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1.
Plant Biotechnol J ; 22(2): 427-444, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38032727

RESUMO

Plants are sessile and therefore have developed an extraordinary capacity to adapt to external signals. Here, the focus is on the plasticity of the plant cell to respond to new intracellular cues. Ketocarotenoids are high-value natural red pigments with potent antioxidant activity. In the present study, system-level analyses have revealed that the heterologous biosynthesis of ketocarotenoids in tomato initiated a series of cellular and metabolic mechanisms to cope with the formation of metabolites that are non-endogenous to the plant. The broad multilevel changes were linked to, among others, (i) the remodelling of the plastidial membrane, where the synthesis and storage of ketocarotenoids occurs; (ii) the recruiting of core metabolic pathways for the generation of metabolite precursors and energy; and (iii) redox control. The involvement of the metabolites as regulators of cellular processes shown here reinforces their pivotal role suggested in the remodelled 'central dogma' concept. Furthermore, the role of metabolic reprogramming to ensure cellular homeostasis is proposed.


Assuntos
Carotenoides , Solanum lycopersicum , Carotenoides/metabolismo , Solanum lycopersicum/genética , Reprogramação Metabólica , Plantas/metabolismo , Homeostase
3.
FEBS Lett ; 596(15): 1865-1870, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35490366

RESUMO

Achieving gain-of-function phenotypes without inserting foreign DNA is an important challenge for plant biotechnologists. Here, we show that a gene can be brought under the control of a promoter from an upstream gene by deleting the intervening genomic sequence using dual-guide CRISPR/Cas9. We fuse the promoter of a nonessential photosynthesis-related gene to DIACYLGLYCEROL ACYLTRANSFERASE 2 (DGAT2) in the lipase-deficient sugar-dependent 1 mutant of Arabidopsis thaliana to drive ectopic oil accumulation in leaves. DGAT2 expression is enhanced more than 20-fold and the triacylglycerol content increases by around 30-fold. This deletion strategy offers a transgene-free route to engineering traits that rely on transcriptional gain-of-function, such as producing high lipid forage to increase the productivity and sustainability of ruminant farming.


Assuntos
Arabidopsis , Sistemas CRISPR-Cas , Arabidopsis/genética , Arabidopsis/metabolismo , Edição de Genes , Fusão Gênica , Genômica , Transgenes
4.
Metab Eng ; 70: 196-205, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35121114

RESUMO

Sinapine (sinapoylcholine) is an antinutritive phenolic compound that can account for up to 2% of seed weight in brassicaceous oilseed crops and reduces the suitability of their protein-rich seed meal for use as animal feed. Sinapine biosynthesis draws on hydroxycinnamic acid precursors produced by the phenylpropanoid pathway. The 4-vinyl derivatives of several hydroxycinnamic acids have industrial applications. For example, 4-vinyl phenol (4-hydroxystyrene) is a building block for a range of synthetic polymers applied in resins, inks, elastomers, and coatings. Here we have expressed a modified bacterial phenolic acid decarboxylase (PAD) in developing seed of Camelina sativa to redirect phenylpropanoid pathway flux from sinapine biosynthesis to the production of 4-vinyl phenols. PAD expression led to a ∼95% reduction in sinapine content in seeds of both glasshouse and field grown C. sativa and to an accumulation of 4-vinyl derivatives of hydroxycinnamic acids, primarily as glycosides. The most prevalent aglycone was 4-vinyl phenol, but 4-vinyl guaiacol, 6-hydroxy-4-vinyl guaiacol and 4-vinylsyringol (Canolol) were also detected. The molar quantity of 4-vinyl phenol glycosides was more than twice that of sinapine in wild type seeds. PAD expression was not associated with an adverse effect on seed yield, harvest index, seed morphology, storage oil content or germination in either glasshouse or field experiments. Our data show that expression of PAD in brassicaceous oilseeds can supress sinapine accumulation, diverting phenylpropanoid pathway flux into 4-vinyl phenol derivatives, thereby also providing a non-petrochemical source of this class of industrial chemicals.


Assuntos
Ácidos Cumáricos , Sementes , Colina/análogos & derivados , Colina/metabolismo , Ácidos Cumáricos/metabolismo , Sementes/metabolismo
5.
Sci Rep ; 8(1): 17346, 2018 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-30478395

RESUMO

Seeds exhibit wide variation in the fatty acid composition of their storage oil. However, the genetic basis of this variation is only partially understood. Here we have used a multi-parent advanced generation inter-cross (MAGIC) population to study the genetic control of fatty acid chain length in Arabidopsis thaliana seed oil. We mapped four quantitative trait loci (QTL) for the quantity of the major very long chain fatty acid species 11-eicosenoic acid (20:1), using multiple QTL modelling. Surprisingly, the main-effect QTL does not coincide with FATTY ACID ELONGASE 1 and a parallel genome wide association study suggested that LYSOPHOSPHATIDYLCHOLINE ACYLTRANSFERASE 2 (LPCAT2) is a candidate for this QTL. Regression analysis also suggested that LPCAT2 expression and 20:1 content in seeds of the 19 MAGIC founder accessions are related. LPCAT is a key component of the Lands cycle; an acyl editing pathway that enables acyl-exchange between the acyl-Coenzyme A and phosphatidylcholine precursor pools used for microsomal fatty acid elongation and desaturation, respectively. We Mendelianised the main-effect QTL using biparental chromosome segment substitution lines and carried out complementation tests to show that a single cis-acting polymorphism in the LPCAT2 promoter causes the variation in seed 20:1 content, by altering the LPCAT2 expression level and total LPCAT activity in developing siliques. Our work establishes that oilseed species exhibit natural variation in the enzymic capacity for acyl editing and this contributes to the genetic control of storage oil composition.


Assuntos
Arabidopsis/genética , Ácidos Graxos/metabolismo , Óleos de Plantas/metabolismo , Sementes/genética , 1-Acilglicerofosfocolina O-Aciltransferase/genética , 1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Mapeamento Cromossômico , Elongases de Ácidos Graxos/genética , Elongases de Ácidos Graxos/metabolismo , Ácidos Graxos/química , Ácidos Graxos/genética , Ácidos Graxos Monoinsaturados/metabolismo , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Estudo de Associação Genômica Ampla , Óleos de Plantas/química , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Locos de Características Quantitativas , Sementes/metabolismo
6.
Proc Natl Acad Sci U S A ; 114(41): 10876-10881, 2017 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-28973873

RESUMO

Ketocarotenoids are high-value pigments used commercially across multiple industrial sectors as colorants and supplements. Chemical synthesis using petrochemical-derived precursors remains the production method of choice. Aquaculture is an example where ketocarotenoid supplementation of feed is necessary to achieve product viability. The biosynthesis of ketocarotenoids, such as canthaxanthin, phoenicoxanthin, or astaxanthin in plants is rare. In the present study, complex engineering of the carotenoid pathway has been performed to produce high-value ketocarotenoids in tomato fruit (3.0 mg/g dry weight). The strategy adopted involved pathway extension beyond ß-carotene through the expression of the ß-carotene hydroxylase (CrtZ) and oxyxgenase (CrtW) from Brevundimonas sp. in tomato fruit, followed by ß-carotene enhancement through the introgression of a lycopene ß-cyclase (ß-Cyc) allele from a Solanum galapagense background. Detailed biochemical analysis, carried out using chromatographic, UV/VIS, and MS approaches, identified the predominant carotenoid as fatty acid (C14:0 and C16:0) esters of phoenicoxanthin, present in the S stereoisomer configuration. Under a field-like environment with low resource input, scalability was shown with the potential to deliver 23 kg of ketocarotenoid/hectare. To illustrate the potential of this "generally recognized as safe" material with minimal, low-energy bioprocessing, two independent aquaculture trials were performed. The plant-based feeds developed were more efficient than the synthetic feed to color trout flesh (up to twofold increase in the retention of the main ketocarotenoids in the fish fillets). This achievement has the potential to create a new paradigm in the renewable production of economically competitive feed additives for the aquaculture industry and beyond.


Assuntos
Aquicultura , Carotenoides/biossíntese , Engenharia Metabólica/métodos , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Solanum lycopersicum/metabolismo , Solanum lycopersicum/genética , Solanum lycopersicum/crescimento & desenvolvimento , Pigmentação , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento
7.
Science ; 356(6343): 1175-1178, 2017 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-28596311

RESUMO

Plants form beneficial associations with arbuscular mycorrhizal fungi, which facilitate nutrient acquisition from the soil. In return, the fungi receive organic carbon from the plants. The transcription factor RAM1 (REQUIRED FOR ARBUSCULAR MYCORRHIZATION 1) is crucial for this symbiosis, and we demonstrate that it is required and sufficient for the induction of a lipid biosynthetic pathway that is expressed in plant cells accommodating fungal arbuscules. Lipids are transferred from the plant to mycorrhizal fungi, which are fatty acid auxotrophs, and this lipid export requires the glycerol-3-phosphate acyltransferase RAM2, a direct target of RAM1. Our work shows that in addition to sugars, lipids are a major source of organic carbon delivered to the fungus, and this is necessary for the production of fungal lipids.


Assuntos
Ácidos Graxos/metabolismo , Micorrizas/metabolismo , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Simbiose , Ácidos Graxos/biossíntese , Regulação Fúngica da Expressão Gênica/genética , Regulação da Expressão Gênica de Plantas/genética , Interações Hospedeiro-Parasita/fisiologia , Medicago truncatula/microbiologia , Medicago truncatula/fisiologia
8.
Plant Physiol ; 173(3): 1594-1605, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28108698

RESUMO

Plants modify the polyunsaturated fatty acid content of their membrane and storage lipids in order to adapt to changes in temperature. In developing seeds, this response is largely controlled by the activities of the microsomal ω-6 and ω-3 fatty acid desaturases, FAD2 and FAD3. Although temperature regulation of desaturation has been studied at the molecular and biochemical levels, the genetic control of this trait is poorly understood. Here, we have characterized the response of Arabidopsis (Arabidopsis thaliana) seed lipids to variation in ambient temperature and found that heat inhibits both ω-6 and ω-3 desaturation in phosphatidylcholine, leading to a proportional change in triacylglycerol composition. Analysis of the 19 parental accessions of the multiparent advanced generation intercross (MAGIC) population showed that significant natural variation exists in the temperature responsiveness of ω-6 desaturation. A combination of quantitative trait locus (QTL) analysis and genome-wide association studies (GWAS) using the MAGIC population suggests that ω-6 desaturation is largely controlled by cis-acting sequence variants in the FAD2 5' untranslated region intron that determine the expression level of the gene. However, the temperature responsiveness of ω-6 desaturation is controlled by a separate QTL on chromosome 2. The identity of this locus is unknown, but genome-wide association studies identified potentially causal sequence variants within ∼40 genes in an ∼450-kb region of the QTL.


Assuntos
Arabidopsis/genética , Ácidos Graxos Ômega-3/biossíntese , Ácidos Graxos Ômega-6/biossíntese , Estudo de Associação Genômica Ampla/métodos , Locos de Características Quantitativas/genética , Temperatura , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Regulação da Expressão Gênica de Plantas , Lipídeos/análise , Fosfatidilcolinas/análise , Fosfatidilcolinas/metabolismo , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sementes/enzimologia , Sementes/genética , Sementes/metabolismo , Triglicerídeos/análise , Triglicerídeos/metabolismo
9.
Nat Commun ; 6: 6659, 2015 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-25858700

RESUMO

Gluconeogenesis is a fundamental metabolic process that allows organisms to make sugars from non-carbohydrate stores such as lipids and protein. In eukaryotes only one gluconeogenic route has been described from organic acid intermediates and this relies on the enzyme phosphoenolpyruvate carboxykinase (PCK). Here we show that two routes exist in Arabidopsis, and that the second uses pyruvate, orthophosphate dikinase (PPDK). Gluconeogenesis is critical to fuel the transition from seed to seedling. Arabidopsis pck1 and ppdk mutants are compromised in seed-storage reserve mobilization and seedling establishment. Radiolabelling studies show that PCK predominantly allows sugars to be made from dicarboxylic acids, which are products of lipid breakdown. However, PPDK also allows sugars to be made from pyruvate, which is a major product of protein breakdown. We propose that both routes have been evolutionarily conserved in plants because, while PCK expends less energy, PPDK is twice as efficient at recovering carbon from pyruvate.


Assuntos
Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Gluconeogênese/genética , Fosfoenolpiruvato Carboxilase/metabolismo , Piruvato Ortofosfato Diquinase/metabolismo , Plântula/metabolismo , Sementes/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Carboidratos/biossíntese , Carbono/metabolismo , Ácidos Dicarboxílicos/metabolismo , Metabolismo dos Lipídeos/genética , Mutação , Fosfoenolpiruvato Carboxilase/genética , Piruvato Ortofosfato Diquinase/genética , Ácido Pirúvico/metabolismo , Plântula/genética , Plântula/crescimento & desenvolvimento , Sementes/genética , Sementes/crescimento & desenvolvimento , Transdução de Sinais
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