METHOD OF IMPROVING CHLOROPLAST FUNCTION AND INCREASING SEED YIELD

Abstract

Ascorbate protects tissues against damage caused by reactive oxygen species (ROS) produced through normal metabolism or generated from stress. The inositol route to AsA involves four enzymes: myo-inositol oxygenase, glucuronate reductase, gluconolactonase (GNL), and L-gulono-1,4-lactone oxidase (GulLO). Eighteen putative GNLs were identified in Arabidopsis, one of which, AtGNL, is interesting because it possesses a chloroplastic signal peptide. Knockouts on this gene had lower foliar AsA and stunted growth compared to controls. The functional gene restored the phenotype of the knockouts, and those plants had higher AsA content, enhanced photosynthetic capacity, and higher seed yield.

Claims

1. A method of increasing production of L-ascorbic acid (AsA) in a plant or enhancing the photosynthetic capacity of the plant or enhancing the seed yield of the plant, the method comprising: increasing the level of AsA in the plant by expressing gluconolactonase (GNL).

2. The method of claim 1 further comprising: Over-expressing the GNL.

3. The method of claim 1 wherein the plant exhibits increased production of AsA.

4. The method of claim 1 where the plant exhibits enhanced photosynthetic capacity and/or enhanced seed yield.

5. The method of the claim 1 wherein the GNL is At1g56500.

6. The method of claim 2 wherein the plant is Arabidopsis.

7. A method for increasing production of L-ascorbic acid (AsA) or enhancing the photosynthetic capacity of the plant or enhancing the seed yield of the plant, the method comprising: genetically engineering said plant to over-express a gluconolactonase (GNL) wherein overexpression of said GNL in said genetically engineered plant results in increased vitamin C content in said genetically engineered plant as compared to a control plant of the same species lacking said over-expressing said GNL.

8. The method of claim 6 wherein said step of genetically engineering includes i) isolating and purifying the GNL; and ii) transforming said plant with said GNL.

9. The method of claim 8 wherein said step of genetically engineering said plant results in increasing production of AsA compared to a control plant of the same species lacking the GNL.

10. The method of claim 8 wherein said step of genetically engineering said plant results in enhancing the photosynthetic capacity of the plant and/or enhanced seed yield of the plant compared to a control plant of the same species lacking said over-expressing GNL.

11. The method of the claim 7 wherein the GNL is At1g56500.

12. The method of the claim 9 wherein the GNL is At1g56500

13. The method of the claim 10 wherein the GNL is At1g56500

14. The method of claim 9 wherein the plant is Arabidopsis.

15. The method of claim 10 wherein the plant is Arabidopsis.

16. A plant genetically engineered to over-express a gluconolactonase (GNL), the plant comprising: Over-expressed GNL wherein said genetically engineered transgenic plant has increased vitamin C content as compared to a control plant of the same species lacking the GNL.

17. The plant of claim 16 wherein said plant exhibits increased production of AsA compared to a control plant of the same species lacking the GNL.

18. The method of claim 16 wherein said plant exhibits increased seed yield compared to a control plant of the same species lacking the GNL.

19. The plant of claim 16 wherein the GNL is At1g56500

20. The plant of claim 16 wherein the plant is Arabidopsis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In the following drawings, which form a part of the specification and which are to be construed in conjunction therewith, and in which like reference numerals have been employed throughout wherever possible to indicate like parts in the various views:

[0051] FIG. 1 is a chemical structure view of one embodiment of the present invention;

[0052] FIG. 2 is a metabolic network view thereof;

[0053] FIG. 3 is a flowchart view thereof;

[0054] FIG. 4 is a metabolic network view thereof;

[0055] FIG. 5 is a table view thereof;

[0056] FIG. 6 is a schematic view thereof;

[0057] FIG. 7 is a blot view thereof;

[0058] FIG. 8 is a construct view thereof;

[0059] FIG. 9 is a table view thereof;

[0060] FIG. 10 is a blot view thereof;

[0061] FIG. 11 is a blot view thereof;

[0062] FIG. 12 is an analytical view thereof;

[0063] FIG. 13 is a table view thereof;

[0064] FIG. 14 is a graph view thereof;

[0065] FIG. 15 is a graph view thereof;

[0066] FIG. 16 is a graph view thereof;

[0067] FIG. 17 is a table view thereof;

[0068] FIG. 18 is a graph view thereof;

[0069] FIG. 19 is a photographic and chart view thereof;

[0070] FIG. 20 is a photographic view thereof;

[0071] FIG. 21 is a chart view thereof;

[0072] FIG. 22 is a table view thereof;

[0073] FIG. 23 is a chart view thereof;

[0074] FIG. 24 is a chart view thereof;

[0075] FIG. 25 is a metabolic network view thereof;

[0076] FIG. 26 is a chart view thereof;

[0077] FIG. 27 is a table view thereof;

[0078] FIG. 28 is a chart view thereof;

[0079] FIG. 29 is a table view thereof;

[0080] FIG. 30 is a chart view thereof;

[0081] FIG. 31 is a table view thereof;

[0082] FIG. 32 is a photographic view thereof;

[0083] FIG. 33 is a phylogenetic view thereof;

[0084] FIGS. 34A and 34B are table views thereof;

[0085] FIG. 35 is a chart view of one embodiment of the present invention;

[0086] FIGS. 36A, 36B, and 36C are a genetic sequencing view of one embodiment of the present invention (SEQ ID NO: 1); and

[0087] FIG. 37 is a genetic sequencing view of one embodiment of the present invention (SEQ ID NO: 2).

DETAILED DESCRIPTION

[0088] Arabidopsis thaliana ecotype Columbia wild type seeds (Col-0, stock #CS60000), SALK_026172, and SALK_011623 were obtained from the Arabidopsis Biological Resource Center (ABRC, Columbus, Ohio). Seeds were sterilized with 70% (v/v) ethanol for 10 min followed by 50% (v/v) sodium hypochlorite containing 0.05% (v/v) Tween-20 for 15 min. Next, seeds were washed 6 times with sterile water. Finally, seeds were transferred to a petri dish containing medium which consisted of Murashige-Skoog (MS) salts (Murashige and Skoog, 1962), MS vitamins, and 3% (w/v) sucrose, at pH 5.6. The medium was supplemented with 0.04% (w/v) MgSO.sub.4.7H.sub.2O. The seeds were vernalized for 3 days at 4 C. Plates were transferred to a growth chamber and incubated at 23 C., 65% humidity, 16:8 h photoperiod and 200 mol/m.sup.2/s light intensity. After establishment, seedlings were transferred to PM-15-13 AIS MIX Arabidopsis soil (Lehle-Seeds, Round Rock, Tex.) in 2 inch pots. Pots were covered with a dome for one week and after that plants were grown until they reached maturity.

[0089] Nicotiana benthamiana seeds were obtained from The Department of Plant Pathology, Physiology and Weed Science at Virginia Polytechnic Institute and State University (Blacksburg, Va.). Seeds were sown in 4.5 inch pots containing Pro-mix BX soil (Premier Horticulture Ltd, Canada) with fertilizer Osmocote 14-14-14 (Scotts, Canada). Vermiculite was overlaid on top of the seeds. The pots were covered with a dome for one week. Plants were grown in an environmental control chamber with the following conditions: 25 C. (day)/21 C. (night) temperature, 65% relative humidity, 16:8 h photoperiod, and 150 mol/m.sup.2/s light intensity.

2.2.2. Constructs of Interest

[0090] Two gene constructs were made for this project. The first construct is one where the cDNA encoding a putative AtGNL was placed under the control of the 35S promoter and the tobacco etch virus (TEV) enhancer (AtGNL-6HIS:pBIB-Kan), (Becker, 1990). In this construct a histidine tag was added to the C-terminus of the protein of interest to allow detection using antibodies and to facilitate purification. The second construct is one where the putative promoter region of the AtGNL (a 1000 bp fragment preceding the ATG) was cloned and fused to the GUS reporter gene to better understand the spatial and temporal expression of this gene (pAtGNL:pCAMBIA1305.1). FIG. 8 shows the Constructs of interest. (A) The At1g56500-HIS:pBIB-Kan construct containing At1g56500 (AtGNL) with a six histidine (6-HIS) tag and adjacent neomycin phosphotransferase II (nptII) selectable marker. (B) The pAt1g56500:pCAMBIA1305.1 construct containing the AtGNL promoter with the GUS-PLUS reporter gene and the hygromycin phosphotransferase (hph) selectable marker. NOS-P: promoter of nopaline synthase gene, 35S-T: terminator of the 35S cauliflower mosaic virus gene; TEV: tobacco etch virus translational enhancer; LB and RB: left and right T-DNA borders, respectively.

2.2.3. Development of Transgenic Lines

[0091] To study the expression of AtGNL in Arabidopsis thaliana, stable transgenic plants were developed by the floral dip method (Clough and Bent, 1998). Wild type CS60000 and knockouts: SALK_026172 and SALK_011623, were transformed with the Agrobacterium tumefaciens GV3101 strain carrying the construct of interest. The T0 seeds were planted on MS medium plus kanamycin. The antibiotic resistant seedlings were transferred to soil and grown to maturity under the above stated conditions. The presence of the transgene of interest was established via PCR using gene specific primers, and genomic cDNA as a template. To develop homozygous versions of the knockout lines, over-expresser (wild type plus AtGNL), and restored lines (knockouts plus AtGNL) T1 plants that were high AsA expressers were identified. The seeds of those plants were sterilized with 70% (v/v) ethanol for 10 min followed by 50% (v/v) sodium hypochlorite containing 0.05% (v/v) Tween-20 for 15 min. Next, seeds were washed 6 times with sterile water. Finally, seeds were transferred to petri dishes containing Murashige and Skoog (MS) medium which consists of salts, MS vitamins, and 3% (w/v) sucrose at pH 5.6. The medium was supplemented with 0.04% (w/v) MgSO.sub.4.7H.sub.2O, and 50 mg/L kanamycin. Plated seeds were vernalized for 3 days at 4 C. After establishment, seedlings were transferred to soil and grown under the above stated conditions until they reached maturity. This process was repeated until plants with a 100% germination score in the presence of antibiotic selection were developed.

2.2.4. Ascorbate Measurements

[0092] In Arabidopsis, in planta AsA concentration changes throughout the day as well as during development (Tamaoki et al., 2003; Zhang et al., 2009). Fifty mg of leaf tissue were collected between 9:00 am and 11:00 am. Tissue was frozen immediately in liquid nitrogen and stored at 80 C. until analyzed. Reduced, oxidized, and total AsA were measured using a 96-well plate format as described by Haroldsen et al., (2011). Briefly, frozen tissue was pulverized in 6% (w/v) meta-phosphoric acid, and centrifuged at 13,000 rpm for 15 min. Reduced AsA was determined by measuring the decrease in absorbance at 265 nm after addition of 0.5 unit of ascorbate oxidase to 300 L of the reaction medium containing the plant extract and 100 mM phosphate buffer at pH 6.9. Oxidized ascorbate was measured in a 300 L reaction mixture with 10 L of 40 mM dithiothreitol (DTT) after incubation in the dark for 20 min at room temperature. The reaction was followed by measuring absorption at 265 nm. Calculations were made based on a standard curve made with pure L-ascorbic acid run in parallel. Ten biological replicates were measured in analytical triplicate and reported as mol per gram fresh weight (mol/g FW).

2.2.5. Transient Expression of AtGNL in Nicotiana benthamiana

[0093] In order to test its function the putative GNL recombinant protein was produced in Nicotiana benthamiana. This plant is a widely used platform for the production of active proteins, including antibodies, enzymes and other proteins that require post-translational modifications (Klimyuk et al., 2012; Leuzinger et al., 2013). To find the optimal expression time, five week old N. benthamiana plants were vacuum infiltrated with the At1g56500-6HIS.pBIB-kan construct as described by Medrano et al., 2009. Leaf tissue was harvested at 24, 48, 72, and 96 h post infiltration for further analysis. The optimum time for tissue collection based on Western blot data, was found to be at 48 h post infiltration (data not shown). In subsequent experiments all leaves were collected at 48 h post-infiltration, frozen immediately in liquid nitrogen and stored at 80 C. until further processing. Plants infiltrated with the empty pBIB-kan binary vector (EV) were used as controls for these experiments.

[0094] In order to confirm the expression of the AtGNL in N. benthamiana, Western blot experiments were carried out. Crude extracts were made by grinding frozen tissue in the presence of two volumes of SDS buffer containing 150 mM Tris-HCl pH 6.8, 5 mM EDTA pH 8.0, 30% (v/v) glycerol, 6% (w/v) SDS. The homogenate extract was then centrifuged at 13,000g for 15 min, and the supernatant was recovered. Proteins were separated via SDS-PAGE. Six L of plant extract were mixed with 2.5 L of SDS loading buffer (4) and 1 L of DTT, incubated for 10 min at 70 C. and separated by SDS-PAGE on 10% precast mini-gels (Expedeon, San Diego, Calif.) with a Tris-MOPS buffer. Subsequently, separated proteins were electro blotted onto a nitrocellulose membrane, using transblotting buffer containing: 25 mM Tris base, 192 mM glycine, and 20% methanol. Recombinant AtGNL-6HIS was detected using an anti-HIS (C-term)/AP antibody at a 1:2,000 v/v dilution (Invitrogen, Carlsbad, Calif.) and CDP-start, a chemiluminescent substrate for alkaline phosphatase detection (Roche Diagnostics, Indianapolis, Ind.).

2.2.6. Recombinant AtGNL Purification

[0095] Recombinant AtGNL protein was purified from N. benthamiana leaves. Five grams of leaf tissue were pulverized in liquid nitrogen and proteins were extracted with 10 mL of buffer A (75 mM sodium phosphate dibasic, 25 mM sodium phosphate monobasic, 150 mM NaCl, 10 mM sodium metabisulfite, and 0.6% (v/v) protease inhibitor cocktail, pH 7.4). The extract was then centrifuged at 13,000g for 15 min. The supernatant obtained after centrifugation was loaded onto a nickel affinity column (HIS60 Ni Superflow) and incubated for 1 h at 4 C. Then, the column was washed with 50 mM sodium phosphate pH 7.4, 300 mM NaCl, 40 mM imidazole buffer and the bound proteins were eluted with 250 mM of imidazole. The eluate from the nickel column was concentrated using an AMICON 30K ultra centrifugal filter (Millipore, Billerica, Mass.). Total soluble protein concentration was estimated by the Bradford method (Bradford, 1976) using Coomassie blue G-250 dye (Thermo Scientific) and bovine serum albumin (Pierce, Rockford, Ill.) as a standard. Protein fractions from the purification procedure were separated by SDS-PAGE and the AtGNL was detected by Western blot and silver staining using Pierce Silver Stain Kit (Thermo Scientific).

2.2.7. Recombinant AtGNL Enzyme Assay

[0096] The lactonase activity was assayed in vitro based on the decrease in absorbance (405 nm) of the p-nitrophenol pH indicator that resulted from the enzymatic opening of the lactone ring when D-glucono--lactone was used as substrate in the presence of the AtGNL as previously described (Ishikawa et al., 2008). Enzyme preparations were made fresh for individual experiments at room temperature.

[0097] In order to establish the optimal enzyme activity for AtGNL, several conditions were tested. The optimum concentration for enzyme activity was 30 g per reaction. One mL of the reaction typically contained: 10 mM PIPES pH 6.5, 5 mM D-glucono--lactone, 75 M MnCl.sub.2, 2.5 mM p-nitrophenol, and an aliquot of the purified enzyme. An equal amount of boiled enzyme was used as control for these experiments.

[0098] In order to examine the specificity of the AtGNL enzyme for D-glucono--lactone, multiple substrates were tested in the lactonase assay. The substrates used in this experiment were: D-glucono--lactone (D-GuIL), L-galactono--lactone (L-GaIL), L-galactonic acid (L-GalA), L-gulono--lactone (L-GulL), and L-gulonic acid (L-GulA). The L-GaIA and L-GuIA were prepared by the hydrolysis of L-GaIL and L-GuIL, respectively. For hydrolysis 20 mL of 0.3 M NaOH were added to 100 L of 10 mM L-GaIL or L-GuIL, the mixture was vigorously agitated by vortexing for 20 s, and 20 L of 0.3 M HCl were added to neutralize the solution (Ishikawa et al., 2008). For enzyme kinetic experiments, individual reactions were monitored for 15 min at different substrate concentrations. Analysis was done using the GraphPadPrism 6.2 software.

2.2.8. High Throughput Phenotyping

[0099] To characterize the phenotype of the over-expresser (L60, L61, L62), knockout lines (SALK_026172 and SALK_011623), and restored lines (L89, L90, L100, L128, L129, L130), under low, normal, and high light conditions a high throughput phenotyping platform (Scanalyzer HTS instrument, Lemnatec, Germany) and the LemnaControl software were used. This instrument is equipped with a robotic arm that holds visible (VIS, a.k.a. RGB), fluorescence (FLUO), and near infrared (NIR) high-resolution cameras. This system empowers unbiased, non-invasive, automated, and effective characterization of plant phenotypes. The cameras in the system are as follows: VIS camera, piA2400-17gc CCD (Basler, Ahrensburg, Germany) with resolution of 24542056 pixels; FLUO camera, scA1600-14gc CCD (Basler, Ahrensburg, Germany) with resolution 16241234 pixels; and NIR camera, Goldeye GIGE P-008 SWIR (Allied Vision Technologies, Stadtroda, Germany) with resolution 320256 pixels and with spectral sensitivity between 900 and 1700 nm.

[0100] In the greenhouse plants were grown in PM-15-13 AIS MIX soil (Lehle-Seeds, Round Rock, Tex.) in Quickpot 15 trays in a greenhouse during Mar. 12-30 2015 in Jonesboro, Ark., USA (latitude 29.4889 and longitude 98.3987). Growth conditions were as follows: 22 C.-26 C. temperature, 16:8 h photoperiod, 55% humidity and three different light conditions: low (35-110 mol/m.sup.2/s), medium (110-350 mol/m.sup.2/s) and high light (350-700 mol/m.sup.2/s). Light intensity was recorded four times per day (9:00 am, 12:00 pm, 3:00 pm, and 6:00 pm) to cover the entire sunlight period.

[0101] Images of AtGNL lines were captured every two days between 16 days and 26 days after germination, to cover the full vegetative growth. Images (5670 images=7 lines15 biological replicates3 light treatment6 times points3 cameras) were analyzed using the LemnaGrid Software. The analysis of the RGB images was done as previously described by Arvidsson et al., (2011). Multiple phenotypic parameters were calculated for each plant including: projected leaf area (cm.sup.2), convex hull area (cm.sup.2), caliper length (a.k.a. rosette diameter, mm) and compactness (measure of the bushiness of the plant). From the RGB images the relative area of the plants displaying normal green color versus the area with detectable yellow color (chlorosis) were calculated. The analysis of the NIR images was similar to the color classification of VIS camera, using the acquired gray-scale images, where high water content corresponds to darker tones while low water corresponds to lighter gray tones. The software used this information to calculate the relative area with low, medium, and high water content. The fluorescence camera acquires red-scale images and in this case the red tones were divided into four equidistant bins, and the software calculated the relative area with zero, low, medium, and high fluorescence. Quantitative data obtained from the images were analyzed.

2.2.9. Photosynthetic Efficiency

[0102] In order to determine photosynthetic efficiency of photosystem II (/II), linear electron flow (LEF), and non-photochemical quenching (NPQt) of the knockout lines (SALK_026172 and SALK_011623), over-expresser (L61), restored lines (L100 and L129), empty vector, and wild type controls growing under low and normal light conditions were analyzed using a MultispeQ. This is a hand held fluorometer developed by the Kramer Laboratory at Michigan State University. Ten biological replicates were chosen randomly at the same time of day for measurements. Data were visualized in an Android tablet (Samsung Galaxy Tab 4) and analyzed in the PhotosynQ website (www.photosynq.org).

2.2.10. PromoterAtGNL:GUS Expression in Arabidopsis thaliana

[0103] To study the expression of At1g56500 in different plant tissues, Arabidopsis thaliana var. Columbia was transformed by the floral dip method (Clough and Bent, 1998) with Agrobacterium tumefaciens GV3101 carrying the construct of interest (pAtGNL:pCAMBIA1305.1). A different set of plants was also transformed with bacteria carrying the empty vector control (pCAMBIA1305.1). T0 seeds were selected with hygromycin and the antibiotic resistant seedlings were transferred to soil and grown to maturity under the above mentioned conditions. The presence of the transgene of interest was established via PCR using gene specific primers, and genomic cDNA as a template. Seeds of the PCR positive plants were sterilized and transferred to a petri dish containing MS media with 20 mg/L hygromycin. Plated seeds were vernalized for 3 days at 4 C. and then transferred to an environmentally controlled chamber. Hygromycin resistant seedlings were transferred to soil and grown until maturity.

[0104] Explants (seedlings, leaves, flowers, and fruits) were cut from plants 4, 8, 12, and 30 days after germination. Next, the explants were incubated in fresh and cold phosphate buffer pH 7.0 with 4% formaldehyde at room temperature for 30 min. The explants were washed several times with cold phosphate buffer for 1 h, then vacuum infiltrated with X-Gluc substrate solution containing: 1 mg 5-bromo-4-chloro-3-indolyl -D-glucuronide in 100 L of methanol, 1 mL 2 phosphate buffer, 20 L 0.1 M potassium ferrocyanide, 20 L 0.1 M potassium ferricyanide, 10 L 10% (w/v) solution of Triton X-100, and 850 L of water. Tissues were incubated in darkness at room temperature overnight until a distinct blue staining appeared. Finally, explants were incubated in 70% ethanol until the chlorophyll was removed. Photographs were taken with AxioCam MRc camera connected to a Stemi 2000-C stereo microscope (Zeiss).

2.2.11. Phylogeny

[0105] In order to identify a functional GNL in Arabidopsis thaliana (AtGNL), known GNLs, and putative GNLs from other organisms were compared using the TAIR database (www.arabidopsis.org). The AtGNL was obtained from TAIR database based on highest protein homolog. That sequence was then converted to FASTA format using the EMBL-EBI (www.ebi.ac.uk/tools/stc/readseq/). The MEGA6 software enabled reading and comparing the AtGNL with known and putative GNL sequences (Tamura et al., 2013).

2.2.12. Statistical Analysis

[0106] Data was analyzed by SAS software 9.4 (SAS Institute, 2016). Analysis of variance was carried out by ANOVA procedure. Least squares means (LS-means) were calculated to evaluate AsA content per line, at =0.05.

2.3. Results and Discussion

2.3.1. Purification and Characterization of Recombinant At1g56500

[0107] To demonstrate the GNL activity of Arabidopsis thaliana gluconolactonase in vitro, an N. benthamiana-based transient expression system was used. Plants were vacuum infiltrated with the Agrobacterium tumefaciens LBA4404 strain carrying the At1g56500-6HIS construct. The protein accumulation is highest at 48 h post infiltration (data not shown).

[0108] In order to establish a protein purification protocol for AtGNL, several extraction buffers were tested to identify those that allow recovery of the highest amount of protein. FIG. 9 shows the list of buffers tested for protein purification.

[0109] Protein fractions from the various purification procedures were separated by SDS-PAGE and AtGNL was detected by Western blot. FIG. 10 shows the AtGNL protein extracted with different buffers. Western blot of total protein extracted from N. benthamiana leaves with different buffers as described in FIG. 7. M: marker, lane 1: crude extract with buffer-1, lane 2: crude extract with buffer-2, lane 3: crude extract with buffer-3, lane 4: crude extract with buffer-4, lane 5: crude extract with buffer-5, lane 6: sample extracted in buffer-5 and resuspended in buffer-4, lane 7: crude extract in buffer-5, lane 8: sample extracted in buffer-5 and resuspended in buffer-3, lane 9: sample extracted in buffer-5 and resuspended in buffer-6, lane 10: crude extract in buffer-6.

[0110] The optimal buffer to recover more recombinant protein was buffer 6.

[0111] In order to establish the optimal concentration for washing and elution buffer, several imidazole concentrations were tested. FIG. 11 shows the AtGNL protein eluted with different imidazole concentrations. Western blot of AtGNL protein with different concentration of imidazole. M: marker, B6: crude extract in buffer 6, FT flow through, W: wash buffer. As illustrated in FIG. 11, 40 mM and 250 mM were best for washing and eluting conditions, respectively.

[0112] FIG. 12 shows the Purification of the AtGNL:pBIB-kan-6HIS expressed in N. benthamiana leaves. M: marker, lane 1: crude extract, lane 2: flow through, lane 3: wash, lane 4: enzyme, lane 5: concentrated enzyme. FIG. 12 illustrates the result of the purification of AtGNL from N. benthamiana tissue using nickel affinity chromatography. Western blot results showed the presence of AtGNL in the crude extract and flow through or wash indicating protein had a partial binding to the cation column. The silver-stained gel indicates that the protein preparation contained mostly the protein of interest with a few minor contaminants.

2.3.2. Enzyme Activity of Recombinant At1g56500

[0113] Once an effective purification procedure was developed, the next step was to standardize the assay to test the AtGNL activity. Gluconolactonase (GNL, EC 3.1.1.17) catalyzes the hydrolysis of D-glucono--lactone (D-GulL) to D-gluconic acid (Ogawa et al., 2002). The lactonase activity was assayed in vitro based on the decrease in absorbance (405 nm) of the p-nitrophenol pH indicator that resulted from the enzymatic opening of the lactone ring when D-glucono--lactone (D-GuIL) was used as substrate in the presence of the AtGNL as previously described (Hucho and Wallenfels, 1972). The enzymatic activity was assayed at 25 C. with 10 mM PIPES pH 6.5, 5 mM D-GuIL, 75 M MnCl.sub.2, 2.5 mM p-nitrophenol, and 30 g of the purified enzyme (AtGNL) in 1 mL of reaction (Ishikawa et al., 2008). With the exception of D-GuTL, the recombinant AtGNL did not exhibit activity with any of the substrates tested. FIG. 13 shows the Substrate preference of AtGNL.

[0114] All enzymes work with a range of temperatures specific to the organism from which they are extracted. The effect of temperature on the AtGNL activity was also determined. The activity of the AtGNL enzyme was highest at temperatures between 25 C. and 35 C. The activity drastically decreased when the temperature was increased to 40 C. FIG. 14 shows the Effects of temperature and pH on the activity of the AtGNL enzyme. (A) pH effect on GNL activity. (B) Temperature effect on GNL activity. Measurements were made in duplicate. Values are meansSD. Ogawa et al., (2002) reported that the GNL enzyme from A. niger had higher activity at 30 C., while the activity of the GNL from P. aeruginosa is optimal at 24 C. (Tarighi et al., 2008), which are similar to the AtGNL.

[0115] Kondo et al., (2006), reported that the activity of the rat GNL was highest at pH 6.4, while Tarighi et al., (2008) demonstrated that the optimal activity of the P. aeruginosa GNL was at pH 7.2. In contrast, in this study the A. thaliana GNL enzyme had a higher activity at pH 6.0, and the activity decreased by 4-fold when the pH was increased to 6.3 (FIG. 14). Lower pH values were not tested because PIPES buffer cannot dissolve at pHs lower than 6.0.

[0116] To assess if the AtGNL activity had a preference for a particular divalent ion, various cofactors were tested. Ishikawa et al., (2008) reported that the GNL enzyme from E. gracilis had a higher activity using ZnCl.sub.2 as a cofactor and that this activity decreased around 4-fold when changed to MnCl.sub.2. In these experiments, no significant difference in GNL activity among the tested cofactors was observed (FIG. 15). Increasing the substrate concentration increased the rate of reaction or enzyme activity. In order to identify the optimal concentration of the D-glucono--lactone, multiple substrate concentrations were tested. FIG. 15 shows the Effects of cofactor and substrate on the activity of the AtGNL enzyme. (A) Cofactors effect on GNL activity. (B) D-GuIL substrate concentration effect on GNL activity. Measurements were made in duplicate. Values are meansSD. The 3 mM of D-glucono--lactone was the most effective substrate concentration for this assay.

[0117] Enzyme kinetic analysis was performed with D-GuIL at a concentration of 1 mM to 50 mM of substrate. The enzyme activity with 5 mM of D-GuIL at pH 6.0 was 10.54 mol min.sup.1 mg.sup.1 of protein, V.sub.max=1.16110.sup.6 (38.7 mol min.sup.1 mg.sup.1 of protein) and K.sub.m=2.989. FIG. 16 shows the Enzyme kinetics of the recombinant At1g56500 enzyme. (A) Michaelis-Menten. (B) Double reciprocal Lineweaver-Burke. Measurements were made in duplicate. Values are meansSD. FIG. 17 summarizes the comparison between the kinetic parameters of the AtGNL with the one of known GNLs. Based on these results the G. oxidans GNL is the most similar to the Arabidopsis GNL.

2.3.3. Characterization of the Phenotype of Gluconolactonase Lines with a Scanalyzer HTS Platform

[0118] Seeds expressing the AtGNL-6HIS:pBIB-kan (AtGNL) and empty pBIB-Kan (control) were screened in the Lorence Laboratory (unpublished). One hundred and thirty primary transformants that were PCR positive were screened to identify high AsA expressers. After four rounds of screening, three lines per group were selected for further analysis: over-expressers (WT+AtGNL), restored 1 (SALK_026172+AtGNL), and restored 2 (SALK_011623+AtGNL).

[0119] Homozygous lines (T5), plants with 100% germination in the presence of antibiotic selection were developed for over-expresser (L60, L61, L62), restored-1 lines (L89, L90, L100), and restored-2 lines (L128, L129, L130). FIG. 18 shows the total foliar AsA level of AtGNL lines under normal light conditions. (A) Over-expressers and wild type (WT). (B) Restored lines and knockout control (S_026172). (C) Restored lines and knockout control (S_011623). Asterisks indicate significant differences between controls and high AsA lines as determine by Turkey multiple comparisons test, =1, ****=P<0.0001, ***=P<0.0002, **=P<0.0017, *=P<0.0155. Values are meansSD, n=15.

[0120] The phenotype of these homozygous lines was analyzed using a Scanalyzer HTS instrument under normal conditions as described in materials and methods. Plant images were captured every two days from 16 to 26 days after germination. Representative images of homozygous AtGNL lines and their respective controls are shown in FIG. 19. From these images the projected leaf area as an indicator of plant growth was measured. FIG. 19 shows the Phenotype of AtGNL lines grown under normal conditions. (A) Representative images of AtGNL lines acquired with the visible camera (aka RGB). (B) Growth curves of AtGNL lines compared with their respective controls. Values are meansSE, n=15. There is a strong correlation between higher biomass and projected leaf area. Restored-1 and restored-2 lines had more biomass compared to their controls SALK_026172, SALK_011623, respectively, and those restored lines presented higher projected leaf area compared with their controls.

[0121] Based on these results, foliar AsA level, and the phenotype analysis, further studies were done only with the lines that had the highest foliar AsA content and fastest growth and higher biomass and projected leaf area.

[0122] Over-expresser L61 (OE) and the empty vector control (EV); restored-1 L100 (R-1) and its control SALK_026172 (KO-1), restored-2 L129 (R-2), and its control SALK_011623 (KO-2), and wild type (WT) control were selected for further analysis. First, the effect of low and high light conditions on the selected plants was assessed. Routinely, plants are grown in environmental control chambers. A first attempt to study light effects was accomplished by growing plants in multiple chambers: (315=7 lines15 biological replicates3 light treatments). However, it was very difficult to achieve uniform conditions, with the only variable being the light intensity. To solve this problem, the experiment was conducted in the greenhouse. Two different density meshes were used to diffuse the light to the plants. FIG. 20 shows the experimental set up for studying the effect of light on the phenotype of AtGNL lines. Light intensity was measured four times per day (9:00 am, 12:00 pm. 3:00 pm, and 6:00 pm) to cover the sunlight period. The light intensity for these three treatment was defined as: low light (35-110 mol/m.sup.2/s), normal light (110-350 mol/m.sup.2/s), and high light (350-700 mol/m.sup.2/s).

[0123] Normal outdoor light on a sunny summer day is around 1000-2000 mol/m.sup.2/s (Mishra et al., 2012) However, because A. thaliana is a spring understory plant, anything above 350 mol/m.sup.2/s is considered high light.

[0124] Fifty mg of leaf tissue were collected at developmental stage 6.3 as defined by Boyes et al., (2001) between 9:00-11:00 am. Reduced, oxidized, and total AsA were measured via an enzyme-based method as previously described (Haroldsen et al., 2011). The results indicate that the over-expresser and the restored lines had a higher foliar AsA than their respective controls growing under similar conditions. FIG. 21 shows the Total foliar AsA levels of AtGNL lines under low, normal and high light conditions. (A) Over-expressers and wild type (WT). (B) Restored lines and knockout control (SALK_026172). (C) Restored lines and knockout control (SALK_011623). S026172 had a lower significant difference compared with wild type control at high light treatment. Each line was compared to the control (WT), analyzed by t-tests (LDS) at =0.05. Significant differences are indicated by ***. WT: wild type, EV: empty vector, OE: over-expresser, KO: knockout, R: restored. n=15. The statistical analysis of total foliar AsA content in the three different light treatments in AtGNL lines shown in FIG. 22 validates this conclusion. Two-way ANOVA (=0.05). The table in FIG. 22 indicates lines are significantly different and that there is a significant interaction between the lines and light treatments.

[0125] The projected leaf area results showed the same trend, where over-expressers and restored lines were bigger than their controls, with KO-1 being the worst performer at all light conditions tested. FIG. 23 shows the Projected leaf area of AtGNL lines grown under low, normal, high light conditions. (A) Low light. (B) Normal light. (C) High light. Values are means of 15 biological replicates. WT: wild type, EV: empty vector, OE: over-expresser, KO: knockout, R: restored. n=15. There is a strong correlation between foliar AsA level and projected leaf area: over-expresser and restored lines had higher foliar AsA levels and higher projected leaf area compared with their respective controls.

[0126] In planta chlorophyll, fluorescence measured with the fluorescence camera can serve as an indicator of whether the plants are under stress. These plants were grown under the normal light regime. High fluorescence in plants is opposite of high photosynthetic efficiency (Lichtenthaler, 1988). FIG. 24 shows the Chlorophyll fluorescence patterns of AtGNL lines. Relative in planta chlorophyll content measured with the FLUO camera. Values are means of 15 biological replicates. WT: wild type, EV: empty vector, OE: over-expresser, KO: knockout, R: restored. n=15. The knockout KO-1 line showed high fluorescence compared to the other lines under high light conditions. This knockout line has a lower AsA level in the leaves, lower biomass, and projected leaf area, and also higher fluorescence compared with rest of the lines. Overall these results show that the AtGNL enzyme is essential to support normal AsA content in leaves and normal growth and development in Arabidopsis.

2.3.4. Photosynthetic Efficiency of AtGNL Lines Under Low and Normal Light Conditions

[0127] Genes involved in the AsA metabolic network have been identified that were down and up regulated in response to light. FIG. 25 shows the Effect of darkness on the expression of genes in the AsA metabolic network. Microarray data deposited at Genevestigator was mined. Genes that are down regulated in darkness are shown in red, while genes that are up-regulated are shown in green. Yellow color indicates genes isoforms that are upregulated under dark conditions. The D-mannose/L-galactose, L-gulose and D-glucuronate pathway are repressed under low light conditions while the myo-inositol pathway keeps working. Suza and Lorence, unpublished. These results indicate that the L-gulose, D-mannose/L-galactose, and D-galacturonate pathways are down regulated under darkness, while the myo-inositol route is up regulated. If the transcripts are down regulated, the enzymes are expected to be down regulated as well. Because photosynthetic efficiency is a measure of light stress and redox potential, photosynthetic efficiency was analyzed for AtGNL lines growing under low and normal light conditions. These photosynthetic efficiency measurements were done with a MultispeQ, a hand-held device developed in the Kramer Laboratory (Michigan State).

[0128] Photosynthetic efficiency is the fraction of light (photons) that plants obtain from the sun to convert into chemical energy during photosynthesis. Under normal light conditions there was no penalty in the photosynthetic efficiency of plants lacking AtGNL expression. FIG. 26 shows the Photosynthetic efficiency of AtGNL lines under low and normal light conditions. Each line was compared to the control (WT), analyzed by t-tests (LDS) at =0.05. Significant differences are indicated by ***. WT: wild type, EV: empty vector, OE: over-expresser, KO: knockout, R: restored. n=10. In contrast, the over-expressers and restored lines displayed enhanced efficiency indicating a positive impact on photosynthesis due to higher AtGNL expression. When plants were grown under low light, results were quite different. In this case a lower photosynthetic efficiency was detected in EV and KO-1 compared to the WT control. This indicates a penalty in photosynthetic efficiency due to lack of AtGNL expression. FIG. 27 shows the Statistical analysis of photosynthetic efficiency of AtGNL lines grew at low and normal light conditions. Two-way ANOVA (=0.05). Photosynthetic efficiency as response to light conditions. The table indicates the treatment light has a significant effect pvalue=0.001. The lines also has a significant effect pvalue<0.0001, and there is a significant interaction between light and lines pvalue=0.1773.

[0129] In addition to photosynthetic efficiency two other parameters related to photosynthesis were measured: linear electron flow (LEF) and non-photochemical quenching (NPQt). The linear electron flow rate (LEF) has a direct correlation to photosynthetic efficiency. LEF facilitates the movement of H.sup.+ ions across the thylakoid membrane to create an electrochemical gradient that is used by ATP-synthase to produce energy (ATP). FIG. 28 shows the Linear electron flow of AtGNL lines under low and normal light conditions. Each line was compared to the control (WT), analyzed by t-tests (LDS) at =0.05. Significant differences are indicated by ***. WT: wild type, EV: empty vector, OE: over-expresser, KO: knockout, R: restored. n=10. FIG. 28 shows that under low light conditions both knockouts had lower LEF values than the controls, while KO-1 was the line with the worst performance under normal light conditions. FIG. 29 shows the Statistical analysis of linear electron flow of AtGNL lines grown at low and normal light conditions. Two-way ANOVA (=0.05). LEF as response to light conditions. The table indicates the treatment light has a significant effect pvalue=0.0006. The lines also has a significant effect pvalue<0.0001, and there is a significant interaction between light and lines pvalue=0.0091. These data highlights the importance of the AtGNL enzyme for efficient ATP production in the chloroplasts.

[0130] Plants exhibit phenotypic plasticity and respond to differences in environmental conditions by acclimation. In a recent study, Arabidopsis plants grown under field conditions were compared with plants grown indoors. Indoor-grown plants had larger leaves, modified leaf shapes and longer petioles and less NPQt, while field-grown plants had a high capacity to perform state transitions (Mishra et al., 2012). If photosynthesis is inefficient, excess light energy is dissipated as heat to avoid damaging the photosynthetic apparatus. When plants are under abiotic stress, such as low light intensity, the photosynthetic efficiency and the NPQt are opposite. The KO-1 line had high NPQt, indicating inefficient photosynthesis at both low and normal light conditions. FIG. 30 shows the Non-photochemical quenching coefficient of AtGNL lines under low and normal light conditions. Each line was compared to the control (WT), analyzed by t-tests (LDS) at =0.05. Significant differences are indicated by ***. WT: wild type, EV: empty vector, OE: over-expresser, KO: knockout, R: restored. n=10. Statistical analysis indicates the KO-1 line had a highly significant difference compared to the WT control. FIG. 31 shows the Statistical analysis of non-photochemical quenching of AtGNL lines grown at low and normal light conditions. Two-way ANOVA (=0.05). Non-photochemical quenching as response to light conditions. The table indicates the treatment light has a significant effect pvalue=0.001. The lines also has a significant effect pvalue<0.0001, and there is a significant interaction between light and lines pvalue=0.4475. Overall, the over-expressers, restored lines, wild type, and empty vector lines had higher values of photosynthetic efficiency and LEF compared with KO lines under normal and low light conditions, while the NPQt values were opposite with the KO-1 having the highest value. These results show that AtGNL expression is essential to maintain high photosynthetic efficiency, high electron flow to make ATP (high LEF) and less loss of energy in the form of heat (NPQt).

2.3.5. Temporal and Spatial Expression of AtGNL Using the GUS Reporter Gene

[0131] To examine the expression of AtGNL within tissues, ten transgenic plants expressing GUS driven by the AtGNL promoter (pAt1g56500:pCAMBIA1305.1) and empty vector pCAMBIA1305.1 (control) were generated. In the empty vector the GUS-PLUS gene is under the control of the 35S constitutive promoter.

[0132] AtGNL, empty vector and wild type plants were treated with the X-Gluc substrate. As illustrated in FIG. 32 GUS activity was evident in plants expressing the AtGNL promoter in all developmental stages from cotyledons to roots, although much less staining was observed in 4-day-old seedlings compared with the controls. The oldest seedlings stained most intensely, especially at the leaf tips and margins. FIG. 32 shows the Temporal and spatial expression of AtGNL using the GUS-PLUS reporter gene. The AtGNL is expressed in the whole plant and at all developmental stages, indicating that the GNL enzyme is important in the physiological development of the plant from beginning to maturity.

2.3.6. Phylogenetic Three of Putative Plant Gluconolactonases

[0133] A phylogenetic tree for At1g56500 (AtGNL) was generated. At1g56500 (AtGNL) was compared with known GNLs and with putative GNLs for many other organisms. After all protein sequences with significant sequence similarity to AtGNL were retrieved, the protein sequences were aligned using the MEGA6 software (Tamura et al., 2013). Only the sequences that had between 90 and 100% of identity with the AtGNL protein of interest were included in this analysis. To develop an updated phylogenetic tree, a BLASTP search was done against the Arabidopsis protein database (www.arabidopsis.org) using the A. thaliana gluconolactonase At1g56500 (AtGNL) protein sequence. This enzyme has been characterized in A. niger (Ogawa et al., 2002), E. gracillis (Ishikawa et al., 2008), P. aeruginosa (Tarighi et al., 2008), R. norvegicus (Kondo et al., 2006), Z. mobilis (Pedruzzi et al., 2007), and now also in Arabidopsis (this work). The BLASTP result revealed the presence of 37 candidates in different organisms with 90-100% identity to the AtGNL query. FIG. 33 shows the Phylogenetic analysis of known and putative GNLs. Phylogenetic analyses were conducted in MEGA6 (Tamura et al., 2013). Five branches can be distinguished in this phylogenetic tree where the AtGNL groups with proteins from plant species including plant crops of agricultural importance including Cucumis sativus (cucumber), Cucumis melo (melon), Citrus sinensis (orange), Vitis vinifera (grapes), Theobroma cacao (cacao), Glycine max (soybean), and Fragaria vesca (strawberry), trees such as Populus trichocarpa (poplar) and Prunus mume (Chinese plum), and energy crops such as Jatropha curcas. The sequence similarity between At1g56500 and putative GNLs from other plants is remarkable. In a second group we can see known GNLs from mammalian species including R. novergicus, H. sapiens, and putative GNLs from Ovis aries, Canis familiaris and others. In the third and fourth branches we can see known and putative GNLs from bacteria and fungi. In a fifth group we find putative GNLs from A. thaliana. The analyses showed that At1g56500 has high similarity with the protein sequences listed in FIGS. 34A and 34B. FIGS. 34A and 34B show the List of known and putative GNLs included in the phylogenetic analysis. Underlined species indicate the GNL enzymes that have been characterized biochemically.

[0134] The constitutive expression of the gene of interest (GNL) leads to higher seed yield in plants, such as Arabidopsis. Such higher seed yield is shown in FIG. 35.

[0135] FIGS. 36A, 36B, and 36C show the GNL DNA sequence.

[0136] FIG. 37 shows the GNL amino acid sequence. In one embodiment, the GNL over-expressed in the plant may include at least 70% of the sequence shown in FIG. 36 or FIG. 37.

2.4. Conclusions

[0137] The evidence presented in this document allows the following conclusions:

[0138] We successfully developed a purification protocol for AtGNL recombinant protein, and characterized this enzyme in detail including temperature, pH, cofactor requirement, and substrate concentration preferences, as well as its kinetic parameters.

[0139] The AtGNL enzyme had highest activity at temperatures between 25 C. to 35 C., while the bacteria P. aeruginosa has optimal temperature activity at 24 C. The optimum temperature of the AtGNL is consistent with the preferred growth temperature of Arabidopsis.

[0140] The optimal pH for AtGNL enzymatic activity was 6.0, and this activity decreased 4 when the pH was slightly increased (pH 6.3). In contrast, mammalian GNLs isolated from rats, mice, and humans have an optimal activity at pH 6.4. Our result is consistent with the prevalent pH in the chloroplasts (Alberts et al., 2002), the organelle where this protein resides.

[0141] The enzyme characterized in this work is very specific with the D-GuIL substrate. In contrast the recombinant GNLs from E. gracilis and R. norvegicus are promiscuous as they displayed activity with additional substrates (Kondo et al., 2006; Ishiwaka et al., 2008b).

[0142] GNL enzymes require a divalent cofactor for activity. The AtGNL displayed similar activity when incubated with MnCl.sub.2, MgCl.sub.2, or ZnCl.sub.2. In contrast, other GNLs such as the E. gracilis isoform prefer ZnCl.sub.2 and activity dropped significantly with other cofactors (Ishikawa et al., 2008). The ability of AtGNL to work with MnCl.sub.2 is consistent with an enzyme that is active in the chloroplasts, as Mn is abundant in that organelle (Alberts et al., 2002).

[0143] Based on optimum pH, optimum T, and kinetic parameters of the known GNLs the one that is the most similar to the one here characterized is the G. oxydans GNL.

[0144] After demonstrating GNL enzyme activity in vitro, the present invention provides a method of controlling the role of this enzyme in AsA biosynthesis in planta. As shown in FIG. 18 when constitutively expressed in wild type, this enzyme leads to over-expressers with up to 3-fold increase in foliar AsA content. Two T-DNA insertion knockouts in this gene (SALK lines) had reduced AsA content compared to the WT control. When the functional gene was inserted into the knockout background this led to plants with restored AsA content. Overall this data indicates that AtGNL is functional in planta.

[0145] Previous results in the Lorence Laboratory obtained using manual phenotyping showed that plants with high AsA accumulate more biomass, delayed aging and are tolerant to abiotic stresses (Lisko et al., 2013). To determine if the AtGNL lines display differences in growth rate and biomass accumulation, a powerful high throughput phenotyping instrument was used to characterize the phenotype of lines with normal (WT), low (knockouts) and high (over-expresser and restores lines) GNL expression. A clear penalty in the growth rate and biomass accumulation of the knockouts exists. The restored lines grew as well or better than the controls. This result indicates that AtGNL is key to the plant to support normal growth and development.

[0146] Based on the fact that AtGNL is a chloroplastic enzyme (FIG. 7), to gain insights about the physiological role of this protein in supporting the function of this organelle, the ability of plants with low, normal, and high AtGNL expression to adapt to light stress conditions was analyzed. It is well established that there is higher ROS production during low and high light exposure (Sharma et al., 2012). Arabidopsis over-expressers and restored lines where AtGNL expression is enhanced display higher AsA content compared to controls.

[0147] Detailed characterization of the phenotype of the AtGNL lines under normal, low and high light conditions showed that over-expressers and restored lines grew better and accumulated more biomass than their respective controls (FIG. 23). The line with the poorest performance was KO-1, the genotype with the lowest AsA content. Interestingly in planta chlorophyll fluorescence analysis show that KO-1 displayed high chlorophyll fluorescence, an indicator of stress. Further analysis will have to be done to determine why KO-1 has a stronger phenotype compared to KO-2.

[0148] Plant growth and yield depend on plants maintaining high photosynthetic efficiency. To determine if the stunted growth we measured in the knockouts is due to deficiencies in photosynthesis, measurements were done with a hand-held fluorometer. Interestingly, results show that over-expressers and restored lines displayed enhanced photosynthesis compared to controls, while KO-1 and KO-2 have decreased LEF and therefore decreased ability to make ATP. Overall KO-1 display the poorest performance for all photosynthetic parameters here measured including non-photochemical quenching.

[0149] In order to analyze the expression of the AtGNL in plants tissues we developed transgenic A. thaliana lines expressing the GUS-PLUS reporter gene under the control of the AtGNL promoter (pAtGNL). The results show that AtGNL is expressed in all tissues examined: seedlings, leaves, stems, and siliques, except roots. This expression pattern suggests that AtGNL is a constitutive enzyme.

[0150] AtGNL is an important enzyme to sustain sufficient AsA content and to maintain plant growth and efficient photosynthesis. In order to gain insights about the conservation of this enzyme in evolution, a phylogenetic tree of known and putative GNLs was developed (FIG. 33). At least 37 candidates with 90-100% sequence identity to the AtGNL at the amino acid level exist. This analysis indicates the presence of GNLs in a wide array of plants including crops of agricultural importance, mammals, bacteria, and fungi. Interestingly it appears to be a GNL in Sellaginella moellendorffii, an ancient vascular plant that is widely used as a model to study the evolution of plants as a whole (Banks et al., 2011).

[0151] From the foregoing, it will be seen that the present invention is one well adapted to obtain all the ends and objects herein set forth, together with other advantages which are inherent to the structure.

[0152] It will be understood that certain features and sub-combinations are of utility and may be employed without reference to other features and sub-combinations. This is contemplated by and is within the scope of the claims.

[0153] As many possible embodiments may be made of the invention without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.