Extraction of Total RNA from Leaves of
            <i>Eucalyptus</i>
            and Other Woody and Herbaceous Plants Using Sodium Isoascorbate

Suzuki, Yasuyuki; Hibino, Takashi; Kawazu, Tetsu; Wada, T.; Kihara, Tomonori; Koyama, Hideki

doi:10.2144/03345st05

Cited by 24 publications

(10 citation statements)

References 10 publications

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“…We used the QIAgen Plant RNeasy kit to extract RNA (QIAGEN, Valencia California). We followed the manufacturer’s instructions, but added 50 μl of 20% polyvinylpyrrolidone (PVP) and 108 mg of sodium isoascorbate (Na-iASC) to the lysis buffer to remove phenolic compounds and polysaccharides that interfere with RNA extraction and downstream applications [35-37]. We used the Oligotex Direct mRNA Mini Kit (Qiagen, Valencia California) to purify mRNA from these samples, following the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Differences in gene expression within a striking phenotypic mosaic Eucalyptus tree that varies in susceptibility to herbivory

et al. 2013

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BackgroundLong-lived trees can accumulate mutations throughout their lifetimes that may influence biotic and abiotic interactions. For example, some Eucalyptus trees display marked variation in herbivore defence within a single canopy. These “mosaic” trees support foliage with distinct chemotypes which are differentially favoured by insect and vertebrate herbivores, resulting in susceptible and resistant branches within a single canopy. These mosaic trees provide a unique opportunity to explore the biosynthesis and genetic regulation of chemical defences in the foliage. The biosynthesis of the principal defence compounds, terpenoid-dominated essential oils, is well understood. However, the regulation of the genes involved and thus the control of phenotypic variation within a single tree canopy remains a mystery.ResultsWe sequenced the transcriptomes of the leaves of the two different chemotypes of a chemically mosaic Eucalyptus melliodora tree using 454 pyrosequencing technology. We used gene set enrichment analysis to identify differentially expressed transcripts and found the proportion of differentially expressed genes in the resistant and susceptible foliage similar to the transcript difference between functionally distinct tissues of the same organism, for example roots and leaves. We also investigated sequence differences in the form of single nucleotide polymorphisms and found 10 nucleotides that were different between the two branches. These are likely true SNPs and several occur in regulatory genes.ConclusionWe found three lines of evidence that suggest changes to a ‘master switch’ can result in large scale phenotypic changes: 1. We found differential expression of terpene biosynthetic genes between the two chemotypes that could contribute to chemical variation within this plant. 2. We identified many genes that are differentially expressed between the two chemotypes, including some unique genes in each branch. These genes are involved in a variety of processes within the plant and many could contribute to the regulation of secondary metabolism, thus contributing to the chemical variation. 3. We identified 10 SNPs, some of which occur in regulatory genes that could influence secondary metabolism and thus contribute to chemical variation. Whilst this research is inherently limited by sample size, the patterns we describe could be indicative of other plant genetic mosaics.

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Section: Methodsmentioning

confidence: 99%

Differences in gene expression within a striking phenotypic mosaic Eucalyptus tree that varies in susceptibility to herbivory

et al. 2013

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“…Total RNA was extracted according to Suzuki et al (2003) with slight modifications. The concentration of Tris-HCl in the extraction buffer was changed from 100 mm to 50 mm.…”

Section: Rna Analysis and Cloning Of E Globulus Rbcs Cdnasmentioning

confidence: 99%

Differences in Rubisco content and its synthesis in leaves at different positions in Eucalyptus globulus seedlings

Suzuki

Kihara-Doi

Kawazu

et al. 2010

Plant Cell & Environment

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The dynamics of ribulose 1·5-bisphosphate carboxylase/ oxygenase (Rubisco) content and turnover during leaf development are not well understood in woody plants. Rubisco synthesis, N influx and the mRNA levels of Rubisco-encoding genes were determined as a function of leaf position in 4.5-month-old Eucalyptus globulus seedlings. Rubisco concentration was slightly higher in the top leaves as leaf expansion progressed and was almost maximal in the uppermost fully expanded leaves. Rubisco concentration remained almost constant in the fully expanded leaves at the top and middle positions and then became slightly low at the lowest positions. Rubisco synthesis was active only in the top leaves. These results suggest that Rubisco turnover rate is low in the middle leaves, leading to the maintenance of Rubisco contents, and that Rubisco degradation primarily occurs in the lowest leaves. Changes in the RBCS and rbcL mRNA levels were roughly parallel with Rubisco synthesis, but N influx was more closely correlated with Rubisco synthesis. These results suggest that N influx rather than the transcript abundance of Rubisco-encoding genes is of primary importance in regulating the rate of Rubisco synthesis. Additionally, expression of RBCS multigene family in E. globulus leaves was discussed.

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“…( A ) Expression of AtALMT1 in the roots of Col-0 and transgenic Col-0 carrying CaMV 35S-driven AtALMT1 (35S- AtALMT1 /Col-0) with or without Al. RNA extraction and cDNA synthesis were performed as described by Suzuki et al 12 The primer pairs 5′-GGCCGACCGT GCTATACGAG-3′ and 5′-CATGAGTCCT GTGAACTCCC-3′, and 5′-TCGTAAGTAC AATCAGGATA AGATG-3′ and 5′-CACTGAAACA AGAAAAACAA ACCCT-3′ were used for the semiquantitative RT-PCR of AtALMT1 , and the internal standard UBQ1 , respectively. ( B ) Malate exudation from the roots of Col-0 and 35S- AtALMT1 /Col-0.…”

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confidence: 99%

Overexpression ofAtALMT1in theArabidopsis thalianaecotype Columbia results in enhanced Al-activated malate excretion and beneficial bacterium recruitment

Lakshmanan

Kobayashi

Asai

et al. 2013

Plant Signaling & Behavior

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AtALMT1 (Arabidopsis thaliana ALuminum activated Malate Transporter 1) encodes an Arabidopsis thaliana malate transporter that has a pleiotropic role in Arabidopsis stress tolerance. Malate released through AtALMT1 protects the root tip from Al rhizotoxicity, and recruits beneficial rhizobacteria that induce plant immunity. To examine whether the overexpression of AtALMT1 can improve these traits, the gene, driven by the cauliflower mosaic virus 35S promoter, was introduced into the Arabidopsis ecotype Columbia. Overexpression of the gene enhanced both Al-activated malate excretion and the recruitment of beneficial bacteria Bacillus subtilis strain FB17. These findings suggest that overexpression of AtALMT1 can be used as an approach to enhance a plant's ability to release malate into the rhizosphere, which can enhance plant tolerance to some environmental stress factors.

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Extraction of Total RNA from Leaves of Eucalyptus and Other Woody and Herbaceous Plants Using Sodium Isoascorbate

Cited by 24 publications

References 10 publications

Differences in gene expression within a striking phenotypic mosaic Eucalyptus tree that varies in susceptibility to herbivory

Differences in gene expression within a striking phenotypic mosaic Eucalyptus tree that varies in susceptibility to herbivory

Differences in Rubisco content and its synthesis in leaves at different positions in Eucalyptus globulus seedlings

Overexpression ofAtALMT1in theArabidopsis thalianaecotype Columbia results in enhanced Al-activated malate excretion and beneficial bacterium recruitment

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