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WO2006114434A1 - Utilisation de genes du complexe elongator pour renforcer la tolerance de vigueur et au stress dans des cellules eucaryotes - Google Patents

Utilisation de genes du complexe elongator pour renforcer la tolerance de vigueur et au stress dans des cellules eucaryotes Download PDF

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WO2006114434A1
WO2006114434A1 PCT/EP2006/061854 EP2006061854W WO2006114434A1 WO 2006114434 A1 WO2006114434 A1 WO 2006114434A1 EP 2006061854 W EP2006061854 W EP 2006061854W WO 2006114434 A1 WO2006114434 A1 WO 2006114434A1
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elongator
stress
protein
genes
plants
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Maria Van Lijsebettens
Dirk Gustaaf INZÉ
Hilde Nelissen
Delphine Fleury
Marc De Block
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Vib Vzw
Universiteit Gent
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to the use of proteins of the Elongator complex, and genes encoding those proteins to modulate vigour and stress tolerance in eukaryotic cells, preferably plant cells. More specifically, it relates to overexpressing of genes of the Elongator complex, and particularly overexpressing of EL03, to obtain increased vigour and stress tolerance.
  • the "histone code” represents the interplay between the different post-translational modifications of the core histones, including methylation and acetylation and provides a signal for downstream processes, such as transcription and DNA repair.
  • Elongator is a histone acetyl transferase (HAT) complex, consisting of six subunits (ELP1 to ELP6), that co-purifies with the elongating RNA Polymerase Il (RNAP II) in yeast and humans.
  • HAT histone acetyl transferase
  • the Elongator complex was mainly described in yeast, allowing us to look for analogies between the unicellular yeast and the multicellular plant. Loss of Elongator function in yeast results in a delayed activation of gene expression, thermosensitivity, sensitivity towards several drugs and a slow growth in changing conditions (Otero et al., 1999; Wittschieben et al., 1999; Frohloff et al., 2001 ). Some of these phenotypes suggest that also in yeast the Elongator complex is involved in the communication with the environment.
  • Elongator complex In human, the information on the Elongator complex is not as extensive as in yeast, but a mutation in the ELP1 component is thought to be responsible for the severe hereditary disorder familial disautonomia (Slaugenhaupt and Gusella, 2002). This neurodevelopmental genetic disorder affects the development and maintenance of sensory and autonomic neurons, resulting in a wide range of pathologies primarily because of a malperception of stimuli from the environment and during development (Axelrod, 2004).
  • the elongata (elol, elo2, elo3, and elo4) mutations were identified in a large-scale screening of EMS-mutagenized Arabidopsis thaliana (L.) Heynh for mutants with abnormally shaped leaves (Berna et al., 1999) and were then mapped at low resolution by linkage analysis (Robles and Micol, 2001).
  • the elo mutants have a narrow leaf phenotype similar to that of the null mutant drl1-2, which in turn was allelic to elo4, which was therefore named drl1-4 (Nelissen et al., 2003).
  • DRL1 was shown to be the orthologue of the yeast KTI12 protein, a putative regulator of the Elongator complex (Fitchner et al., 2002). Homologues of the six structural components of Elongator are present in the Arabidopsis genome (Nelissen et al., 2003).
  • a first aspect of the invention is the use of a protein of the elongator complex and/or a nucleotide sequence encoding said protein to modulate vigour and stress tolerance in eukaryotic cells.
  • a plant is vigorous, versus a less performing plant, when the plant has a high energy use efficiency (hereafter expressed as TTC-reducing capacity) preferentially combined with a high energy production (hereafter expressed as NAD(P)H, ATP content).
  • TTC-reducing capacity a high energy use efficiency
  • NAD(P)H, ATP content a high energy production
  • the high productive energy metabolism (low TTC-reducing capacity and high NAD(P)H and ATP content) of vigorous plants results in high seed yield and/or biomass production and often broadens the optimal growth windows for many if not all growth factors as temperature, water availability, light availability
  • Polypeptide refers to a polymer of amino acids and does not refer to a specific length of the molecule. This term also includes post-translational modifications of the polypeptide, such as glycosylation, phosphorylation and acetylation.
  • Nucleotide sequence “DNA sequence” or “nucleic acid molecule(s)” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA, and RNA.
  • the proteins of the Elongator complex are known by the person skilled in the art, and are orthologues of the Arabidopsis thaliana ELOI (At3g11220), ELO2 (At5g13680), ELO3 (At5g50320), ELO4/DRL1 (At1g13870) genes (Nelissen et al., 2003), the Arabidopsis orthologues of yeast ELP2 (At1g49540), ELP5 (At2g18410) and ELP6 (At4g10090).
  • said eukaryotic cells are plant cells.
  • the protein of the Elongator complex is Elo3p, and the gene ELO3.
  • One preferred embodiment is the use of proteins of the elongator complex whereby said stress is oxidative stress, even more preferable, said stress is high light stress.
  • Another preferred embodiment is the use of proteins of the elongator complex whereby said stress is oxidative stress
  • the use is overexpression of ELO3 nucleic acid, to obtain increased vigour and stress tolerance.
  • Overexpression as used here means the expression level of the Elo3p in the ELO3 overexpressing cell is significantly higher than the expression level of Elo3p in a control cell that is genetically identical to the ELO3 overexpressing cell except for the modification needed for the overexpression, grown under the same conditions.
  • Coding sequence is a nucleotide sequence, which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences.
  • the boundaries of the coding sequence are determined by a translation start codon at the 5'- terminus and a translation stop codon at the 3'-terminus.
  • a coding sequence can include, but is not limited to mRNA, cDNA, recombinant nucleotide sequences or genomic DNA, while introns may be present as well under certain circumstances.
  • Operably linked refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a promoter sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the promoter sequence.
  • Another aspect of the invention is a method to obtain stress tolerant eukaryotic cells, by inducing overexpression of a gene encoding a protein of the Elongator complex.
  • said eukaryotic cells are plant cells.
  • the protein of the Elongator complex is Elo3p, and the gene ELO3.
  • One preferred embodiment is a method according to the invention whereby said stress is oxidative stress, even more preferably said stress is high light stress.
  • Another preferred embodiment is a method according to the invention whereby said stress is salt stress
  • transgenic stress resistant plant comprising one or more overexpressed Elongator complex genes.
  • said transgenic plant comprises at least an overexpressed ELO3 gene.
  • said transgenic plant is genetically identical to the non-transgenic, non-stress resistant parental plant, except for the modification needed for the overexpression of the Elongator complex genes.
  • said stress is selected form the group consisting of light stress and salt stress.
  • FIGURES Figure 1 Number of genes specifically differentially expressed in shoot apex of Elongator mutants (elo2, elo3 and drl1-2) compared to the wild-type Lerfrom microarrays method using ATH 1 chips.
  • the raw data were preprocessed by the GCRMA method using affy package of Bioconductor.
  • the difference of expression for a gene was significant at p ⁇ 5 % according to the Bayesian t test between the wild-type and each mutant after p correction by Holm's method using limma package of Bioconductor.
  • Figure 2 Genes down-regulated (A) and up-regulated (B) in shoot apex of Elongator mutants compared to the wild-type Ler using semi-quantitative RT-PCR method.
  • the star corresponds to significant difference at p ⁇ 5% between the wild-type Ler and each mutant from anova and
  • Figure 3 Leaf pigments concentration in Elongator mutants and the wild-type Ler.
  • Chlorophylls a (A) and b (B), carotenoids (C) and anthocyanins (D). Comparison were made between genotypes according to anova and Duncan's test at p ⁇ 5% with 4 replicates per genotype. Bars represent the standard deviation of mean.
  • FIG. 4 Jasmonic acid (JA) and methyl-jasmonate (JA-Me) concentration in aboveground organs of the wild-type Ler and elo2 mutant grown in in vitro conditions. ** and *** mean a significant difference between Ler and elo2 respectively at p ⁇ 0.01 and p ⁇ 0.001 according to the T test with 3 replicates for JA and 5 replicates for JA-Me. Bars represent the standard deviation of mean.
  • FIG. 5 High light experiment. CoI-O grown under normal light conditions (A.) and with high light treatment (B). The EL03 overexpression line B6 grown under normal light conditions (C) and with high light treatment (D).
  • Figure 6 TTC reducing capacity of the EL03 overexpressing transgenic lines. The concentration of reduced TTC was calculated taking the OD485 values and the chlorophyll pigment background into account and is expressed as the percentage TTC reducing capacity relative to the untransformed control.
  • Figure 7 Graphical representation of the values for the fresh weight, the TTC reducing capacity and the NADH content. For each assay the standard error is indicated. HL, high light conditions; LL, low light conditions.
  • Figure 8 Primary root growth of the ELP3B6.4 line and CoI on GM+V and GM+V+NaCI.
  • the medium used contained V ⁇ x MS salts (micro and macro elements), 1g/l sucrose, 0.5 g/l MES, pH 6.0, 6 g/l plant tissue culture agar. Sixty seeds were sowed in per 150 x 25 mm round dish, sealed with Urgopore tape and placed in darkness at 4 0 C for 3 days. The growth chamber conditions were: 16/8 hrs (d/n) with white light (Neon tubes, cool white), 100 ⁇ Em- 2 h-1 PAR and 20 0 C.
  • V ⁇ x MS salts micro and macro elements
  • 1g/l sucrose sucrose
  • 0.5 g/l MES pH 6.0
  • 6 g/l plant tissue culture agar 6 g/l plant tissue culture agar.
  • Sixty seeds were sowed in per 150 x 25 mm round dish, sealed with Urgopore tape and placed in darkness at 4 0 C for 3 days.
  • the growth chamber conditions were: 16/8 hrs (d/n) with white light (N
  • the shoot apex of plants (comprising shoot apex meristem, first and second leaf primordia at petiole-less stage) were harvested at growth stage 1.0 (Boyes et al., 2001), removing the cotyledons and the hypocotyls when the two first leaves appeared, in the lab conditions under additional light, at 20 0 C and at least 4 hours after the beginning of the photoperiod when photosynthetic activity of plants is stable .
  • the age of the plants at the harvesting step were between 8 and 15 days after sowing depending of the delay of mutant development.
  • the RNA was extracted according to the Trizol method (Gibco-BRL).
  • the experimental design comprised 3 replicates of each genotype, one replicate corresponding to one RNA extraction on a independent pool of plants.
  • RNA was fragmented with alkaline hydrolysis, resuspended with control spikes in 300 ⁇ l hybridization buffer (Eukaryotic Hybridization Control Kit, cat# 900299, Affymetrix, High Wycombe, UK) and hybridized at 45 0 C.
  • the genechips were washed and stained in the GeneChip Fluidics Station 400 (Affymetrix, UK) using EukGE- WS2 protocol, and subsequently scanned with the GeneChip Scanner (Affymetrix, UK). Image analysis was performed by Affymetrix's Microarray Suite 5.0.1 software.
  • the procedure includes a background adjustment step using estimators derived from a statistical model describing the relationship between perfect matches, mismatches and probe of the same G-C content, and to do a log normalization step (Wu et al., 2005).
  • the model is based on simple hybridization theory from molecular biology and specific experiments.
  • the final step in the pre-processing is to summarize the data combining the 11 probes pair intensities for a given gene to define an expression value representing the amount of the corresponding mRNA.
  • the genes were ranked in order of evidence for differential expression (DE) between the mutant and the wild-type using an empirical Bayesian t test performed with the limma package of Bioconductor. This method consists to combine at the gene level with means and standard deviation from the 3 replicates to form a statistic B which is a Bayes log posterior log-odds that each gene is DE (Lonnstedt and Speed, 2002; Smyth et al., 2003). Because of the few number of replicates, a stringent method to multiple testing (Holm's method) was used to control for family-wise error rate, which is the probability of at least one false positive among the genes selected as differentially expressed and so, to correct the P value. The cut-off value of p was
  • the semi-quantitative RT-PCR was done according to the QIAGEN OneStep RT-PCR Kit method.
  • the primers that amplified the target gene (At2g18600, At3g45140, At5g42650, At2g41410, At3g10190, At4g02280, At4g38210, At2g40610, At4g23990 and At4g25420) and the control (At3g18780) genes in the forward direction were both labelled by mixing 1 ⁇ M of primer, 1x of T 4 buffer, 0.2 unit of T4 polynucleotide kinase and 20 pmol of radiolabeled [v- 33 P]ATP at 6000 Ci/mmol in a final volume of 10 ⁇ l and incubated at 37°C for 40 min. for kination and at 80 0 C for 10 min. to stop the reaction.
  • the RT-PCR reaction was prepared as follows: 1x QIAGEN OneStep RT-PCR buffer, 0.4 mM dNTP, 0.6 ⁇ M R primer target gene, 1.5 ⁇ l labelled F primer of target gene, 0.08 to 0.24 ⁇ M R primer of control gene, 0.2 to 0.6 ⁇ l labelled F primer of control gene, 1x Enzymes mix, 5.2 units RNase Inhibitor, 200 ng RNA in a final volume of 25 ⁇ l.
  • the cDNA synthesis and the amplification were done in one step: 1 cycle at 50 0 C for 30 min., 1 cycle at 95°C for 15 min., 20 to 35 cycles at 94°C for 30 sec, 55 0 C for 30 sec. and 72°C for 1 min., and 1 cycle at 72°C for 10 min.
  • Loading buffer (20 ⁇ l) was added to the RT-PCR mix which was denaturated at 95 0 C for 3 min.
  • a 4 ⁇ l sample was loaded onto a 4.5 % denaturing polyacrylamide gel in vertical electrophoresis.
  • the samples were visualized after drying the gel and exposition using the phospholmager (model no. 445S1 , Molecular Dynamics, Sunnyvale, CA).
  • Quantitative data analysis gel intensity analysis and normalization was performed with AFLP-QUANTARPRO Software (Key gene, Wageningen, The Netherlands) (Breyne et al., 2003).
  • the gene expression data were statistically analyzed using SPSS (Chicago, IL, USA) by one-way analyses of variance with one fixed factor (genotypes) and 3 replicates, Bartlett's test to check the homogeneity of variances, and Tukey's pairwise comparisons between genotypes.
  • SPSS Chomicgo, IL, USA
  • the Ler, elol, elo2, elo3, elo4 and drl1-2 in vitro aboveground plants were harvested at first and second expanded leaves stage between 12 days and 20 days old, excluding the cotyledons and the hypocotyl (20-30 mg fresh weight).
  • the frozen tissues were grinded with metal balls in a high frequency shaking Retsch MM200 instrument (Retsch GmbH & Co. KG, Haan, Germany). Four pools of 10 plants from 4 dishes were used as 4 independent replicates for the pigments extraction.
  • the chlorophylls a and b were extracted by adding 3.5 ml of 80% acetone to the samples, centrifugation for 10 minutes at 5000 rpm and measuring the absorption using a spectrophotometer at 663 nm, 646 nm and 470 nm.
  • concentrations of chlorophyll a, b, carotenoids and anthocyanins were calculated per gram of fresh weight and the data analyzed statistically with SPSS by one-way analyses of variance with one fixed factor (genotypes), Bartlett's test to check the homogeneity of variances, and Tukey's pairwise comparisons between genotypes.
  • the Ler and elo2 in vitro aboveground plants were harvested at stage 1.04 between 12 days and 20 days old (about 200 mg of fresh tissue) (Boyes et al., 2001).
  • the frozen tissues were grinded with metal balls in a high frequency shaking Retsch instrument. Between 3 and 5 pools of plants were used as 3 to 5 independent replicates for the hormones extraction.
  • JA and Me-JA were separated using solid phase extraction as described for auxins by Prinsen et al. 2000. After solid phase extraction, JA was methylated with diazomethane before measuring by GC-MS (TRIO 2000, Waters-micromass, Manchester, UK) using electron impact mode. (El(+) GC-MS) and measured as Me-JA (diagnostic ions Me-JA: 224 amu; O 18 -Me-JA: 226 amu). The concentrations of jasmonic acid and methyl-jasmonate were calculated per gram of fresh weight and the data analyzed statistically with SPSS by t test to compare the wild-type Lerand the elo2 mutant.
  • Overexpression lines The Arabidopsis open reading frames (including ATG and stop codon) were amplified for ELO1 , ELO2, ELO3 and DRL1 and the homologues of ELP2, ELP5 and ELP6 introducing the attB1 and attB2 recombination sites via the primers. These PCR fragments were cloned into the pDONR 207 vector using the GATEWAYTM recombination strategy (Invitrogen) to obtain ENTRY clones.
  • GATEWAYTM recombination strategy Invitrogen
  • the ENTRY clones were then recombined with the pK7WG2 vector (Karimi et al., 2002) to obtain a DESTINATION vector in which the ORF under the control of a 35S promotor is present between the T-DNA borders, together with a Kanamycin resistance gene.
  • These constructs were introduced into Agrobacterium tumefaciens and subsequently CoI-O plants were transformed with the Agrobacterium tumefaciens suspension through floral dip.
  • the T 0 seeds were grown in high density on growth medium containing Kanamycin (50 ⁇ g/ml), Nystatin (50 ⁇ g/ml) and Carbenicillin (250 ⁇ g/ml) to select the transformants. These Ti transformants were transferred to soil to obtain T 2 seeds. The experiments described here were all done using the T 2 population, except for the ELP3B6.4, which is a homozygous F3.
  • the level of overexpression of the ELO3 gene for the ELP3B6.4 line in comparison to the CoI control was done using real-time quantitative PCR.
  • the RNA of the two lines was prepared using the Rneasy kit (Qiagen) and the cDNA synthesis was performed using the Superscript first strand synthesis kit (Invitrogen).
  • the quantitative PCRs were done on the iCycler real-time PCR machine with gene-specific primers, designed using the Beacon Designer software. De PCR products were detected through the measurement of the increase of fluorescence, caused by the binding of the SYBR GREEN I (Eurogentec) dye to double stranded DNA.
  • the level of expression of the ELO3 gene was normalized according to the expression levels of three house keeping genes using the GeNorm software (ME ⁇ SSOJJSSDL ⁇ I ⁇ B ⁇ MOOCO ⁇ )-
  • the ELO3 gene was 13,56 times higher expressed in the ELP3B6.4 overexpression line in comparison to CoI. High light experiment
  • the high light experiments on the ELP3B6.4 line were carried out by growing the plants on germination medium including vitamins (using 1 ,7 % glucose), each petri dish was divided into two: on one half ELP3B6.4 was sown and on the other half CoI. After an overnight vernalization, the plates were incubated in the tissue culture room under low light conditions (50 ⁇ Em ⁇ h "1 PAR) for 13 days. After 13 days, half of the experiment was moved to the tissue culture room in Bayer Cropscience and submitted to high light (250 ⁇ Em "2 h "1 PAR) for 48 h. The other half of the experiment stayed in PSB under the "normal” light conditions (50 ⁇ Em "2 h "1 PAR).
  • NADH, TTC, chlorophyll meaning that for 1 NADH determination only the plants from 1 plate were taken, both for ELP3B6.4 and CoI, allowing to examine the plate effect and to do more repeats in order to do statistics on the data.
  • 15 biological repeats were taken for each condition (50 ⁇ Em "2 h "1 PAR and 250 ⁇ Em "2 h "1 PAR) and for each line (ELP3B6.4 and CoI).
  • TTC assay A vigour assay was performed on the ELO3 overexpressing lines (27 in total) grown under normal in vitro growth conditions in comparison to CoI-O as described by De Block and De Brouwer, 2002 and De Block et al., 2005. The overexpressing lines were grown on Kanamycin containing germination medium, while the CoI-O control plants were grown on normal germination medium. The assay was performed on the vegetative part of 17 day old seedlings. This vigour assay relates to the capacity of cells to reduce 2,3,5-triphenyltetrazolium chloride (TTC). The reduced TTC was extracted and the absorption of the extract was measured at 485 nm.
  • TTC 2,3,5-triphenyltetrazolium chloride
  • Plants (ELP3B6.4 overexpression line and CoI control) were grown on germination medium including vitamins. After an overnight vernalization, the plates were incubated in the tissue culture room for 6 days. After 6 days, the seedlings were transferred to germination medium containing 10OmM NaCI (half of the experiment) and germination medium without salt as a control (half of the experiment). The plantlets were placed upside down allowing to monitor the growth after transfer by measuring the length of the primary root from the downward curling on (method described in Zhu et al., 1998). Plants were kept on the salt containing and control medium for 6 days, after which the plates were scanned. From the scanned images, the root lengths were measured using the ImageJ software and data analysis was done in Excel.
  • Example 1 Transcript profiling of elo2, elo3 and drl 1-2 mutants
  • a total of 1494 genes were detected for their difference of expression between at least one mutant Elongator and Ler.
  • 655 genes are also differentially regulated in the ang4 mutant, so appear unrelated to the Elongator function, and 839 genes are specifically regulated in Elongator mutants.
  • the elo3 mutant with the strongest phenotype had the highest number of DE genes ( Figure 1 ).
  • Some genes known to be regulated during development as cellulose synthase genes and expansins are differentially expressed in Elongator mutants (Table 1).
  • the dwarf gene coding for the gibberelin-20-oxydase, enzyme of gibberellins synthesis pathway is down-regulated, although two ethylene response genes are up-regulated.
  • genes related to jasmonic acid synthesis, to defence response, to proteolysis via ubiquitin complex are up-regulated in Elongator mutants.
  • Seven genes related to photosynthesis are down-regulated in the mutants, and two genes related to light perception pigments synthesis are up-regulated.
  • Two calmodulin genes, with function in signal transduction in the cell are over-expressed in Elongator mutants.
  • the ATCLH1 gene is a chlorophyllase the first enzyme involved in chlorophyll degradation.
  • This gene also called coronatine-induced protein 1 (COR11) was 20 fold change over- expressed in elo2, elo3 and drl1-2 mutants. Its expression is induced rapidly by methyl- jasmonate, a known promoter of senescence and chlorophyll degradation.
  • the At1gO5385 photosystem Il 11 kDa protein-related
  • At1g14150 oxygen evolving enhancer 3, PsbQ
  • PSII K genes were down-regulated in elo2, elo3 and drl1-2 mutants.
  • At1g03630 gene coding for a protochlorophyllide reductase C and At2g28800 gene for the chloroplast membrane protein ALBINO3 were under-expressed respectively in elo3 and in elo2. All these observations suggested a decrease of chlorophyll content by induction of degradation pathway via the gene CORH and consequently a dysfunction of the photosystems
  • Table 1 Microarray data for selected differentially expressed genes in Elongator mutant compared to the wild-type Ler. Data were performed on microarrays ATH 1 experiment with RNA from shoot apex of young plants grown in in vitro conditions. The p values are calculated according to a Bayesian test of linear model and corrected by Holm's method. fold change expression p elo3 elo2 elo3 drl1-2 AGI Annotation class
  • VSP1 vegetative storage protein 1
  • At1g19670 coronatine-responsive protein CORH: Chlorophyllase light
  • the genes involved in jasmonate synthesis coding the lipoxygenase (LOX2) and the allene oxyde synthase (AOS), are over- expressed in elol (significant only for LOX2), elo2, elo3 and drl1-2.
  • the gene At2g18600 coding a putative RUB1 protein, related to SCF proteasome complex is up-regulated in elol, elo2, elo3 and drl1-2. This result was also observed growing plants in soil conditions (data not shown) showing the putative implication of the SCF proteasome complex in Elongator function in plants.
  • the gene coding the expansin EXP8 appears down-regulated specifically in elo2 mutant whereas the gene coding expansin EXP20 is up-regulated in all Elongator mutant except elo2.
  • Two calmodulins genes are also up-regulated in elo2, elo3 and drl1-2 mutants.
  • the sucrose synthase gene (At4g02280) appeared significantly over-expressed only in the strongest alleles elo3 and drl1-2.
  • Example 3 Pigments content is modified by Elongator mutation Because some genes related to chlorophylls and anthocyanins metabolism appeared differentially regulated in Elongator mutants, the pigments content of young plants were measured to observe the effects of the gene expression at the metabolic level. An analysis of variance showed significant less chlorophylls a and b content in the mutants compared to Ler ( Figure 3). The concentration of chlorophylls a and b were respectively 0.63-0.78 ⁇ g/mg and 0.12-0.20 ⁇ g/mg for the mutants compared to 0.90 and 0.25 ⁇ g/mg in Ler plants.
  • the carotenoids content was also significantly lower but only in the elo2, elo3 and drl1-2 mutants with a mean value from 0.19 to 0.24 ⁇ g/mg rather than 0.28 ⁇ g/mg in the wild-type.
  • the anthocyanins concentration was significantly higher in elo2 and drl1-2 mutants compared to Ler but was not affected in the weakest alleles elol and elo4.
  • the lower content of chlorophylls and carotenoids, and higher content of anthocyanins in Elongator mutants suggests that the Elongator system acts as regulator of light inducible genes, positive for the photosynthesis pigments (chlorophylls and carotenoids) and negative for anthocyanins biosynthesis.
  • the light inducible genes may be a potential downstream target of Elongator in mediating light control of gene expression. This modified production of pigments could be also a consequence of the jasmonate over-production as the gene expression profile suggests it.
  • Example 5 Elongator overexpression protects against high light stress.
  • Example 6 Overexpression of Elongator enhances plant vigour .
  • a vigour assay (De Block and De Brouwer, 2002; De Block et al., 2005) was performed on the ELO3 overexpressing lines grown under normal in vitro growth conditions in comparison to CoI-O.
  • the OD485 values of all the EL03 overexpressing lines were lower than those of the CoI-O control.
  • the values obtained are summarized in Figure 6. These data show that the EL03 overexpressing lines have less reduced TTC. This indicates that these lines have a higher energy-use efficiency (De Block et al., 2005), resulting in a higher vigour.
  • Example 7 Results and statistical analysis of the high light experiment
  • the ELO3 gene was 13,56 times higher expressed in the ELP3B6.4 overexpression line in comparison to CoI.
  • the values for NADH and TTC could not be determined since they were so low, results that were also seen in Bayer when testing plants grown under normal light conditions. So for the plants grown under 50 ⁇ Em "2 h "1 PAR only the data of the weight were used for statistical analysis.
  • the results and a statistical analysis are summarized in table2.
  • Genome Biol 5 R80 Hoekstra M F, Liskay RM, Ou AC, DeMaggio AJ, Burbee DG, Heffron F (1991). HRR25, a putative protein kinase from budding yeast: association with repair of damaged DNA. Science.
  • a novel histone acetyltransferase is an integral subunit of elongating RNA polymerase Il holoenzyme. MoI Cell. 4:123-128.

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Abstract

La présente invention se rapporte à l'utilisation de protéines du complexe Elongator et de gènes codant ces protéines pour moduler la tolérance de vigueur et au stress dans des cellules eucaryotes, de préférence des cellules végétales. Cette invention concerne plus spécifiquement la surexpression de gènes du complexe Elongator et en particulier la surexpression d'EL03 pour obtenir une plus grande tolérance de vigueur et au stress.
PCT/EP2006/061854 2005-04-28 2006-04-26 Utilisation de genes du complexe elongator pour renforcer la tolerance de vigueur et au stress dans des cellules eucaryotes WO2006114434A1 (fr)

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WO2017011462A1 (fr) * 2015-07-15 2017-01-19 University Of Florida Research Foundation, Inc. Utilisation des gènes elongator pour améliorer la résistance des plantes aux maladies

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WO2003066852A2 (fr) * 2002-02-07 2003-08-14 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Modulation de croissance des plantes

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WO2003066852A2 (fr) * 2002-02-07 2003-08-14 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Modulation de croissance des plantes

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Title
NELISSEN HILDE ET AL: "The elongata mutants identify a functional Elongator complex in plants with a role in cell proliferation during organ growth", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 102, no. 21, May 2005 (2005-05-01), pages 7754 - 7759, XP002392006, ISSN: 0027-8424 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017011462A1 (fr) * 2015-07-15 2017-01-19 University Of Florida Research Foundation, Inc. Utilisation des gènes elongator pour améliorer la résistance des plantes aux maladies
US10829779B2 (en) 2015-07-15 2020-11-10 University Of Florida Research Foundation, Incorporated Use of elongator genes to improve plant disease resistance

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