Functional Diversification of the Dihydroflavonol 4-Reductase from Camellia nitidissima Chi. in the Control of Polyphenol Biosynthesis

Plant secondary metabolism is complex in its diverse chemical composition and dynamic regulation of biosynthesis. How the functional diversification of enzymes contributes to the diversity is largely unknown. In the flavonoids pathway, dihydroflavonol 4-reductase (DFR) is a key enzyme mediating dihydroflavanol into anthocyanins biosynthesis. Here, the DFR homolog was identified from Camellia nitidissima Chi. (CnDFR) which is a unique species of the genus Camellia with golden yellow petals. Sequence analysis showed that CnDFR possessed not only conserved catalytic domains, but also some amino acids peculiar to Camellia species. Gene expression analysis revealed that CnDFR was expressed in all tissues and the expression of CnDFR was positively correlated with polyphenols but negatively with yellow coloration. The subcellular localization of CnDFR by the tobacco infiltration assay showed a likely dual localization in the nucleus and cell membrane. Furthermore, overexpression transgenic lines were generated in tobacco to understand the molecular function of CnDFR. The analyses of metabolites suggested that ectopic expression of CnDFR enhanced the biosynthesis of polyphenols, while no accumulation of anthocyanins was detected. These results indicate a functional diversification of the reductase activities in Camellia plants and provide molecular insights into the regulation of floral color.

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Metabolic Engineering of Yeast and Plants for the Production of the Biologically Active Hydroxystilbene, Resveratrol

Journal of Biomedicine and Biotechnology ◽

10.1155/2012/579089 ◽

2012 ◽

Vol 2012 ◽

pp. 1-14 ◽

Cited By ~ 58

Author(s):

Philippe Jeandet ◽

Bertrand Delaunois ◽

Aziz Aziz ◽

David Donnez ◽

Yann Vasserot ◽

...

Keyword(s):

Antioxidant Activities ◽

Stilbene Synthase ◽

Molecular Engineering ◽

Biologically Active ◽

The Other ◽

Alternative Mechanism ◽

Comprehensive Review ◽

Transgenic Lines ◽

Key Enzyme ◽

Recombinant Microorganisms

Resveratrol, a stilbenic compound deriving from the phenyalanine/polymalonate route, being stilbene synthase the last and key enzyme of this pathway, recently has become the focus of a number of studies in medicine and plant physiology. Increased demand for this molecule for nutraceutical, cosmetic and possibly pharmaceutic uses, makes its production a necessity. In this context, the use of biotechnology through recombinant microorganisms and plants is particularly promising. Interesting results can indeed arise from the potential of genetically modified microorganisms as an alternative mechanism for producing resveratrol. Strategies used to tailoring yeast as they do not possess the genes that encode for the resveratrol pathway, will be described. On the other hand, most interest has centered in recent years, onSTSgene transfer experiments from various origins to the genome of numerous plants. This work also presents a comprehensive review on plant molecular engineering with theSTSgene, resulting in disease resistance against microorganisms and the enhancement of the antioxidant activities of several fruits in transgenic lines.

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Branched-chain amino acid aminotransferase 2 regulates ferroptotic cell death in cancer cells

Cell Death and Differentiation ◽

10.1038/s41418-020-00644-4 ◽

2020 ◽

Author(s):

Kang Wang ◽

Zhengyang Zhang ◽

Hsiang-i Tsai ◽

Yanfang Liu ◽

Jie Gao ◽

...

Keyword(s):

Cell Death ◽

Amino Acid ◽

Cancer Cells ◽

Ectopic Expression ◽

Branched Chain Amino Acid ◽

Pancreatic Cancer Cells ◽

Key Enzyme ◽

Branched Chain

Abstract Ferroptosis, a form of iron-dependent cell death driven by cellular metabolism and iron-dependent lipid peroxidation, has been implicated as a tumor-suppressor function for cancer therapy. Recent advance revealed that the sensitivity to ferroptosis is tightly linked to numerous biological processes, including metabolism of amino acid and the biosynthesis of glutathione. Here, by using a high-throughput CRISPR/Cas9-based genetic screen in HepG2 hepatocellular carcinoma cells to search for metabolic proteins inhibiting ferroptosis, we identified a branched-chain amino acid aminotransferase 2 (BCAT2) as a novel suppressor of ferroptosis. Mechanistically, ferroptosis inducers (erastin, sorafenib, and sulfasalazine) activated AMPK/SREBP1 signaling pathway through iron-dependent ferritinophagy, which in turn inhibited BCAT2 transcription. We further confirmed that BCAT2 as the key enzyme mediating the metabolism of sulfur amino acid, regulated intracellular glutamate level, whose activation by ectopic expression specifically antagonize system Xc– inhibition and protected liver and pancreatic cancer cells from ferroptosis in vitro and in vivo. On the contrary, direct inhibition of BCAT2 by RNA interference, or indirect inhibition by blocking system Xc– activity, triggers ferroptosis. Finally, our results demonstrate the synergistic effect of sorafenib and sulfasalazine in downregulating BCAT2 expression and dictating ferroptotic death, where BCAT2 can also be used to predict the responsiveness of cancer cells to ferroptosis-inducing therapies. Collectively, these findings identify a novel role of BCAT2 in ferroptosis, suggesting a potential therapeutic strategy for overcoming sorafenib resistance.

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Heterologous Expression of a Soybean Gene RR34 Conferred Improved Drought Resistance of Transgenic Arabidopsis

Plants ◽

10.3390/plants9040494 ◽

2020 ◽

Vol 9 (4) ◽

pp. 494 ◽

Cited By ~ 1

Author(s):

Duong Hoang Trong Nghia ◽

Nguyen Nguyen Chuong ◽

Xuan Lan Thi Hoang ◽

Nguyen Cao Nguyen ◽

Nguyen Huu Cam Tu ◽

...

Keyword(s):

Response Regulator ◽

Ectopic Expression ◽

Plant Performance ◽

Transcriptional Analysis ◽

Antioxidant Enzyme Activities ◽

Transgenic Lines ◽

Detached Leaves ◽

Two Component Systems ◽

Soybean Gene ◽

Response To Water

Two-component systems (TCSs) have been identified as participants in mediating plant response to water deficit. Nevertheless, insights of their contribution to plant drought responses and associated regulatory mechanisms remain limited. Herein, a soybean response regulator (RR) gene RR34, which is the potential drought-responsive downstream member of a TCS, was ectopically expressed in the model plant Arabidopsis for the analysis of its biological roles in drought stress response. Results from the survival test revealed outstanding recovery ratios of 52%–53% in the examined transgenic lines compared with 28% of the wild-type plants. Additionally, remarkedly lower water loss rates in detached leaves as well as enhanced antioxidant enzyme activities of catalase and superoxide dismutase were observed in the transgenic group. Further transcriptional analysis of a subset of drought-responsive genes demonstrated higher expression in GmRR34-transgenic plants upon exposure to drought, including abscisic acid (ABA)-related genes NCED3, OST1, ABI5, and RAB18. These ectopic expression lines also displayed hypersensitivity to ABA treatment at germination and post-germination stages. Collectively, these findings indicated the ABA-associated mode of action of GmRR34 in conferring better plant performance under the adverse drought conditions.

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CDPK6 phosphorylates and stabilizes MYB30 to promote hyperoside biosynthesis that prolongs the duration of full-blooming in okra

Journal of Experimental Botany ◽

10.1093/jxb/eraa174 ◽

2020 ◽

Vol 71 (14) ◽

pp. 4042-4056

Author(s):

Qing Yang ◽

Biying Dong ◽

Litao Wang ◽

Zhihua Song ◽

Lili Niu ◽

...

Keyword(s):

Ectopic Expression ◽

Fold Increase ◽

Bimolecular Fluorescence Complementation ◽

Dependent Manner ◽

Flavonoid Pathway ◽

Calcium Dependent ◽

Key Enzyme ◽

Dependent Protein ◽

The Relationship

Abstract The flowers of okra (Abelmoschus esculentus) open and wilt within only a few hours, and this is accompanied by accumulation of hyperoside, a secondary metabolite in the flavonoid pathway. However, little is known about the relationship between flavonoids and flowering. Here, we found that exogenous application of hyperoside extended the duration of the full-blooming period by more than 3-fold, and this was accompanied by a 14.7-fold increase in the expression of CALCIUM-DEPENDENT PROTEIN KINASE6 (AeCDPK6). Gene expression profiling indicated that the transcription factor AeMYB30 was co-expressed with AeCDPK6, and detailed protein interaction and phosphorylation experiments together with yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated an interaction between AeMYB30 and AeCDPK6. AeCDPK6 specifically phosphorylated AeMYB30S191, leading to increased protein stability and prevention of degradation. Furthermore, AeMYB30 directly bound to the promoter of AeUF3GaT1, a key enzyme in the hyperoside biosynthesis pathway. Analysis of transgenic plants showed that AeCDPK6 was required for the hyperoside-induced phosphorylation of AeMYB30 to enhance its stability and transcriptional activity. Ectopic expression of AeCDPK6 promoted hyperoside accumulation and prolonged the full-blooming period in an AeMYB30-dependent manner. Our results indicate the role of AeCDPK6–AeMYB30 in the molecular mechanism by which hyperoside regulates the period of full blooming in okra, a plant with a short duration of flowering.

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Ectopic expression ofGcm1induces congenital spinal cord abnormalities

Development ◽

10.1242/dev.129.16.3957 ◽

2002 ◽

Vol 129 (16) ◽

pp. 3957-3964 ◽

Cited By ~ 1

Author(s):

Brahim Nait-Oumesmar ◽

Barbara Stecca ◽

Girish Fatterpekar ◽

Thomas Naidich ◽

Joshua Corbin ◽

...

Keyword(s):

Spinal Cord ◽

Transgene Expression ◽

Ectopic Expression ◽

Spinal Column ◽

Life Time ◽

Tail Bud ◽

Neural Tubes ◽

Motor Disabilities ◽

Transgenic Lines ◽

Stimulation Of

Brief ectopic expression of Gcm1 in mouse embryonic tail bud profoundly affects the development of the nervous system. All mice from 5 independently derived transgenic lines exhibited either one or both of two types of congenital spinal cord pathologies: failure of the neural tube to close (spina bifida) and multiple neural tubes (diastematomyelia). Because the transgene is expressed only in a restricted caudal region and only for a brief interval (E8.5 to E13.5), there was no evidence of embryonic lethality. The dysraphisms develop during the period and within the zone of transgene expression. We present evidence that these dysraphisms result from an inhibition of neuropore closure and a stimulation of secondary neurulation. After transgene expression ceases, the spinal dysraphisms are progressively resolved and the neonatal animals, while showing signs of scarring and tissue resorption, have a closed vertebral column. The multiple spinal cords remain but are enclosed in a single spinal column as in the human diastematomyelia. The animals live a normal life time, are fertile and do not exhibit any obvious weakness or motor disabilities.

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Senescence-associated down-regulation of 1-aminocyclopropane-1-carboxylate (ACC) oxidase delays harvest-induced senescence in broccoli

Functional Plant Biology ◽

10.1071/fp05076 ◽

2005 ◽

Vol 32 (10) ◽

pp. 891 ◽

Cited By ~ 22

Author(s):

Nigel E. Gapper ◽

Simon A. Coupe ◽

Marian J. McKenzie ◽

Richard W. Scott ◽

Mary C. Christey ◽

...

Keyword(s):

Ethylene Production ◽

Acc Oxidase ◽

Ethylene Biosynthesis ◽

Northern Analysis ◽

Marker Genes ◽

Total Protein Content ◽

Protein Accumulation ◽

Delayed Senescence ◽

Transgenic Lines ◽

Key Enzyme

To gain an in-depth understanding of the role of ethylene in post harvest senescence, we used broccoli (Brassica oleracea var. italica) as our model species. The senescence-associated asparagine synthetase (AS) promoter from asparagus was used to drive the expression of an antisense 1-aminocyclopropane-1-carboxylate oxidase (ACO) cDNA from broccoli, BoACO2, to reduce ethylene production following harvest. Physiological analyses revealed that transgenic broccoli lines harbouring the antisense BoACO2 gene construct (designated as AS-asACO) displayed delayed senescence in both detached leaves and detached heads as measured by hue angle. Harvested floret tissue from these plants also showed a delayed loss of chlorophyll, lower protease activity and higher total protein content, and changes in transcript levels of senescence marker genes when compared with wild type and transgenic lines transformed with an empty T-DNA. Genes that were down-regulated included those coding for cysteine protease (BoCP5), metallothionein-like protein (BoMT1), hexokinase (BoHK1), invertase (BoINV1) and sucrose transporters (BoSUC1 and BoSUC2). Northern analysis for BoACO1 and BoACO2, ACO assays and western analysis, revealed reduced ACO transcript, enzyme activity and protein accumulation, as well as reduced ethylene production in the transgenic AS-asACO lines when compared with controls, confirming that a key enzyme regulating ethylene biosynthesis was reduced in these plants. This, together with the changes observed in gene expression, confirm a significant role for ethylene in regulating the events leading to senescence in broccoli following harvest.

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The Ectopic Expression of a Pectin Methyl Esterase Inhibitor Increases Pectin Methyl Esterification and Limits Fungal Diseases in Wheat

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-01-11-0021 ◽

2011 ◽

Vol 24 (9) ◽

pp. 1012-1019 ◽

Cited By ~ 97

Author(s):

Chiara Volpi ◽

Michela Janni ◽

Vincenzo Lionetti ◽

Daniela Bellincampi ◽

Francesco Favaron ◽

...

Keyword(s):

Cell Wall ◽

Fungal Pathogens ◽

Ectopic Expression ◽

Bipolaris Sorokiniana ◽

Specific Protein ◽

Pectin Methyl Esterase ◽

Primary Role ◽

Methyl Esterification ◽

Pectic Enzymes ◽

Transgenic Lines

Cell wall pectin methyl esterification can influence plant resistance because highly methyl-esterified pectin can be less susceptible to the hydrolysis by pectic enzymes such as fungal endopolygalacturonases (PG). Pectin is secreted into the cell wall in a highly methyl-esterified form and, here, is de-methyl esterified by pectin methyl esterase (PME). The activity of PME is controlled by specific protein inhibitors called PMEI; consequently, an increased inhibition of PME by PMEI might modify the pectin methyl esterification. In order to test the possibility of improving wheat resistance by modifying the methyl esterification of pectin cell wall, we have produced durum wheat transgenic lines expressing the PMEI from Actinidia chinensis (AcPMEI). The expression of AcPMEI endows wheat with a reduced endogenous PME activity, and transgenic lines expressing a high level of the inhibitor showed a significant increase in the degree of methyl esterification. These lines showed a significant reduction of disease symptoms caused by the fungal pathogens Bipolaris sorokiniana or Fusarium graminearum. This increased resistance was related to the impaired ability of these fungal pathogens to grow on methyl-esterified pectin and to a reduced activity of the fungal PG to hydrolyze methyl-esterified pectin. In addition to their importance for wheat improvement, these results highlight the primary role of pectin despite its low content in the wheat cell wall.

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Transgene coding of a key enzyme of the glycolytic pathway helps to decrease sugar content in potato tubers

Czech Journal of Genetics and Plant Breeding ◽

10.17221/147/2011-cjgpb ◽

2012 ◽

Vol 48 (No. 1) ◽

pp. 42-45 ◽

Cited By ~ 3

Author(s):

O. Navrátil ◽

P. Bucher ◽

J. Vacek

Keyword(s):

Cold Storage ◽

Reducing Sugars ◽

Sugar Content ◽

Field Trials ◽

Sugar Accumulation ◽

Parental Genotype ◽

Potato Tubers ◽

Bacterial Gene ◽

Transgenic Lines ◽

Key Enzyme

Cold-stored potato tubers gradually accumulate reducing sugars. A proposed reason is a cold-induced blocking of glycolysis. The introduction and expression of the bacterial gene Lbpfk coding for cold-tolerant phosphofructokinase might counteract this effect. We have recently introduced this gene into several Czech potato cultivars. The obtained transgenic lines were then tested for three years in field trials. In 17 transgenic lines derived from two of the cultivars we have investigated the accumulation of reducing sugars during two and four months of cold storage. Although in all transgenic lines the sugar content still increased between the 2<sup>nd</sup> and the 4<sup>th</sup> month of cold-storage, the level of reducing sugars was in all transgenic lines after both two and four months of cold storage considerably lower than in the original cultivars. The extent of sugar accumulation was also influenced by the parental genotype. No significant differences in sugar accumulation were observed between the transgenic lines from the same parent. 

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Cloning and functional characterization of pig CMP-N-acetylneuraminic acid hydroxylase for the synthesis of N-glycolylneuraminic acid as the xenoantigenic determinant in pig–human xenotransplantation

Biochemical Journal ◽

10.1042/bj20090835 ◽

2010 ◽

Vol 427 (1) ◽

pp. 179-188 ◽

Cited By ~ 37

Author(s):

Kwon-Ho Song ◽

Yun-Jeong Kang ◽

Un-Ho Jin ◽

Yong-Il Park ◽

Sung-Min Kim ◽

...

Keyword(s):

Small Intestine ◽

Functional Characterization ◽

Ectopic Expression ◽

Vascular Endothelial ◽

Reading Frame ◽

Transfected Cells ◽

Acetylneuraminic Acid ◽

Rejection Response ◽

Key Enzyme ◽

Human Xenotransplantation

In the present study, the pig CMP-N-acetylneuraminic acid hydroxylase gene (pcmah), a key enzyme for the synthesis of NeuGc (N-glycolylneuraminic acid), was cloned from pig small intestine and characterized. The ORF (open reading frame) of pcmah was 1734 bp, encoding 577 amino acids and consisting of 14 exons. Organ expression pattern analysis reveals that pcmah mRNA is mainly expressed in pig rectum, tongue, spleen and colon tissues, being the most highly expressed in small intestine. In the ectopic expression of pcmah, when pig kidney PK15 cells and human vascular endothelial ECV304 cells were transfected with the cloned pcmah, the NeuGc contents of these transfectants were greater in comparison with vector transfectants used as controls. In addition, in the functional analysis of NeuGc, HSMC (human-serum-mediated cytotoxicity) was elevated in the ectopic NeuGc-expressing pcmah-transfected cells compared with controls. Moreover, binding of human IgM to the pcmah-transfected cells was significantly increased, whereas binding of IgG was slightly increased, indicating that the human IgM type was a major anti-NeuGc antibody. Furthermore, pcmah silencing by shRNA (short hairpin RNA) resulted in a decrease in NeuGc content and xenoantigenicity in PK15. From the results, it was concluded that the pcmah gene was capable of synthesizing the NeuGc acting as a xenoantigen in humans, confirming the NeuGc-mediated rejection response in pig–human xenotransplantation.

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Transgenic lines of Begonia maculata generated by ectopic expression of PttKN1

Biologia ◽

10.2478/s11756-011-0008-3 ◽

2011 ◽

Vol 66 (2) ◽

Cited By ~ 1

Author(s):

Quan-le Xu ◽

Jiang-ling Dong ◽

Nan Gao ◽

Mei-yu Ruan ◽

Hai-yan Jia ◽

...

Keyword(s):

Transgenic Plants ◽

Ectopic Expression ◽

Class I ◽

Wild Type Plant ◽

Wild Type ◽

Knox Gene ◽

Knox Genes ◽

Pttkn1 Gene ◽

Transgenic Lines ◽

First Time

AbstractKNOX (KNOTTED1-like homeobox) genes encode homeodomain-containing transcription factors which play crucial roles in meristem maintenance and proper patterning of organ initiation. PttKN1 gene, isolated from the vascular cambium of hybrid aspen (Populus tremula × P. tremuloides), is a member of class I KNOX gene family. In order to understand the roles of PttKN1 gene in meristem activity and morphogenesis as well as to explore the possibility to generate novel ornamental lines via its ectopic expression, it was introduced into the genome of Begonia maculata Raddi by Agrobacterium tumefasciens-mediated gene transformation here. Four types of transgenic plants were observed, namely coral-like (CL) type, ectopic foliole (EF) type, phyllotaxy-irregular (IP) type and cup-shaped (CS) type, which were remarkably different from corresponding wild type and were not also observed in the regenerated plantlets of wild type plant. Among these four types of transgenic plants, the phenotype of coral-like was observed for the first time in the transformants ectopically expressed KNOX genes. The observation of scanning electron microscope (SEM) showed ectopic meristems on the adaxial leaf surface of the transformants. Interestingly, the plantlets with ectopic foliole could generate new ectopic folioles from the original ectopic folioles again, and the plants regenerated from the EF-type transformants could also maintain the original morphology. The same specific RT-PCR band of the four types of transgenic plantlets showed that PttKN1 was ectopically expressed. All these data demonstrated that the ectopic expression of PttKN1 caused a series of alterations in morphology which provided possibilities producing novel ornamental lines and that PttKN1 played important roles in meristem initiation, maintenance and organogenesis events as other class I KNOX genes.

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