Validation of reference genes for gene expression analysis of response to anthocyanin induction in cell cultures of Vitis davidii (Rom. Caill.) Foëx

Lai, Chengchun; Pan, Hong; Xiangui, Huang; Fan, Li‐Hua; Duan, Chang‐Qing; Li, Shaozhen

doi:10.1007/s11627-018-9937-7

Cited by 6 publications

(6 citation statements)

References 60 publications

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“…The red callus of spine grape was used in this study. This red callus was derived from immature embryos of spine grape and was subjected to long-term culture on solid MS medium (Murashige and Skoog medium) supplemented with 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), sucrose 30 g/L, and agar 6 g/L (pH 5.8), and the callus subculture cycle was 20 days …”

Section: Methodsmentioning

confidence: 99%

Light Quality Modulates Growth, Triggers Differential Accumulation of Phenolic Compounds, and Changes the Total Antioxidant Capacity in the Red Callus of Vitis davidii

Lai

Pan

Zhang

et al. 2022

J. Agric. Food Chem.

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Light quality is one of the key elicitors that directly affect plant cell growth and biosynthesis of secondary metabolites. In this study, the red callus of spine grape was cultured under nine light qualities (namely, dark, white, red, yellow, blue, green, purple, warm-yellow, and warm-white light). The effects of different light qualities were studied on callus growth, accumulation of phenolic compounds, and total antioxidant capacity of the red callus of spine grape. The results showed that blue and purple light induced increased red coloration in the callus, whereas yellow light induced the greatest callus proliferation. Among all of the light quality treatments, darkness treatment downregulated the contents of phenolic compounds, whereas blue light was the treatment most conducive to the accumulation of total phenolics. White, blue, and purple light induced increased anthocyanin accumulation. Mixed-wavelength light was beneficial to the accumulation of flavonoids. Blue and purple light were conducive to the accumulation of proanthocyanidins. A further study showed that cyanidin 3-glucoside (Cy3G) and peonidin 3-glucoside (P3G) were the main anthocyanin components in the callus, and blue, purple, and white light treatments promoted their accumulation, whereas flavan-3-ols and flavonols were the main components of non-anthocyanin phenolics, and their accumulation changed in response to not only light quality but also culture duration. The total antioxidant capacity of the callus cultures changed significantly in response to different light qualities. These results will provide evidence for an abiotic elicitor strategy to stimulate callus growth and enhance the accumulation of the main phenolic compounds in the red callus of spine grape.

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Section: Methodsmentioning

confidence: 99%

Light Quality Modulates Growth, Triggers Differential Accumulation of Phenolic Compounds, and Changes the Total Antioxidant Capacity in the Red Callus of Vitis davidii

Lai

Pan

Zhang

et al. 2022

J. Agric. Food Chem.

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“…For example, the three least stable candidates were EF-hand, actin, and NAD5 based on geNorm; actin, EF-hand, and NAD5 by NormFinder; and NAD5, GDT1, and EF-Hand by BestKeeper (Table 3). It is important to note that both actin and NAD5, two conventional reference genes commonly reported in past gene expression literature [4,[91][92][93][94], ranked among the least stable by all three algorithms, with the exception that actin was ranked in the middle by BestKeeper.…”

Section: Expression Stability Of Reference Gene Candidatesmentioning

confidence: 98%

“…NAD5 and actin were two of the conventional reference genes used in gene expression studies involving RT-qPCR [4,[91][92][93][94]. They were regarded as the most suitable reference genes in the context of GLRaV-3 infection in grapevine leaf [4].…”

Section: Novel Reference Genes Outperformed Traditional Reference Genes In Transcripts Quantificationmentioning

confidence: 99%

Genome-wide screening of novel RT-qPCR reference genes for study of GLRaV-3 infection in wine grapes and refinement of an RNA isolation protocol for grape berries

2021

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Background Grapevine, as an essential fruit crop with high economic values, has been the focus of molecular studies in diverse areas. Two challenges exist in the grapevine research field: (i) the lack of a rapid, user-friendly and effective RNA isolation protocol for mature dark-skinned berries and, (ii) the lack of validated reference genes that are stable for quantification of gene expression across desired experimental conditions. Successful isolation of RNA with sufficient yield and quality is essential for downstream analyses involving nucleic acids. However, ripe berries of dark-skinned grape cultivars are notoriously challenging in RNA isolation due to high contents of polyphenolics, polysaccharides, RNase and water. Results We have optimized an RNA isolation protocol through modulating two factors at the lysis step that could impact results of RNA isolation - 2-ME concentration and berry mass. By finding the optimal combination among the two factors, our refined protocol was highly effective in isolating total RNA with high yield and quality from whole mature berries of an array of dark-skinned wine grape cultivars. Our protocol takes a much shorter time to complete, is highly effective, and eliminates the requirement for hazardous organic solvents. We have also shown that the resulting RNA preps were suitable for multiple downstream analyses, including the detection of viruses and amplification of grapevine genes using reverse transcription-polymerase chain reaction (RT-PCR), gene expression analysis via quantitative reverse transcription PCR (RT-qPCR), and RNA Sequencing (RNA-Seq). By using RNA-Seq data derived from Cabernet Franc, we have identified seven novel reference gene candidates (CYSP, NDUFS8, YLS8, EIF5A2, Gluc, GDT1, and EF-Hand) with stable expression across two tissue types, three developmental stages and status of infection with grapevine leafroll-associated virus 3 (GLRaV-3). We evaluated the stability of these candidate genes together with two conventional reference genes (actin and NAD5) using geNorm, NormFinder and BestKeeper. We found that the novel reference gene candidates outperformed both actin and NAD5. The three most stable reference genes were CYSP, NDUFS8 and YSL8, whereas actin and NAD5 were among the least stable. We further tested if there would be a difference in RT-qPCR quantification results when the most stable (CYSP) and the least stable (actin and NAD5) genes were used for normalization. We concluded that both actin and NAD5 led to erroneous RT-qPCR results in determining the statistical significance and fold-change values of gene expressional change. Conclusions We have formulated a rapid, safe and highly effective protocol for isolating RNA from recalcitrant berry tissue of wine grapes. The resulting RNA is of high quality and suitable for RT-qPCR and RNA-Seq. We have identified and validated a set of novel reference genes based on RNA-Seq dataset. We have shown that these new reference genes are superior over actin and NAD5, two of the conventional reference genes commonly used in early studies.

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“…Anthocyanins are natural, water-soluble glycosylated subclass of flavonoids, providing a diverse color range from orange, red, purple to blue in fruit plants and crops [57,58]. They fulfill a range of specific and vital roles in plants, such as protection (from ultraviolet irradiation, pathogen attack, photo-oxidative injury, and temperature) and are used to attract pollinators and facilitate seeds dispersal [59,60].…”

Section: Metabolic Engineering Of Anthocyanins Pathway In Plant In VImentioning

confidence: 99%

“…The anthocyanin biosynthesis pathway has been studied in several plant in vitro model systems, in order to understand and fine-tune its molecular regulation, e.g., V. vinifera [65], A. thaliana [61], Daucus carota [66], Hypericum perforatum [67], Ocimum basilicum [68], and Panax sikkimensis [69] investigating the effect of elicitors [14], precursors [13], plant growth regulators [61], nutrient supply [70,71], and light [72]. In synchronized grape cell cultures, the CHS enzyme [73], as well as, DFR has been recognized as essential contributors for the anthocyanins biosynthesis [57]. The significance of F3H gene was proven in purple-colored carrot callus.…”

Section: Metabolic Engineering Of Anthocyanins Pathway In Plant In VImentioning

confidence: 99%

Plant In Vitro Systems as a Sustainable Source of Active Ingredients for Cosmeceutical Application

Marchev

Georgiev

2020

Molecules

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Cosmeceuticals are hybrids between cosmetics and pharmaceuticals which are being designed for a dual purpose: (1) To provide desired esthetical effects and (2) simultaneously treat dermatological conditions. The increased demand for natural remedies and the trends to use natural and safe ingredients resulted in intensive cultivation of medicinal plants. However, in many cases the whole process of plant cultivation, complex extraction procedure, and purification of the targeted molecules are not economically feasible. Therefore, the desired production of natural cosmetic products in sustainable and controllable fashion in the last years led to the intensive utilization of plant cell culture technology. The present review aims to highlight examples of biosynthesis of active ingredients derived through plant in vitro systems with potential cosmeceutical application. The exploitation of different type of extracts used in a possible cosmeceutical formulation, as well as, their activity tested in in vitro/in vivo models is thoroughly discussed. Furthermore, opportunities to manipulate the biosynthetic pathway, hence engineering the biosynthesis of some secondary metabolites, such as anthocyanins, have been highlighted.

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Validation of reference genes for gene expression analysis of response to anthocyanin induction in cell cultures of Vitis davidii (Rom. Caill.) Foëx

Cited by 6 publications

References 60 publications

Light Quality Modulates Growth, Triggers Differential Accumulation of Phenolic Compounds, and Changes the Total Antioxidant Capacity in the Red Callus of Vitis davidii

Light Quality Modulates Growth, Triggers Differential Accumulation of Phenolic Compounds, and Changes the Total Antioxidant Capacity in the Red Callus of Vitis davidii

Genome-wide screening of novel RT-qPCR reference genes for study of GLRaV-3 infection in wine grapes and refinement of an RNA isolation protocol for grape berries

Plant In Vitro Systems as a Sustainable Source of Active Ingredients for Cosmeceutical Application

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