Patent Application
20160272992
This application relates generally to plant biology and more specifically to the use of SMR5, possibly in combination with SMR4 and/or SMR7, to modulate ROS and oxidative stress response in plants. More specifically, it relates to an SMR5 knock out or knock down to improve the oxidative stress tolerance in plants.
Being immobile, plants are continuously exposed to changing environmental conditions that can impose biotic and abiotic stresses. One of the consequences observed in plants subjected to altered growth conditions is the disruption of the reactive oxygen species (ROS) homeostasis (Mittler et al., 2004). Under steady-state conditions, ROS are efficiently scavenged by different non-enzymatic and enzymatic antioxidant systems, involving the activity of catalases, peroxidases, and glutathione reductases. However, when stress prevails, the ROS production rate can exceed the scavenging mechanisms, resulting into a cell- or tissue-specific rise in ROS. These oxygen derivatives possess a strong oxidizing potential that can damage a wide diversity of biological molecules, including the electron-rich bases of DNA, which results into single- and double-stranded breaks (Amor et al., 1998; Dizdaroglu et al., 2002; Roldan-Mona and Ariza, 2009). Hydrogen peroxide (H2O2) is a major ROS compound and is able to transverse cellular membranes, migrating into different compartments. This feature grants H2O2 not only the potential to damage a variety of cellular structures, but also to serve as a signaling molecule, allowing the activation of pathways that modulate developmental, metabolic and defense pathways (Mittler et al., 2011). One of the signaling effects of H2O2 is the activation of a cell division arrest by cell cycle checkpoint activation (Tsukagoshi, 2012), however, the molecular mechanisms involved remain unknown.
Cell cycle checkpoints adjust cellular proliferation to changing growth conditions, arresting it by the inhibition of the main cell cycle controllers: the heterodimeric complexes between the cyclin-dependent kinases (CDK) and the regulatory cyclins (Lee and Nurse, 1987; Norbury and Nurse, 1992). The activators of these checkpoints are the highly conserved ATAXIA TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED (ATR) kinases that are recruited in accordance with the type of DNA damage (Zhou and Elledge, 2000; Abraham, 2001; Bartek and Lukas, 2001; Kurz and Lees-Miller, 2004). ATM is activated by double-stranded breaks (DSBs); whereas ATR is activated by single-stranded breaks or stalled replication forks, causing inhibition of DNA replication. In mammals, ATM and ATR activation result in the phosphorylation of the Chk2 and Chk1 kinases, respectively. In mammals, both kinases subsequently phosphorylate p53, a critical transcription factor responsible to conduct DNA damage responses (Chaturvedi et al., 1999; Shieh et al., 2000; Chen and Sanchez, 2004; Rozan and El-Deiry, 2007). p53 seemingly appears to have no plant ortholog, although an analogous role for p53 is suggested for the plant-specific SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1) transcription factor that is under direct post-transcriptional control of ATM (Yoshiyama et al., 2009; Yoshiyama et al., 2013). Another distinct feature relates to the inactivation of CDKs in response to DNA stress. CDK activity is in part controlled by its phosphorylation status at the N-terminus, determined by the interplay of the CDC25 phosphatase and the antagonistic WEE1 kinase, acting as the “on” and “off” switches of CDK activity, respectively (Francis, 2011). Whereas in mammals and budding yeast, the activation of the DNA replication checkpoint, leading to a cell cycle arrest, is predominantly achieved by the inactivation of the CDC25 phosphatase, as plant cells respond to replication stress by transcriptional induction of WEE1 (De Schutter et al., 2007). In absence of WEE1, Arabidopsis thaliana plants become hypersensitive to replication inhibitory drugs such as hydroxyurea (HU), which causes a depletion of dNTPs because of an inhibition of the ribonucleotide reductase (RNR) protein. However. WEE1-deficient plants respond similarly to control plants exposed to other types of DNA damage (De Schutter et al., 2007; Dissmeyer et al., 2009). Other, yet to be identified pathways controlling cell cycle progression under DNA stress, operating independently of WEE1, may exist.
There are several potential candidates to operate in checkpoint activation upon DNA stress mainly belonging to the family of CDK inhibitors (CKIs). CKI proteins are mostly low molecular weight proteins that inhibit cell division by their direct interaction with the CDK and/or cyclin subunit (Sherr and Roberts, 1995; De Clercq and Inzé, 2006). The first identified class of plant CKIs was the ICK/KRP (interactors of CDK/Kip-related protein) protein family comprising seven members in A. thaliana, all sharing a conserved C-terminal domain being similar to the CDK-binding domain of the animal CIP/KIP proteins (Wang et al., 1998; Wang et al., 2000; De Veylder et al., 2001). The TIC (tissue-specific inhibitors of CDK) is the most recently suggested class of CKIs (DePaoli et al., 2012) and encompasses SCI1 in tobacco, the only tissue-specific CKI reported so far (DePaoli et al., 2011). SCI1 shares no outstanding sequence similarity with the other classes of CKIs in plants, and has been suggested to connect cell cycle progression and auxin signaling in pistils (DePaoli et al., 2012). The third class of CKIs is the plant-specific SIAMESE/SIAMESE-RELATED (SIM/SMR) gene family. SIM has been identified as a cell cycle inhibitor with a role in trichome development and endocycle control (Churchman et al., 2006). Based on sequence analysis, five additional gene family members have been identified in A. thaliana, and together with EL2 from rice, been suggested to act as cell cycle inhibitors modulated either by biotic and abiotic stresses (Peres et al., 2007). Plants subjected to treatments inducing DSBs showed a rapid and strong induction of specific family members (Culligan et al., 2006; Adachi et al., 2011).
Surprisingly, it was found that three SMR genes (SMR4, SRM5 and SMR7) are transcriptionally activated by DNA damage. Even more surprisingly, the SMR5 gene encodes for a novel protein not described earlier. Cell cycle inhibitory activity was demonstrated by overexpression analysis, whereas knockout data illustrated that both SMR5 and SMR7 are essential for DNA cell cycle checkpoint activation in leaves of plants grown in the presence of HU. Remarkably, it was found that SMR induction mainly depends on ATM and SOG1, rather than ATR as would be expected for a drug that triggers replication fork defects. Correspondingly, it was demonstrated that the HU-dependent activation of SMR genes is triggered by ROS rather than replication problems, linking SMR genes with cell cycle checkpoint activation upon the occurrence of DNA damage-inducing oxidative stress.
A first aspect of the disclosure is the use of SMR5, or a homologue, orthologue or paralogue thereof, to modulate ROS signaling and/or oxidative stress response in plants. In a preferred embodiment, this use is combined with the use of SMR4 and/or SMR7. The “use of an SMR,” as used herein, comprises the use of the gene, and/or the use of the protein encoded by the gene. Preferably, the use of SMR5 is the use of a gene encoding a protein selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6 of the incorporated herein Sequence Listing. In one preferred embodiment, the use of SMR5 is the use of a gene encoding a protein preferably consisting of SEQ ID NO:2. In another preferred embodiment, the use of SMR5 is the use of a gene encoding a protein preferably consisting a of a sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:6. “Homologues” of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived. Orthologues and paralogues encompass evolutionary concepts used to describe the ancestral relationships of genes. Paralogues are genes within the same species that have originated through duplication of an ancestral gene; orthologues are genes from different organisms that have originated through speciation, and are also derived from a common ancestral gene.
Preferably, the use is a down-regulation of the expression of the protein, and/or the inactivation of the protein. Preferably, the down-regulation is used to improve oxidative stress tolerance in plants. “Improve” as used herein, means that the plants wherein the SMR is down-regulated have a significantly better oxidative stress resistance than the plants with the same genetic background, except for the modifications needed for the down-regulation, grown under the same conditions. Methods for down-regulation are known to the person skilled in the art, and include, but are not limited to, mutations, insertions or deletions in the gene and/or its promoter, the use of anti-sense RNA or RNAi and gene silencing methods. Methods to induce site-specific mutations in plants are known to the person skilled in the art and include Zinc-finger nucleases, transcription activator-like nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas-based RNA guided DNA endonucleases (Gaj et al., 2013). Inactivation of the protein can be obtained, as a non-limiting example, by the use of antigen-binding proteins directed against the protein, or by protein aggregation, as described in WO 2012/123419. The down-regulation of SMR5 can be measured by measuring the activity of its substrate (Cyclin-dependent kinase A, CDKA) as described in De Veylder et al. (1997); a higher CDKA activity points to a down-regulation of SMR5.
A “plant” as used herein may be any plant. Plants include gymnosperms and angiosperms, monocotyledons and dicotyledons, trees, fruit trees, field and vegetable crops and ornamental species. Preferably, the plant is a crop plant including, but not limited to, soybean, corn, wheat, barley and rice.
Another aspect of the disclosure is a genetically modified plant comprising an inactivated SMR5 gene and/or protein. “Inactivated,” as used herein, means that the activity of the inactivated form is significantly lower than that of the active form. “Significantly,” as used herein, means that the activity of the mutant gene or protein is at least 20% lower, preferably at least 50% lower, more preferably at least 75% lower, most preferably at least 90% lower than the wild-type gene or protein. Preferably, the activity of the gene is measured as the amount of messenger RNA. Preferably, the activity of the protein is measured as inhibition of cell division. In one preferred embodiment, the active form of the gene is encoding a protein preferably consisting of SEQ ID NO:2. In another preferred embodiment, the use of SMR5 is the use of a gene encoding a protein preferably consisting of a sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:6. In a preferred embodiment, the plant is a maize plant in which ZmSMRg and/or ZmSMRh are inactivated, preferably as a CRISPR/Cas knock out.
In one preferred embodiment, the gene encoding the SMR5p is disrupted. In another preferred embodiment, the gene encoding the SMR5p is silenced. In still another embodiment, the SMR5p itself is inactivated by protein aggregation.
Preferably, the genetically modified plant further comprises an inactivated SMR4 gene and/or protein, and/or an inactivated SMR7 gene and/or protein.
Still another aspect of the disclosure is a method to increase oxidative stress resistance in a plant comprising the down-regulation of SMR5p expression and/or activity. Preferably, the down-regulation is combined with the down-regulation of SMR4p expression and/or activity, and/or down-regulation of SMR7p expression and/or activity.
In one preferred embodiment, the method comprises a step wherein the plant is transformed with an RNAi construct against one or more of the SMR genes. In one preferred embodiment, the RNAi construct is placed under control of a constitutive promoter. In another preferred embodiment, the RNAi construct is placed under control of an oxidative stress-inducible promoter.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The smr5 (SALK_100918) and smr7 (SALK_128496) alleles were acquired from the Arabidopsis Biological Research Center. Homozygous insertion alleles were checked by genotyping PCR using the primers listed in Table 3. The atm-1, atr-2 and sog1-1 mutants have been described previously (Garcia et al., 2003; Preuss and Britt, 2003; Culligan et al., 2004; Yoshiyama et al., 2009). Unless stated otherwise, plants of Arabidopsis thaliana (L.) Heyhn (ecotype Columbia), were grown under long-day conditions (16 hours of light, 8 hours of darkness) at 22° C. on half-strength Murashige and Skoog (MS) germination medium (Murashige and Skoog, 1962). Arabidopsis plants were treated with HU as described by Cools et al. (2011). For bleomycin treatments, five-day-old seedlings were transferred into liquid MS medium supplemented with 0.3 μg/mL bleomycin. For γ-irradiation treatments, five-day-old in vitro-grown plantlets were irradiated with γ-rays at a dose of 20 Gy. For light treatments, one-week-old seedlings were transferred to continuous high-light conditions (growth rooms kept at 22° C. with 24-hour day/0-hour night cycles and a light intensity of 300-400 μmol m−2 s−1) for 2 days, and subsequently retransferred to low-light conditions. The first leaf pair was harvested and incubated in 100% ethanol for epidermis cell drawing as described by De Veylder et al. (2001).
Genomic DNA was extracted from Arabidopsis leaves with the DNEASY® Plant Kit (Qiagen) and RNA was extracted from Arabidopsis tissues with the RNEASY® Mini Kit (Qiagen). After DNase treatment with the RQ1 RNase-Free DNase (Promega), cDNA was synthesized with the iScript cDNA Synthesis Kit (Bio-Rad). A quantitative RT-PCR was performed with the SYBR® Green kit (ROCHE) with 100 nM primers and 0.125 μL of RT reaction product in a total of 5μL per reaction. Reactions were run and analyzed on the LIGHTCYCLER® 480 (Roche) according to the manufacturer's instructions with the use of the following reference genes for normalization: ACTIN2 (At3g46520), EMB2386 (At1g02780), PACI (At3g22110) and RPS26C (At3g56340). Primers used for the RT-PCR are given in Table 5.
SIM/SMR promoter sequences were amplified from genomic DNA by PCR using the primers described in Table 5. The product fragments were created with the Pfu DNA Polymerase Kit (Promega, Catalog #M7745), and were cloned into a pDONR P4-Plr entry vector by BP recombination cloning and subsequently transferred into the pMK7S*NFml4GW,0 destination vector by LR cloning, resulting in a transcriptional fusion between the promoter of the SMR genes and the nlsGFP-GUS fusion gene (Karimi et al., 2007). For the overexpression constructs, the SMR coding regions were amplified using primers described in Table 5, and cloned into the pDONR221 vector by BP recombination cloning and subsequently transferred into the pK2GW7 destination vector (Kamimi et al., 2002) by LR cloning. All constructs were transferred into the Agrobacterium tumefaciens C58C1RifR strain harboring the pMP90 plasmid. The obtained Agrobacterium strains were used to generate stably transformed Arabidopsis lines with the floral dip transformation method (Clough and Bent, 1998). Transgenic plants were obtained on kanamycin-containing medium and later transferred to soil for optimal seed production. All cloning primers are listed in Table 5.
Complete seedlings or tissue cuttings were stained in multiwell plates (Falcon 3043; Becton Dickinson). GUS assays were performed as described by Beeckman and Engler (1994). Samples mounted in lactic acid were observed and photographed with a stereomicroscope (Olympus BX51 microscope) or with a differential interference contrast (DIC) microscope (Leica).
For leaf measurements, first leaves were harvested at 21 days after sowing on control medium, medium supplemented with 1 mM hydroxyurea or 0.3 μg/mL bleomycin. Leaves were cleared overnight in ethanol, stored in lactic acid for microscopy, and observed with a microscopy fitted with DIC optics (Leica). The total (blade) area was determined from images digitized directly with a digital camera (Olympus BX51 microscope) mounted on a binocular (Stemi SV 11; Zeiss). From scanned drawing-tube images of the outlines of at least 30 cells of the abaxial epidermis located between 25% to 75% of the distance between the tip and the base of the leaf, halfway between the midrib and the leaf margin, the following parameters were determined: total area of all cells in the drawing and total numbers of pavement and guard cells, from which the average cell area was calculated. The total number of cells per leaf was estimated by dividing the leaf area by the average cell area. For confocal microscopy, root meristems were analyzed 2 days after transfer using a Zeiss LSM 510 Laser Scanning Microscope and the LSM Browser version 4.2 software (Zeiss). Plant material was incubated for 2 minutes in a 10 μm PI solution to stain the cell walls and was visualized with a HeNe laser through excitation at 543 nm. GFP fluorescence was detected with the 488-nm line of an Argon laser. GFP and PI were detected simultaneously by combining the settings indicated above in the sequential scanning facility of the microscope. Acquired images were quantitatively analyzed with the ImageJ v1.45s software (on the World Wide Web at rsbweb.nih.gov/ij/) and Cell-o-Tape plug-ins (French et al., 2012). Chlorophyll a fluorescence parameters were measured using the IMAGING PAM M-Series Chlorofyll Fluorescence (Walz) and associated software.
For flow cytometric analysis, root tip tissues were chopped with a razor blade in 300 μL of 45 mM MgCl2, 30 mM sodium citrate, 20 mM MOPS, pH 7 (Galbraith et al., 1991). One microliter of 4,6-diamidino-2-phenylindole (DAPI) from a stock of 1 mg/mL was added to the filtered supernatant. Leaf material was chopped in 200 μL of Cystain UV Precise P Nuclei extraction buffer (Partec), supplemented with 800 μL of staining buffer. The mix was filtered through a 50-μm green filter and read by the C
Plants were germinated on either control medium, medium with 1 mM HU or 6 μM 3-AT. Leaf tissue of 10 plants was ground in 200 μL extraction buffer (60 mM Tris (pH 6.9), 1 mM phenylmethylsulfonylfluoride, 10 mM DTT) on ice. The homogenate was centrifuged at 13,000 g for 15 minutes at 4° C. A total of 45 μg protein extract was mixed with potassium phosphate buffer (50 mM, pH 7.0) (Vandenabeele et al., 2004). After addition of 11.4 μL H2O2 (7.5%), the absorbance of the sample at 240 nm after 0 and 60 seconds was measured to determine catalase activity by H2O2 breakdown (Beers and Sizer, 1952; Vandenabeele et al., 2004).
Seeds were plated on sterilized membranes and grown under a 16-hour light/8-hour dark regime at 21° C. After 2 days of germination and 5 days of growth, the membrane was transferred to MS medium containing 0.3 μg/mL bleomycin for 24 hours. Triplicate batches of root meristem material seedlings were harvested for total RNA preparation using the RNEASY® plant mini kit (Qiagen). Each of the different root tip RNA extracts were hybridized to 12 AFFYMETRIX® Arabidopsis Gene 1.0 ST Arrays according to manufacturer's instructions at the Nucleomics Core Facility (Leuven, Belgium; World Wide Web at nucleomics.be). Raw data were processed with the RMA algorithm (Irizarry et al., 2003) using the AFFYMETRIX® Power Tools and subsequently subjected to a Significance Analysis of Microarray (SAM) analysis with “MultiExperiment Viewer 4” (MeV4) of The Institute for Genome Research (TIGR) (Tusher et al., 2001). The imputation engine was set as 10-nearest neighbor imputer and the number of permutations was 100. Expression values were obtained by log 2-transforming the average value of the normalized signal intensities of the triplicate samples. Fold changes were obtained using the expression values of the treatment relative to the control samples. Genes with Q-values<0.1 and fold change>1.5 or <0.666 were retained for further analysis.
Transcripts induced by bleomycin (Q-value<0.1 and fold change>1.5) were compared with different published DNA stress-related data sets. For γ-irradiation, an intersect of the genes with a significant induction (P-value<0.05, Q-value<0.1, and fold change>1.5) in 5-day-old wild-type seedlings 1.5 hours post-irradiation (100 Gy) was made of two independent experiments (Culligan et al., 2006; Yoshiyama et al., 2009). For replication stress, genes showing a significant induction (P-value (Time)<0.05, Q-value (Time)<0.1 and fold change>1.5) in 5-day-old wild-type root tips after 24 hours of 2-mM hydroxyurea treatment were selected (Cools et al., 2011). Meta-analysis of the SMR genes during various stress conditions and treatments were obtained using GENEVESTIGATOR® (Hruz et al., 2008). Using the “Response Viewer” tool, the expression profiles of genes following different stimuli were analyzed. Only biotic and abiotic stress treatments with a more than 2-fold change in the transcription level (P-value<0.01) for at least one of the SMR genes were taken into account. Fold-change values were hierarchically clustered for genes and experiments by average linkage in MeV from TIGR.
Microarray results have been submitted to MiamExpress (on the World Wide Web at ebi.ac.uk/miamexpress), with accession E-MEXP-3977. Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: SMR4 (At5g02220); SMR5 (At1g07500); SMR7 (At3g27630); ATM (At3g48490); ATR (At5g40820); and SOG1 (At1g25580).
When DNA damage occurs, two global cellular responses are essential for cell survival: activation of the DNA repair machinery and delay or arrest of cell cycle progression. In recent years, gene expression inventories have been collected that focus on the transcriptional changes in response to different types of DNA stress (Culligan et al., 2006; Ricaud et al., 2007; Yoshiyama et al., 2009; Cools et al., 2010). To identify novel key signaling components that contribute to cell cycle checkpoint activation, bleomycin-induced genes were compared to those induced by HU treatment (Cools et al., 2010) and γ-radiation (Culligan et al., 2006; Yoshiyama et al., 2009). Twenty-two genes were up-regulated in all DNA stress experiments and can be considered as transcriptional hallmarks of the DNA damage response (DDR), regardless of the type of DNA stress (
Previously, the existence of one SIM and five SMR genes (SMR1-SMR5) in the A. thaliana genome (Peres et al., 2007) was reported, whereas protein purification of CDK/cyclin complexes resulted in the identification of two additional family members (SMR6 and SMR8) (Van Leene et al., 2010). With the availability of new sequenced plant genomes, the Arabidopsis genome was re-examined using iterative BLAST searches for the presence of additional SMR genes, resulting in the identification of six non-annotated family members, nominated SMR7 to SMR13 (Table 3). With the GENEVESTIGATOR® toolbox (Hruz et al., 2008), the expression pattern of the twelve SIM/SMR genes represented on the AFFYMETRIX® ATHI microarray platform was analyzed in response to different biotic and abiotic stress treatments. Distinct family members were induced under various stress conditions, albeit with different specificity (
To confirm involvement of SIM/SMR genes in the genotoxic stress response, transcriptional reporter lines containing the putative upstream promoter sequences were constructed for all. After selection of representative reporter lines, one-week-old seedlings were transferred to control medium, or medium supplemented with HU (resulting into stalled replication forks) or bleomycin (causing DSBs). Focusing on the root tips revealed distinct expression patterns (
Previously, SIM had been proven to encode a potent cell cycle inhibitor, since its ectopic expression results in dwarf plants holding less cells compared to control plants (Churchman et al., 2006). To test whether the DNA stress-induced SMR genes encode proteins with cell division inhibitory activity, SMR4-, SMR5- and SMR7-overexpressing (SMR4OE, SMR5OE and SMR7OE) plants were generated. For each gene, multiple lines with strong transcript levels were isolated, all showing a reduction in rosette size compared to wild-type plants (
To address the role of the different SMR genes in DNA stress checkpoint control, the growth response to HU treatment of plants being knocked out for SMR5 or SMR7 (
Because of the observed role of the SMR5 and SMR7 genes in DNA stress checkpoint control, the dependence of their expression on the ATM and ATR signaling kinases and the SOG1 transcription factor was analyzed by introducing the SMR5 and SMR7 GUS reporter lines into the atr-2, atm-1 and sog1-1 mutant backgrounds. Both genes were induced in the proliferating leaf upon HU and bleomycin treatment (
To examine whether an increase in H2O2 might trigger expression of SMR genes, SMR5 and SMR7 expression levels were analyzed in plants that are knockout for CAT2 and/or APX1, encoding two enzymes important for the scavenging of H2O2. SMR5 expression levels were clearly induced in the apx1 cat2 double mutant, whereas SMR7 transcriptional activation was observed in the apx1 knockout and apx1 cat2 double mutant (
Sequences of the Arabidopsis and maize SMR proteins were aligned and subsequently clustered. The maize proteins ZmSMRg and ZmSMRh were identified as the closest orthologues of Arabidopsis SMR5. The coding sequence is given in SEQ ID NO:3 (ZmSMRg) and SEQ ID NO:5 (ZmSMRh). The results are given in
The transcriptional induction of the maize SMR genes after HU treatment was measured using qRT-PCR analysis, similar as described for Arabidopsis, and both genes show a strong up-regulation upon HU treatment, both in root tips and in leaves.
Detailed expression analysis of both the ZmSMRg gene and the ZmSMRh gene is carried out using promoter-GUS fusions, transformed into maize. These transformed plants are tested under a variety of stresses including, but not limited to, drought, high light, cold, heat, hydroxyurea and bleomycin treatment.
The ZmSMRg gene and the ZmSMRh gene are knocked out using the CRISPR-Cas technology, generating single and double knock out mutants. These knock out mutants are submitted to oxidative stress as described for Arabidopsis, and the mutants show a significant protection against oxidative stress, when compared to the wild-type grown under the same conditions.
aAccording to Cools et al., 2011
bAccording to Culligan et al., 2006
cAccording to Yoshiyama et al., 2009
Arabidopsis
Arabidopsis
thaliana
Arabidopsis
thaliana
aAccording to Cools et al., 2011
bAccording to Culligan et al., 2006
cAccording to Yoshiyama et al., 2009
| Number | Date | Country | Kind |
|---|---|---|---|
| 13193423.4 | Nov 2013 | EP | regional |
This application is a national phase entry under 35 U.S.C. §371 of International Patent Application PCT/EP2014/074758, filed Nov. 17, 2014, designating the United States of America and published in English as International Patent Publication WO 2015/074992 A1 on May 28, 2015, which claims the benefit under Article 8 of the Patent Cooperation Treaty to European Patent Application Serial No. 13193423.4, filed Nov. 19, 2013.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2014/074758 | 11/17/2014 | WO | 00 |
