido/metabolismo , Sequência de Aminoácidos , Amilopectina/metabolismo , Amilose/análise , Cromatografia em Gel , Cromatografia por Troca Iônica , Clonagem Molecular , DNA Complementar/genética , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Mutação , Pisum sativum/genética , Filogenia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/biossíntese , Plantas Geneticamente Modificadas , Sementes/enzimologia , Sementes/genética , Homologia de Sequência de Aminoácidos , Solanum tuberosum/genética , Amido/metabolismo , Sintase do Amido/genéticaRESUMO
Reductions in activity of SSIII, the major isoform of starch synthase responsible for amylopectin synthesis in the potato tuber, result in fissuring of the starch granules. To discover the causes of the fissuring, and thus to shed light on factors that influence starch granule morphology in general, SSIII antisense lines were compared with lines with reductions in the major granule-bound isoform of starch synthase (GBSS) and lines with reductions in activity of both SSIII and GBSS (SSIII/GBSS antisense lines). This revealed that fissuring resulted from the activity of GBSS in the SSIII antisense background. Control (untransformed) lines and GBSS and SSIII/GBSS antisense lines had unfissured granules. Starch analyses showed that granules from SSIII antisense tubers had a greater number of long glucan chains than did granules from the other lines, in the form of larger amylose molecules and a unique fraction of very long amylopectin chains. These are likely to result from increased flux through GBSS in SSIII antisense tubers, in response to the elevated content of ADP-glucose in these tubers. It is proposed that the long glucan chains disrupt organization of the semi-crystalline parts of the matrix, setting up stresses in the matrix that lead to fissuring.