A micromethod for high throughput RNA extraction in forest trees

Provost, Grégoire Le; Herrera, Raúl; Paiva, Jorge A P; Chaumeil, Philippe; Salin, Franck; Plomion, Christophe

doi:10.4067/s0716-97602007000400003

Cited by 110 publications

(47 citation statements)

References 15 publications

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“…Compared to other protocols developed for RNA extraction from recalcitrant species and/or tissues (Li & Gray 1997, Sanchez & Mariani 2002, Fils-Lycaon et al 1996, MacLean et al 1996, Birtic & Kranner 2006, De Keukeleire et al 2006, Le Provost et al 2007, Fort et al 2008, Wang et al 2008, the presented method combines the advantages of being phenol-free, easily applicable to a wide range of tissues and not requiring ex pensive commercial kits. Moreover, the varying amounts of starting material needed for different tissues (from 20 mg of styles to 1 g of fruit flesh or peel) enabled testing of the protocol's scalability.…”

Section: Discussionmentioning

confidence: 99%

Total RNA extraction from strawberry tree (Arbutus unedo) and several other woody-plants

Zamboni¹,

Pierantoni²,

Franceschi³

2008

iForest

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© iForest -Biogeosciences and Forestry IntroductionRNA extraction from specific plant organs and tissues is a preliminary step for many molecular studies in plant biology. However, isolation of high-quality RNA from some tis sues is often difficult due to the high amounts of polyphenols, polysaccharides and other secondary metabolites they accu mulate. These contaminants tend to co-puri fy and co-precipitate with the RNA in the presence of alcohols, thereby remaining in the final extracts and interfering with such downstream applications as cDNA synthesis, restriction endonuclease enzyme digestion and the establishment of cDNA libraries (Salzman et al. 1999). These problems often occur during RNA extraction from recalcit rant plants and, especially, from reserve-rich organs like fruits and seeds. In addition, the biosynthesis and accumulation of secondary metabolites in plants under biotic and abiotic stress, such as pathogen infection and drought, can be significantly enhanced (Winkel-Shirley 2002, Chaves et al. 2003. While kits supplied by biotechnology com panies extract RNA successfully from many tissues, they proved ineffective on tissues rich in polyphenols or polysaccharides (Kiefer et al. 2000).Several protocols for RNA isolation from tissues of species with high contents of poly phenols or polysaccharides have been repor ted, including methods using soluble polyvinylpyrrolidone and ethanol precipita tion (Salzman et al. 1999), hot borate (Wan & Wilkins 1994), phenol extraction (Kom janc et al. 1999), calcium precipitation (Dal Cin et al. 2005), 2-butoxyethanol (Malnoy et al. 2001, Manning 1991 or cetyl trimethyl ammonium bromide (CTAB) in the extrac tion buffer (Meisel et al. 2005). We tested four of these protocols (Komjanc et al. 1999, Malnoy et al. 2001, Manning 1991, Meisel et al. 2005 and two commercially available RNA extraction kits to secure high-quality RNA in good amounts from an extremely re calcitrant plant such as strawberry tree (Ar butus unedo); the overall results proved un satisfactory for all the methods, although the one described by Meisel et al. gave better results in terms of RNA purity compared with the others. We thus developed and tested a modified protocol that, through a simple optimization of some critical steps, allowed to achieve a great improvement in both RNA yield and purity when extracting from strawberry tree leaves. The effective ness and versatility of this method was sub sequently tested on different tissues (styles, fruit peel, fruit flesh, roots, leaves and seeds) from Pyrus communis, Prunus avium, Prun us persica and Cydonia oblonga, yielding in all cases adequate RNA amounts with a good purity grade. Materials and Methods Plant materialAll plant materials used for RNA extrac tion were grown in the experimental fields of the Department of Fruit Tree and Woody Plant Sciences (Cadriano, Bologna, Italy). The tissues were completely differentiated and healthy. Pear tree styles were harvested immediately before bloom and fruit peel col lected in the course of ripeni...

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Section: Discussionmentioning

confidence: 99%

Total RNA extraction from strawberry tree (Arbutus unedo) and several other woody-plants

Zamboni¹,

Pierantoni²,

Franceschi³

2008

iForest

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“…All collected samples were ground in liquid nitrogen and a total RNA extraction was performed from 2 g of plant material, according to an optimized method from Provost and colleagues [23]. Then, a DNase treatment was carried out following the instructions of the manufacturer (Kit TURBO DNA-free by Life Technologies, Hong Kong, China).…”

Section: Rna Extraction Cdna Synthesis Library Preparation and Sequmentioning

confidence: 99%

Expression Profiling in Pinus pinaster in Response to Infection with the Pine Wood Nematode Bursaphelenchus xylophilus

Gaspar

Trindade

Usié

et al. 2017

Forests

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Abstract:Forests are essential resources on a global scale, not only for the ecological benefits, but also for economical and landscape purposes. However, in recent years, a large number of forest species have suffered a serious decline, with maritime pine being one of the most affected. In Portugal, the maritime pine forest has been devastated by the pine wood nematode (PWN), the causal agent of pine wilt disease. In this study, RNA-Seq data was used to characterize the maritime pine response to infection with PWN, by determining the differentially expressed genes and identifying the regulatory networks and pathways associated. The analyses showed clear differences between an early response that occurs immediately after inoculation and a late response that is observed seven days after inoculation. Moreover, differentially expressed genes related to secondary metabolism, oxidative stress and defense against pathogen infection were identified over different time points. These results provide new insights about the molecular mechanisms and metabolic pathways involved in the response of Pinus pinaster against PWN infection, which will be a useful resource in follow-up studies and for future breeding programs to select plants with lower susceptibility to this disease.

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“…RNAwas extracted from 100 to 150 mg of ground leaf, petiole, and root material by incubation for 10 min with 900 mL of prewarmed hexadecyltrimethylammonium bromide extraction buffer (Chang et al, 1993) at 65°C and 660 rpm. After 2-fold extraction with 900 mL of chloroform-isoamyl alcohol, RNA was precipitated overnight (4°C) from the supernatant by the addition of LiCl to a final concentration of 2 M. Pellets were dissolved in 500 mL of sodium chloride-Tris-EDTA buffer (le Provost et al, 2007; prewarmed to 65°C) containing 0.1% SDS. RNA was extracted with chloroform-isoamyl alcohol and precipitated from the aqueous phase by adjusting the NaCl concentration to 0.67 M, adding 1 volume of isopropanol, and subsequent incubation for 5 h at 220°C.…”

Section: Comparison Of Saponin Levels and In Planta Expression Of Ugtmentioning

confidence: 99%

UDP-Glycosyltransferases from the UGT73C Subfamily in Barbarea vulgaris Catalyze Sapogenin 3-O-Glucosylation in Saponin-Mediated Insect Resistance

et al. 2012

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Triterpenoid saponins are bioactive metabolites that have evolved recurrently in plants, presumably for defense. Their biosynthesis is poorly understood, as is the relationship between bioactivity and structure. Barbarea vulgaris is the only crucifer known to produce saponins. Hederagenin and oleanolic acid cellobioside make some B. vulgaris plants resistant to important insect pests, while other, susceptible plants produce different saponins. Resistance could be caused by glucosylation of the sapogenins. We identified four family 1 glycosyltransferases (UGTs) that catalyze 3-O-glucosylation of the sapogenins oleanolic acid and hederagenin. Among these, UGT73C10 and UGT73C11 show highest activity, substrate specificity and regiospecificity, and are under positive selection, while UGT73C12 and UGT73C13 show lower substrate specificity and regiospecificity and are under purifying selection. The expression of UGT73C10 and UGT73C11 in different B. vulgaris organs correlates with saponin abundance. Monoglucosylated hederagenin and oleanolic acid were produced in vitro and tested for effects on P. nemorum. 3-Ob-D-Glc hederagenin strongly deterred feeding, while 3-O-b-D-Glc oleanolic acid only had a minor effect, showing that hydroxylation of C23 is important for resistance to this herbivore. The closest homolog in Arabidopsis thaliana, UGT73C5, only showed weak activity toward sapogenins. This indicates that UGT73C10 and UGT73C11 have neofunctionalized to specifically glucosylate sapogenins at the C3 position and demonstrates that C3 monoglucosylation activates resistance. As the UGTs from both the resistant and susceptible types of B. vulgaris glucosylate sapogenins and are not located in the known quantitative trait loci for resistance, the difference between the susceptible and resistant plant types is determined at an earlier stage in saponin biosynthesis.

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A micromethod for high throughput RNA extraction in forest trees

Cited by 110 publications

References 15 publications

Total RNA extraction from strawberry tree (Arbutus unedo) and several other woody-plants

Total RNA extraction from strawberry tree (Arbutus unedo) and several other woody-plants

Expression Profiling in Pinus pinaster in Response to Infection with the Pine Wood Nematode Bursaphelenchus xylophilus

UDP-Glycosyltransferases from the UGT73C Subfamily in Barbarea vulgaris Catalyze Sapogenin 3-O-Glucosylation in Saponin-Mediated Insect Resistance

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