Patent Application
20120005774
The present invention relates to the use of trehalase to obtain drought and/or salt resistance in plants. More specifically, it relates to genetically modified constructs, transformed into plants and resulting in overexpression of trehalase, whereby the transformed plants show a significantly better drought resistance during the drought period, and a better recovery when water is supplied after the drought period.
The disaccharide trehalose (α-D-glucopyranosyl-1,1-α-D-glucopyranoside) is a non-reducing sugar found in a variety of organisms including plants. The role of trehalose has often been associated with stress resistance, particularly resistance to desiccation. Jang et al. (2003) describe the construction of transgenic rice plants with increased abiotic stress tolerance by the expression of a fusion of the Escherichia coli genes for trehalose-6-phosphate synthase and trehalose-6phosphate phosphatase. Suarez et al. (2008) obtained improvement of drought tolerance in common beans by overexpression of trehalose-6-phosphate synthase. In those studies, the accumulation of trehalose is promoted by increasing the biosynthesis.
However, apart from the biosynthetic pathway, the level of trehalose may be influenced by trehalose degradation. Trehalase is the only known enzyme in Arabidopsis able to degrade trehalose to two glucose units. The expression of its gene, AtTRE1, is significantly up-regulated during specific plant development stages, such as flowering and senescence, and during stress conditions. Because of the intimate connection between trehalose metabolism and plant vital processes, stringent level control of trehalose, or indirectly of T6P, by trehalase activity might be crucial in the fine regulation of these metabolites/signals and, subsequently, in the control of plant growth and development. Trehalase may also mobilize stress-induced trehalose levels to allow subsequent cellular repair, as is the case in lower organisms. Moreover, high trehalase activity may also prevent perturbations of carbon metabolism by external trehalose, released by pathogenic or symbiotic microbes. However, despite the involvement in multiple processes, little is known about the exact role of trehalase in higher plants.
EP0784095 discloses trehalose accumulation in potato upon inhibition of trehalase, either by addition of the trehalase inhibitor Validamycin A, or by expressing trehalase anti-sense RNA. However, these inhibition experiments were carried out in trehalose-6-phosphate synthase overexpressing plants, making it difficult to make a distinction between the effect of the overexpression of the synthase and that of the inhibition of the trehalase. WO2008095919 discloses the use of overexpression of a trehalase gene to confer nematode resistance to plants. However, no reference to drought or salt tolerance was made. On the contrary, it was indicated that higher trehalose concentration results in increased tolerance to drought and stress.
Surprisingly, we found that overexpression of trehalase resulted in a better tolerance of drought, and a better growth upon rewatering after a period of drought. Knock-out of the trehalase gene caused a decrease in drought tolerance.
A first aspect of the invention is the use of a recombinant trehalase gene to modulate drought and/or salt tolerance in plants. “Modulation drought” and/or “salt tolerance,” as used herein, can be an increase or a decrease in drought and/or salt tolerance. “Trehalase,” as used herein, means a glycoside hydrolase, converting trehalose into glucose. Trehalases are known to the person skilled in the art and include, but are not limited to, bacterial, fungal and plant trehalases. Preferably, trehalase is an Arabidopsis trehalase; even more preferably, it is the Arabidopsis trehalase TRE1 (genbank accession number NM—118536). A “recombinant trehalase gene,” as used herein, means that the nucleic acid encoding trehalase has been isolated, was optionally modified, and introduced into a plant, resulting in modified drought and/or stress tolerance. Recombinant trehalase genes comprise, but are not limited to, knock-out genes, genes with modified, preferably optimized, coding sequence, genes with a modified promoter, antisense genes, and genes encoding RNAi. “Optimized coding sequences,” as used herein, include, as a non-limiting example, codon optimation for better translation, as well as mutations that may increase the specific activity of the protein. Due to the modification, the fragment introduced in the plant may be only a fragment of the trehalase gene. However, modification of the gene is optional. As a non-limiting example of the use of a trehalase gene without modification, the gene of one species, even a non-plant species, may be introduced in the plant. The introduction can be a single copy, or several copies can be introduced. Alternatively, the plant may be transformed with the endogenous gene to obtain a higher copy number of the gene, and so a higher expression of the trehalase protein. Preferably, that use is the overexpression of the trehalase gene, resulting in an improved drought and/or salt tolerance. Preferably, overexpression is resulting in improved drought tolerance. “Drought tolerance,” as used herein, comprises drought tolerance during cultivation, as well as drought tolerance after harvesting, resulting in a better shelf-life of the harvested plant. As a non-limiting example, drought tolerance after harvest may result in a better shelf-life of ornamental flowers, or in a better shelf-life of vegetables, preferably green vegetables such as, but not limited to, lettuce and spinach.
Preferably, overexpression of trehalase is specifically in the stomatal guard cells. “Specific expression in the stomatal guard cells,” as used herein, means that the expression is largely biased toward the stomatal guard cells; preferably, there is expression in the stomatal guard cells and no expression in the mesophyllic or epidermal cells of the plant leafs or plant stem, but there may be expression in parts of the root or the flowers. Such specific overexpression can be realized by the endogenous Arabidopsis thaliana TRE1 promoter.
Another aspect of the invention is the use of trehalase overexpression to obtain drought and/or salt tolerance in plants. Methods to obtain trehalase overexpression are known to the person skilled in the art and include, but are not limited to, increasing the copy number of trehalase genes, placing the trehalase coding sequence under control of a strong promoter, or removing repressing elements from the promoter. Trehalase overexpression may be obtained by mutagenesis and selection of an endogenous trehalase in a plant. Preferably, trehalase overexpression is obtained by isolating a trehalase gene, adapting it for overexpression and transforming it into a plant. Preferably, overexpression is resulting in improved drought tolerance. Preferably, trehalase is placed after a stomata-specific promoter, and overexpression is specifically in the stomatal guard cells.
Still another aspect of the invention is the use of trehalase overexpression to obtain improved growth upon rewatering after a drought period. “Rewatering,” as used herein, means that after a period of drought, the plant is exposed again to water. “Improved growth,” as used herein, includes, amongst others, a shorter adaptation period after the start of the rewatering, and better growth after adaptation.
Still another aspect of the invention is the use of a trehalase according to the invention, preferably the use of a recombinant trehalase gene according to the invention, to modulate the stomatal function. Stomatal function can be modulated by modulating the aperture of the stomatal cells, and/or the number of stomatal cells per leaf surface unit, and/or the stomatal index. Preferably, modulation of the stomatal function is realized by modulating the aperture of the stomata. Preferably, the use of trehalase is overexpression and modulation of the stomatal function is a limitation of the opening of the stomata. It is clear for the person skilled in the art that a limitation of the opening of the stomata, and/or a decrease in number of stomatal guard cells per leaf surface, and/or a decrease of stomatal index, can limit water loss and result in drought resistance, at least as long as a decrease of one factor is not compensated by an increase of another factor.
Materials and Methods to the Examples
Arabidopsis Plants Used in this Study
Colombia
Landsberg erecta
Primers Used in this Study
Plasmids Used in this Study
Trehalase CDS was cloned (source material: siliques and flowers of wild-type Arabidopsis thaliana ecotype Colombia) in plant vector pGFP (Kovtun et al., 1998; 2000; Hwang and Sheen, 2001). pGFP=pUC18 modified for GFP fusion. CDS sequence was blasted against TAIR Arabidopsis Genome Initiative Coding Sequence datasets (www.arabidopsis.org) and mutations, leading to amino acid changes, were corrected by site-directed mutagenesis (sdm).
Transgenic and wild-type Arabidopsis, ecotype Col and ecotype Ler seeds were vapor-phase sterilized according to a protocol adapted from Ye et al. (1999). Seeds were placed in open micro-centrifuge tubes and put inside a desiccator jar, where bleach-chlorine fumes sterilize the seeds for a period of 3 hours-9 hours (bleach+concentrated HCl). Sterile water is added afterwards working in laminar flow, followed by a 48-hour cold treatment under constant light for imbibition and stratification.
Vapor-sterilized seeds were sown, after a 48-hour stratification period, on wet soil and germinated for two days in high humidity. In general, they were grown in 12 hours-12 hours, 16 hours-8 hours day/night cycle or continuous light (21° C., 60% humidity, 100 μmol s−1 m−2).
Vapor-sterilized seeds were sown after a 48-hour stratification period in vertically or horizontally oriented Petri dishes on a full strength (or mentioned otherwise) Murashige and Skoog Salt Mixture including vitamins (MS) medium (4.3 g/l) with MES (0.5 g/l) and solidified with Phytagar (8 g/l) (Duchefa, Haarlem, The Netherlands). The media was enriched with sucrose/glucose (0-6%) or trehalose, depending on the tested conditions, which are specified. Val A was added in a concentration of 10 μM.
For homozygous plant selection, SALK lines and GT lines (with the NTPII marker gene as selection marker) were screened on 1×MS+Kanamycin (50 μg/ml, Sigma-Aldrich). For growth on MS media with selective media, seeds were germinated for seven days on MS and antibiotics, positives were transferred to soil to flower and to set seeds.
RNA Extraction from Plants and cDNA Preparation
Plant samples (seedlings) were harvested and immediately frozen in liquid nitrogen. RNA extraction of plant material was performed with Trizol reagent (Invitrogen), according to the manufacturer's instructions. For each sample, reverse transcription (RT) was performed using 1 μg of total RNA (Reverse Transcription System, Promega).
Plasmid DNA Isolation from Bacterial Cells
Mini preparation—CTAB method (Del Sal et al., 1988)
Mini preparation—GenElute™ Plasmid Miniprep kit (Sigma-Aldrich)
Maxi preparation—Pure Yield Plasmid Maxiprep System (Promega)
Rapid Genomic DNA Isolation from Arabidopsis Plants
After grinding a medium size leaf with screwdriver-pestle in 100 μl TPS buffer (100 mM Tris-HCl, pH 9.5, 1 M KCl, 10 mM EDTA), the samples were incubated at 70° C. for 20-30 minutes. The debris was pelleted by centrifugation at top speed for 10 minutes at RT and supernatans transferred to a new Eppendorf tube. For further PCR reactions, 0.5 to 1 μl supernatans was used in a 20-50 μl reaction.
PCR reactions were performed using different polymerase kits, according to manufacturer's recommendations; High-Fidelity Taq Polymerases (Roche), Phusion High-Fidelity DNA polymerase (FINNZYMES) and Ultra Pfu HF DNA polymerase (Stratagene) for cloning and ExTaq (TAKARA) for diagnostic PCR.
Purification of PCR Products
Equal amount of PCI was added to the PCR product (45 μl-45 μl), vortex, and centrifuged at maximum speed, for 5 minutes. To the transferred supernatans, NaAc 3M (3.6 μl) and ice-cold pure ethanol (109 μl) was added, vortex, and spinned down for 1 minute at maximum speed. After additional wash with ice-cold 80% ethanol, the pellet was dried out and dissolved in 12.5 μl H2O. PCR products could also be purified with using the Wizard SV Gel and PCR Clean-UP System (Promega, USA), according to manufacturer's guidelines.
PCR was performed on cDNA samples equally diluted to 250 ng/μl and a control gene was used as control. The PCR product was run on 1% agarose gel and visualized with SYBR Safe DNA gel stain (Invitrogen).
25 μl real-time PCR reaction was composed of 12.5 μl Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen), 0.5 μl of ROX reference dye, 1.25 μl of each primer of 500 nM stock solution and 5 μl of cDNA mix (100 ng/μl). The PCR program comprised an initial denaturation of 2 minutes at 95° C., amplification by 50 cycles of 15 seconds at 95° C., 1 minute at 58° C. The expression levels were analyzed with ABI prism and were all normalized to ACTIN2 expression.
Site-Directed Mutagenesis (sdm)
Site-directed mutagenesis (sdm) was performed according to the Stratagene's QuickChange Site-Directed Mutagenesis kit. As such, point mutations, insertions and deletions were created in a plasmid with Ultra Pfu HF DNA polymerase (Stratagene).
Three to four week-old Arabidopsis plants (in soil, 12 hours-12 hours day/night cycle, under low light, 50-75 μmol m−2 s−1) with healthy, unstressed, and well-expanded true leaves were used. This technique is detailed described by Yoo et al. (2007) (genetics.mgh.harvard.edu/sheenweb/protocols_reg.html). Briefly, after the removal of cell walls, protoplasts are released, washed and collected. DNA-PEG4000 (platelets, Fluka)-Calcium transfection is performed using 2×104 protoplasts per 10-20 μg of endotoxin-free (maxiprep) plasmid DNA. The transfected protoplasts are incubated for 6 hours in the light before harvesting.
Subcellular localization in mesophyll protoplast cells of the protein of interest can be determined by fluorescence microscopic analysis, using the GFP-tagged vectors.
Crude leaf material (300 mg, mix of plant material/different plants) was homogenized and powdered on dry ice, suspended in 2 ml ice-cold extraction buffer (0.1 M MES/K+, pH 6; 1 mM PMSF 2 mM EDTA, 10 mg/gFW insoluble PVP, polyvinylpyrrolidone). The suspension was vortexed and centrifuged (13000 rpm, cold, 10 minutes). The supernatant was dialyzed (BRL microdialysis system) ON at 4° C. against MES buffer pH 7 with 50 μl CaCl2. Trehalase activity was determined by a method described by Pernambuco et al. (1996) adapted to microtiter plates. 10 μl dialyzed sample was added to 50 μl trehalose buffer (250 mM trehalose, 25 mM MES, pH 7, 50 μl CaCl2). The microtiter plate was incubated for 30 minutes at 30° C. and reaction was stopped by boiling 5 minutes at 95° C. warm waterbath. Glucose was determined using glucose oxidase-peroxidase method by adding 200 μl GOD-PAP (Dialab) (blancs: residual glucose content in each sample determined by boiling samples before trehalose addition). A505 was measured after 15 minutes of incubation at 30° C. The amount of protein was determined using Lowry method with BSA as standard (Lowry et al., 1951).
The transposon insertion Genetrap (GT) line GT 16843 (A. thaliana, ecotype Ler), was originally produced in the Martienssen's Cold Spring Harbor Laboratory (Sundaresan et al., 1995; Martienssen, 1998). Based on the flanking sequences of the insertion site obtained by TAIL-PCR (Liu et al., 1995), line GT—16843 was predicted to carry a unique insertion of a GT transposable Ds element, 395 by downstream of the ATG start codon, in the middle of the first exon of the AtTRE1 gene (
The SALK—151791 line (A. thaliana, ecotype Col) (
Measurements of trehalase activity in different plant tissues demonstrated that SA TRE OX plants exhibited much higher trehalase activity than control wild-type plants. For example, up to 25 times more trehalase activity was observed in adult leaves of TRE OX, compared to wild-type Col leaves (
Trehalose causes dramatic effects on plant metabolism, growth and development. Validamycin A (Val A) specifically inhibits trehalase activity and prohibits the use of trehalose as an extra source of glucose. It is worth noting that, while Val A treatment alters adult plant stress and reproduction responses and even causes phytotoxicity, no phenotypic irregularities are observed in seedlings (Wingler et al., 2000; Müller et al., 2001; Ishikawa et al., 2005; 2007; Ramon et al., 2007).
In our growth assay, no extra sugar was added to the media. As known from literature, gradually increased levels of trehalose in the presence of Val A, cause increased inhibition of root growth and development of wild-type seedlings (
In the absence of Val A, external supplied trehalose can be used as an extra glucose source for growth. On high trehalose concentrations, wild-type seedlings arrest with development shortly after germination, show altered cotyledons extension, and develop only short primary roots (Wingler et al., 2000).
Interestingly, at low trehalose contents, GT tre KO seedlings do not seem to be able to use the extra glucose source for growth (
Mutants in trehalose metabolism have been reported to exhibit sensitivity to supplemented sugars, and T6P has been highlighted as an important regulator for carbohydrate utilization (Schluepmann et al., 2003; Avonce et al., 2004).
When grown on gradually increasing sugar concentrations, SA TRE OX seedlings exhibit similar leaf and hypocotyl development as control seedlings (horizontally orientated plates, 16 hours-8 hours day/night cycle (
Plants engineered in trehalose metabolism typically exhibit dramatic phenotypic irregularities, with altered physiology and morphology. In addition, Val A treatment can alter plant reproduction and can even cause phytotoxicity (Müller et al., 1995; Müller et al., 2001; Ishikawa et al., 2005; 2007).
However, to our surprise, altering endogenous trehalase activities did not seem to cause severe phenotypic irregularities in Arabidopsis. We did notice a slower germination rate of GT tre KO seeds in soil in a 12 hours-12 hours day/night cycle, and this phenotype was abrogated when increasing the light period to 16 hours. Moreover, in a 12 hours-12 hours day/night cycle, leaves of the GT tre KO plants were smaller (
This observation again suggests that growth of plants, altered in trehalase activity, strongly depends on the available energy. GT tre KO plants seem to have problems to cope with low energy conditions, whereas higher growth rates are achieved when more energy is available, e.g., through longer photosynthesis or supplied sugars. In contrast, SA TRE OX plants grow slightly better than controls in low carbohydrate conditions (
Interestingly, GT tre KO plants started flowering earlier than wild-types (16 hours-8 hours day/night cycle) (
Given trehalose-modified plants performed better in multiple stress treatments, we tested trehalase OX and KO plants under variable stress conditions.
When performing heat-stress during germinating (33° C., continuous low light, 1× MS, 1% suc), we noticed a better early seedling development of SA TRE OX seeds, compared to wild-type seeds, in contrast to GT tre KO lines (
These results, together with ample circumstantial evidence of pivotal roles of trehalose metabolism in abiotic stress responses, give us incentives to look closer to more environmental stresses such as drought and dehydration stresses in the vegetative stage. Four-week-old KO and OX plants grown in a 16-hour/8-hour day/night cycle (in soil with controlled watering regime), were subjected to drought stress for two weeks and then rewatered. In contrast to the performances of GT tre KO lines, the SA TRE OX plants withstand and recovered much better the dehydration stress (
Given trehalase controls extracellular trehalose levels, but seems also to regulate plant trehalose (sugar) metabolism, we analyzed the fluorescence of AtTRE1-GFP constructs in Arabidopsis mesophyll protoplasts, to gain more insight in the exact location of trehalase. AtTRE1 seemed to be mainly localized in the plasma-membrane (
The expression was further analyzed by fusing the trehalase promoter to a GUS/GFP construct and analyzing the plants after GUS staining. In leafs and leaf primordia, a clear specificity of the expression is seen in the stomatal guard cells (
To analyze further the effect of modulation of trehalase expression on stomata, mutants of AtTRE1 were grown in soil under normal moist conditions in a growth room. 21 days after sowing, Leaf 1 or Leaf 2 were cut out of the plant and submerged for the abaxial side in aperture buffer (10 mM MES, 10 mM CaCL2, 10 mM KCl, pH 6.1) under light conditions, or closure buffer (10 mM MES, 10 mM CaCL2, pH 6.1) under dark conditions. After two hours, leaves were submerged in aperture buffer containing 20 uM ABA under light conditions for 2 hours, the abaxial epidermis was mounted on a slide with double-sided tape and stomata aperture measurements (width) were recorded under a microscope, n=50. Trehalase overexpression plants showed a decrease in opening of stomata in aperture buffer, whereas in closure buffer, no significant difference between the wild-types, overexpression of KO plant could be detected (
To analyze the effect of trehalase expression on number of stomata, mutants of AtTRE1 were grown on ½ MS, 1% sucrose horizontal plates, under 16 hours light/8 hours dark regime for 21 days. Leaf 1 or Leaf 2 were cut off and cleared in lactic acid for 2 days under constant shaking. Leaves were put on a slide and pictures (0.142 mm2) of 2 areas (tip and base) of each leaf were taken to count stomata and pavement cells, n=10 and to calculate the stomatal index. The stomatal index corresponds to the ratio of stomata per total number of cells in the epidermis (# stomata/(2×# stomata+# pavement cells). Compared with their respective wild-types, there is a slight decrease in the number of stomata (
This is a national phase entry under 35 U.S.C. §371 of International Patent Application PCT/EP2010/051500, filed Feb. 8, 2010, published in English as International Patent Publication WO 2010/089392 A1 on Aug. 12, 2010, which claims the benefit under Article 8 of the Patent Cooperation Treaty to U.S. Provisional Patent Application Ser. No. 61/207,188, filed Feb. 9, 2009.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP10/51500 | 2/8/2010 | WO | 00 | 9/12/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61207188 | Feb 2009 | US |
