5. Materials and Methods
5.4 Ascorbate Measurement
Equipment: spectrophotometer, centrifuge (4°C), oven (37°C), mortar and pestle.
Material: liquid nitrogen, 1.5mL and 2mL microcentrifuge tubes, 96well microplate.
Reagents:
Fresh 1M ascorbate (AsA) and dithiothreitol (DTT).
Procedures:
1. Homogenize tissue (0.5g) by pre-chilled mortar and pestle with liquid nitrogen.
2. Mix 1mL 6% trichloroacetate (TCA) well and transfer the mixture to a 2mL
microcentrifuge tubes.
66
3. Vortex well for 10 second and keep the mixtures on the ice.
4. Delaminate by centrifugating with 13000rpm for 5min at 4°C and transfer the
supernatants to the new 1.5mL microcentrifuge tubes.
5. Transfer 200 μL supernatant to new 2 mL microcentrifuge tubes labeled total AsA.
6. Transfer 200 μL supernatant to new 2 mL microcentrifuge tubes labeled reduced
AsA.
7. Prepare 200 μL 6% TCA as blank and 200 μL AsA standards (0.15-1 mM) in the
mL microcentrifuge tubes.
8. Add 100 μL 75 mM sodium phosphate buffer (pH7.0) to all above microcentrifuge
tubes.
9. Add 100 μL 10 mM DTT to the total AsA microcentrifuge tubes and incubate at
room temperature for 10 min. DTT would reduce the oxidized AsA.
10. Add 100 μL 0.5% N-ethylmaleimide (NEM) to the total AsA tubes for removing the
excess DTT and incubate for 30 second.
11. Add 200 μL ddH2O to the reduced AsA microcentrifuge tubes.
12. Add 500 μL 10%TCA, 400 μL 43% phosphoric acid (H3PO4), 400 μL 4%
α-α‟-bipyridyl and 200 μL 3% ferric chloride (FeCl3) to all microcentrifuge tubes.
67
13. Incubate the microcentrifuge tubes at 37°C for 1hr.
14. Load 200 μL samples, blank and standards in a 96-well microplate and measure the
absorbance at 525nm.
15. Calculate a linear regression curve from the A525 of the AsA standards.
16. Concentration of total AsA and reduced AsA of samples can be estimated through
the linear regression equation.
17. Oxidized AsA can be estimated by subtracting the reduced portion from the total
ascorbate pool.
Principles:
This protocol describes a microplate-adapted colorimetric ascorbate assay, in which
ferric ion (Fe3+) is reduced by ascorbate to the ferrous ion (Fe2+). The ferrous ion reacts with α-α‟-bipyridyl to form a complex with characteristic absorbance at 525 nm.
With the DTT reduction of any dehydroascorbate (DHA) in a sample, total ascorbate can be assayed using the α-α‟-bipyridyl method, and DHA can be estimated by
subtracting the reduced portion from the total ascorbate pool. (Ainsworth and Gillespie,
2007)
68
Notes:
a. Reagents would work at room temperature for avoid the precipitation of α-α‟-bipyridyl and storage them at 4°C.
b. All sample and standards were prone to keep on ice and avoid the light.
c. 10% TCA, 400 μL 43% phosphoric acid (H3PO4), 400 μL 4% α-α‟-bipyridyl and 200 μL 3% ferric chloride (FeCl3) could be mixed in order before use.
d. α-α‟-bipyridyl is prepared in or higher 4% phosphoric acid.
e. Mix the 3% ferric acid after solving 4% α-α‟-bipyridyl completely to avoid precipitation of 4% α-α‟-bipyridyl.
5.5
Hydrogen Peroxide Measurement
Preparation:
Equipments: spectrophotometer, centrifuge (4°C), mortar and pestle.
Materials: 2 mL microcentrifuge tubes 96well microplate, liquid nitrogen.
Reagents:
1. Buffer 1:10mM 3-amino-1.2.4-triazole in 50mM sodium phosphate buffer (pH 6.5).
2. Buffer 2: Titanium (IV) oxysulfate-sulfuric acid solution.
69
3. 30% (9.8M) H2O2.
Procedures:
1. Prepare the hydrogen peroxide standard (9.8 mM~9.8 nM) to confirm the efficiency
of the buffers before assay.
2. Homogenize tissue (0.5 g) with liquid nitrogen in pre-chilled mortar and pestle.
3. Add 1.8 mL Buffer 1 to extract and transfer the mixture to the 2 mL microcentrifuge
tubes.
4. The samples were centrifugated with 13000 rpm for 5 min at 4°C and transfer the
supernatants to the new 1.5 mL microcentrifuge tubes.
5. Repeat the centrifugation.
6. Preparation 200 μL Buffer 2 to the new microcentrifuge tubes.
7. Add 600 μL supernatant and H2O2 (9.8 mM~9.8 nM) to above microcentrifuge
tubes and mix well.
8. All mixtures were centrifugated with 13000 rpm for 5 min at 4°C.
9. Take 500 μL supernatant (yellow or light pink) to the new microcentrifuge tubes
and discard the pellet (white).
10. The supernatants were centrifugated with 13000 rpm for 5 min at 4°C to remove
70
excess Titanium chelate.
11. Load 200 μL samples, blank and standards in 96-well microplate and measure the
absorbance at 410 nm.
12. Calculate a linear regression curve from the A410 of the H2O2 standards.
13. H2O2 amount in each sample can be estimated through the linear regression
equation.
Principles:
Ti (IV) in sulfuric acid is present as [Ti(OH)2]2+ and [Ti(OH)3] + colorless ions.
However, it would be converted into [Ti(O)2OH] + yellow-orange ion. This chelate is
soluble in sulfuric acid and could be separated with excess Ti ion by a simple
centrifugation. The limit of detection of hydrogen peroxide is 10 nM. (Jana and
Choudhuri, 1982)
Notes:
a. The color is dark red when add excess H2O2 as standard, and the optimal standard
concentration ranges from 9.8 mM~9.8 nM and display the colour in yellow.
b. (samples or standard mixing with Buffer1) / (Buffer 2) = 3:1 (v/v).
71
5.6
Hydrogen Peroxide Staining
Preparation:
Equipments: microscope, vacuum dryer, water bath.
Materials: knifes (razor), tweezers, glass slides, 50 mL microcentrifuge tubes.
Reagents:
1. DAB buffer (0.5 mg mL-1): 50 mg 3,3‟-diaminobenzidine in 100 mL 50 mM
sodium phosphate buffer (pH 7.0).
2. Wash buffer: 95 mL 70% ethanol plus 5 mL NH4OH ACS reagent.
3. Ethanol.
Procedures:
1. Samples were sliced to form 2 mm section.
2. Sections were incubated in DAB buffer.
3. Increase the efficiency of the fixation by evacuation for 2 min twice.
4. Keep the sections in the dark at room temperature for 24 h.
5. Boil the sections with ethanol (96%, v/v) until remove the chlorophyll completely.
6. Keep the sections in the 100% ethanol.
7. Photograph the sections.
72
Principles:
The staining was based on the instant polymerization of DAB (to form a reddish-brown
complex which is stable in most solvents), as soon as it comes into contact with H2O2 in
the presence of peroxidase. (ThordalChristensen et al., 1997)
Notes:
Keep the DAB buffer in the dark and storage at 4°C
5.7
Quantitative Analysis of Protein Concentration
Bio-Rad Protein Assay Kit.Preparation:
Equipment: spectrophotometer.
Material: 96 well microplate.
Reagents:
1. Bio-Rad Protein Assay kit.
2. BSA(1 μg μL-1).
Procedures:
1. Mix dye and ddH2O by 4:1 (v/v).
73
2. Add 1,2,4,8,16 μL bovine serum albumin (BSA) and 1 μL~5 μL crude proteins with above mixture to 200μL and incubate at room temperature for at least 5 min.
3. Measure the absorbance at 595 nm and calculate a linear regression curve from the
A595 of the BSA standards.
4. Protein concentration of crude protein can be estimated through the linear regression
equation.
Principles:
This assay is based on the method of Bradford. The absorbance maximum for an acidic
solution of Coomassie Brilliant Blue G-250 dye shifts from 465 nm to 595 nm when
binding to protein occurs. The Coomassie blue dye binds to primarily basic and
aromatic amino acid residues, especially arginine.
Notes:
Absorbance will increase over time; samples should incubate at room temperature for
no more than 1 hr.
5.8 RNA Extraction
Preparation:
74
Equipments: mortar and pistil, centrifuge, water bath (65°C), refrigerator (4,-80°C), 30
mL centrifuge tubes.
Materials: liquid nitrogen, 5 mL tip.
Reagents:
1. Extraction buffer (500 mL): 10 g polyvinylpyrrolidone (PVP, average mol wt 40000)
+ 10 g hexadecyltrimethylammonium bromide (CTAB) + 4.653 g EDTA + 58.44 g
NaCl + 0.25 g spermidine.
2. 10X MOPS (200 mL): 8.372 g MOPS + 0.8203 g sodium acetate + 0.7448 g EDTA.
3. Denature buffer: 600 μL formamide + 210 μL formaldehyde + 10X MOPS buffer + 7 μL ethidium bromide (EtBr; 10 mg mL-1).
4. RNase free ddH2O: 1 mL diethypyrocarbonate (DEPC) in 1 L water and sterilize in
autoclave with 2 atm for 40 min.
Procedures:
1. Grind 1 g sample in the mortar treated with liquid nitrogen.
2. Add 10 mL extraction buffer with fresh 200 μL β-mercaptoethanol into the mortar.
3. After thawing the mixture, transfer the slurry to the centrifugation tube.
4. Incubate the mixture at 65°C in water bath for 10 min.
75
5. Delaminate by centrifugating with 13000rpm at 25°C for 15min.
6. Transferring the supernatant into a new centrifugation tube.
7. Add equal volume chloroform/isoamyl alcohol (24:1) and gently mix it for 1 min.
8. Repeat step 5 to step 7.
9. Delaminate by centrifugating with 13000 rpm at 25°C for 15 min.
10. Transferring the supernatant into a new centrifugation tube.
11. Add one fourth fold volume 10 M lithium chloride (LiCl).
12. Incubate the mixture at 4°C overnight.
13. Collect the RNA by centrifugating with 13000 rpm at 4°C for 15 min.
14. Wash the pellet by 75% ethanol two times.
15. Remove the excess alcohol in the pellet by vacuum dryer.
16. Resuspend the pellet with optimal volume RNase free ddH2O.
17. Check the quality of RNA by electrophoresis.
18. The remaining RNA samples were immediately steeped in liquid nitrogen and keep
in -80°C for long time storage.
19. RNA electrophoresis (e.g. 25mL volume):
(1) 25 g agar + 25 mL RNase free ddH2O.
76
(2) Melt the agar by microwave oven.
(3) Until the mixture cool down to 40~50°C and add 2.5 mL 10X 3-(N-morpholino)
propanesulfonic acid, 4-morpholinepropanesulfonic acid (MOPS) and 0.75 mL 37%
formaldehyde.
(4) Solidify the agar in the suitable tank.
(5) Electrophoresis in 50 mV 1 hr.
(6) Photograph the gel under UV exposure.
Note:
a. There are three methods for RNA precipitation:
(1) LiCl: For precipitation of RNA which molecular weight is longer than 200 bp.
The optimal precipitation condition is at 4°C.
Supernatant: 10M LiCl = 4:1 (v/v)
(2) 100% ethanol: The optimal precipitation condition is at -20°C.
Supernatant: 100% EtOH: 10M NH4OAc =1:2.5:0.25 (v/v/v)
(3) 100% Isopropanol: The optimal precipitation condition is at -20°C.
Supernatant: Isopropanol: 10M NH4OAc =1: 0.25 (v/v)
b. All material including motars, pistils, reagents, etc should be remove the RNase by
77
rinsing with 1‰ DEPC and then sterilize in autoclave with 2 atm for 40 min.
c. The chemicals for extraction buffer should be mixed in order and sterilize in
autoclave with 2 atm for 40 min after stirring with 0.5mL DEPC overnight.
5.9 RT PCR for Gene Expression (One-step RT PCR)
(1) Add the following reagents to a 0.2 mL microcentrifuge tube in order
RNase Free ddH2O 24 μL
10 X One Step RNA PCR Buffer 5 μL
25 mM MgCl2 10 μL
10 mM dNTP 5 μL
10 μM Gene specific forward primer 1 μL
10 μM Gene specific reverse primer 1 μL
RNA (1 μg μL-1) 1 μL
RNase Inhibitor (40 units mL-1) 1 μL
AMV RTase XL (5 units mL-1) 1 μL
AMV-Optimized Taq (5 units mL-1) 1 μL
(2) Setup a PCR reaction following the below condition:
78
50°C 45 min
94°C 2 min
94°C 30 sec
50~60°C 30 sec 12~25 cycles
72°C 30 sec
72°C 10 min
16°C ∞
(2) The expression level of gene in different samples was quantificated by the intensity
of PCR product which displayed in the electrophoresis.
5.10 Construction of Functional Plasmid for Overexpressing the Interested Genes in Arabidopsis
Preparation:
Equipments: Electrophoresis set, centrifuge (4/-20°C), heating plate (65/50/42°C),
refrigerator, oven (80/37°C), PCR machine, electroporation machine and cuvette.
Materials: Ligation Kit, Gel elution Kit, pCAMBIA 2300 vector, 15mL centrifuge tube,
1.5mL microcentrifuge tubes, restriction enzymes,
79
Reagents:
1. LB medium (1L):Mix 15g (Luria-Bertani) medium and 25g agar (for bacteria
culture) and sterilize at 2 atm for 20 min.
2. LB broth (1L):Mix 15g (Luria-Bertani) medium and sterilize at 20 atm for 20 min.
3. Solution 1: 50mM glucose, 10mM EDTA in 25mM Tris-HCl (pH8.0).
4. Solution 2 (Fresh): 880μL ddH2O, 100μL 10%SDS and 20μL10N sodium hydroxide.
Keep at room temperature.
5. Solution 3: 60mL 5M potassium acetate +11.5mL glacial acetic acid and 28.5mL
H2O.
6. RNase: 0.1g RNase A solves in 10μL H2O, and then boils it for 10min. Filter
sterilize the solution by passing it through 0.22 micron filter and storage at -20°C.
7. PCI: phenol/chloroform/isoamylalcohol (25:24:1)
8. CI: chloroform/isoamylalcohol (24:1).
9. X-gal:1gof X-gal in 10 mL of dimethyl formamide (DMF).
10. IPTG: Isopropyl Thiol-D-Galactoside, 0.2g IPTG solves in 10mL H2O and filter
sterilize the solution by passing it through 0.22 micron filter and store them in
-20°C.
80
Procedures:
1. Elute the DNA preduct from the electrophoresis by following the elution kit
protocol.
2. Check and quantify the elution product by electrophoresis.
3. Ligate the DNA fragment with T-A cloning vector (e.g. pGEM-T easy)
DNA fragment 3μL
2X ligation buffer 5μL
pGEM-T easy vector 1μL
T4 DNA ligase 1μL
4. Incubate the mixture at 4°C overnight.
5. Transformation by heat shock method following below steps:
(1) Mix above mixture with 100μL E.coli (DH5α) competence cell and keep on ice
for 30 min.
(2) Heat the above mixture at 42°C for 90 sec.
(3) Keep on ice for 2 min.
(4) Add 100μL LB broth to above mixture and incubate at 37°C for 30 min.
(5) Spread the mixture with 40μL 10% X-gel and 7μL 20% IPTG for Blue White
81
screening on the100ppm ampicillin LB medium plate.
(6) Incubate the plate at 37°C overnight.
6. Subculture several single colony in a new plate and operate a colony-PCR to select
the colonies transformed successfully.
7. Sequence the plasmid to confirm and analyze the interested gene by bioinformatics
approach.
8. Generation of single colony from the accurate colony by streaking plate method.
9. Extraction the plasmids from the colony by following the below steps:
(1) Culture the single colony in 5mL LB broth with 100ppm ampicillin at 37oC
overnight.
(2) Collect the bacteria by centrifugation with 3000 rpm at room temperature for 10
min.
(3) Discard the supernatant and resuspend the pellet with 100μL solution I.
(4) Incubate at room temperature for 5 min.
(5) Add the solution II and votex gently until the mixture becomes clear.
(6) Keep on ice for 5 min.
(7) Add 150μL solution III and vortex gently until appearance of while slurry.
82
(8) Delaminate the above mixture by centrifugating with 13000 rpm at 4oC for 15
min.
(9) Transfer the 400 μL supernatant to a new 1.5 mL microcentifuge tube and add 4 μL RNase (10mg mL-1).
(10) Incubate the mixture at 50 oC for 1 hr.
(11) Add 400 μL PCI and vortex vigorously.
(12) Delaminate the above mixture by centrifugating with 13000 rpm at 4 oC for
15 min.
(13) Transfer 300 μL supernatant to a new microcentrifue tube.
(14) Add 300 μL CI and vortex vigorously.
(15) Delaminate the above mixture by centrifugating with 13000 rpm at 4oC for 5
min.
(16) Transfer 200 μL supernatant to a new centrifugating tube.
(17) Add 50 μL 10M ammonium acetate (NH4OAc; pH 6.5) and 500 μL 100%
ethanol.
(18) Keep at -20 oC for 2 hr.
(19) Collect the plasmid by centrifugating with 13000 rpm at 4oC for 20 min.
83
(20) Remove the excess salt form the plasmid by washing with 70% ethanol twice.
(21) Remove the ethanol from the plasmid by vacuating.
(22) Resuspend the pellet with ddH2O.
10. Digestion of plasmid with optimal restriction enzymes following the below steps:
Plasmid (50μg) 85 μL
Restriction enzymes (less than 5% of total volume) 4 μL
10X Digestion buffer 10 μL
100X BSA 0-1 μL
11. Incubate the above mixture at 37 oC overnight.
12. Check the digestion product by electrophoresis.
13. Elute the DNA product from the electrophoresis by following the elution kit
protocol.
14. Check and quantify the elution product by electrophoresis.
15. Ligate the DNA fragment with pCAPMBIA 2300 vector
DNA fragment 6 μL
2X ligation buffer 9 μL
Vector 1 μL
84
T4 DNA ligase 1 μL
16. Incubate the mixture at 4oC overnight.
17. Transformation, colony PCR and sequencing to confirm the accuracy of the
functional plasmid.
18. Transformation of Agrobacterium tumefaciens by electroporation method.
(1) Keep the electroporation cuvette in the oven (80oC) for 15 min to remove the
excess ethanol.
(2) Capping and keep on ice for 5 min.
(3) Keep the competence cell on ice for 5 min.
(4) Add the 2 μL pure plasmid to the competent cell and incubate on ice for 5 min.
(5) Transfer the above mixture to the electroporation cuvette.
(6) Charge and trigger the pulse.
(7) Add 900 μL LB broth and incubate at 28 oC for 1 hr.
(8) Spread 200 μL mixture on the 100 ppm kanamycin plate and incubate at 28oC
for two day.
19. Subculture several signal colony in a new plate and operate a colony-PCR to select
the colonies transformed successfully.
85
Notes:
a. The pH of the phenol as shipped is 6.6 ± 0.2. After addition of the Tris alkaline
buffer, the pH is confirmed to be 7.9 ± 0.2, and it is suitable for deproteinization of
nucleic acids.
b. The rotational speed when collection of the bacteria by centrifugating should not
higher than 5000 rpm to prevent the damage of bacteria.
c. X-gal is light sensitive and hence store in a glass or polypropylene containers
wrapped with aluminum foil. Stock solution need to be store in -20 oC.
d. The plasmid for electroporation is extracted from Viogene Mini Plus TM Plasmid
DNA Extraction System.
5.11 Agrobacterium Infiltration (Host: Arabidopsis thaliana Col.) Preparation:
Equipment: Oven (28oC), centrifuge.
Materials: Arabidopsis with inflorescence, 50/250 mL centrifuge tubes, triangle flask,
Reagents:
1. Infiltration broth (1L; pH5.7): 2.2g MS with vitamin, 50g sucrose, 0.5g MES, 0.1mL
86
Silweet L-77.
2. 100 ppm kanamycin
Procedure:
1. Subculture the colony containing the plasmid in the kanamycin-selected plate and
incubate it at 28oC for 2 days.
2. Select a single colony from the plate and culture it with 40 mL LB broth in 50 mL
centrifuge tube (Agro in anaerobic) overnight.
3. Transfer the bacteria solution to 500 mL LB and culture it at 28 oC overnight.
4. While the absorbance at 600 nm is higher than 0.8, collect the bacteria by
centrifugating with 5000 rpm at 4oC for 10 min.
5. Resuspend the pellet with 250 mL infiltration broth.
6. Dip the optimal plants in the 250 mL infiltration broth for 45 sec.
7. Repeat step 6.
8. Seal the plant with the plastic wrap at the dark overnight.
9. Remove the plastic wrap.
10. Until the whole plant product the fully mature siliques, collect and sieve the seed in
the 1.5 mL microcentrifuge tubes.
87
11. Remove the water on the surface of the seeds by keep in the humidity-controlled
oven.
Notes:
a. Remove the growing siliques from the Arabidopsis inflorescence before the dipping
could decrease the population of wild-type seeds in the later collection of seeds.
b. 250 mL infiltration broth is enough for dipping 30 plants.
c. Infiltration broth is not necessary to sterilize.
d. Adjust the pH valve of infiltration buffer by 1N potassium hydroxide.
88
References
Agius, F., Gonzalez-Lamothe, R., Caballero, J.L., Munoz-Blanco, J., Botella, M.A., and Valpuesta, V. (2003). Engineering increased vitamin C levels in plants by overexpression of a D-galacturonic acid reductase. Nat Biotechnol 21: 177-181.
Ainsworth, E.A., and Gillespie, K.M. (2007). Measurement of reduced, oxidized and total ascorbate content in plants. Nature Protocols 2: 871-874.
Attolico, A.D., and De Tullio, M.C. (2006). Increased ascorbate content delays flowering in long-day grown Arabidopsis thaliana (L.) Heynh. Plant Physiol Biochem 44: 462-466.
Ausin, I., Alonso-Blanco, C., Jarillo, J.A., Ruiz-Garcia, L., and Martinez-Zapater, J.M. (2004). Regulation of flowering time by FVE, a retinoblastoma-associated protein. Nat Genet 36: 162-166.
Badiani, M., Paolacci, A.R., Miglietta, F., Kimball, B.A., Pinter, P.J., Garcia, R.L., Hunsaker, D.J., LaMorte, R.L., and Wall, G.W. (1996). Seasonal variations of antioxidants in wheat (Triticum aestivum) leaves grown under field conditions.
Aust J Plant Physiol 23: 687-698.
Balasubramanian, S., Sureshkumar, S., Lempe, J., and Weigel, D. (2006). Potent induction of Arabidopsis thaliana flowering by elevated growth temperature.
PLoS Genet 2: e106.
Barth, C., Moeder, W., Klessig, D.F., and Conklin, P.L. (2004). The timing of senescence and response to pathogens is altered in the ascorbate-deficient Arabidopsis mutant vitamin c-1. Plant Physiol 134: 1784-1792.
Blanchard, M.G., and Runkle, E.S. (2006). Temperature during the day, but not during the night, controls flowering of Phalaenopsis orchids. J Exp Bot 57:
4043-4049.
Barth, C., De Tullio, M., and Conklin, P.L. (2006). The role of ascorbic acid in the control of flowering time and the onset of senescence. J Exp Bot 57: 1657-1665.
Blazquez, M.A., Green, R., Nilsson, O., Sussman, M.R., and Weigel, D. (1998).
Gibberellins promote flowering of Arabidopsis by activating the LEAFY promoter. Plant Cell 10: 791-800.
Bond, D.M., Dennis, E.S., Pogson, B.J., and Finnegan, E.J. (2009). Histone acetylation, VERNALIZATION INSENSITIVE 3, FLOWERING LOCUS C, and
89
the vernalization response. Mol Plant 2: 724-737.
Cheong, J.J., Jung, C., Seo, J.S., Han, S.W., Koo, Y.J., Kim, C.H., Song, S.I., Nahm, B.H., and Do Choi, Y. (2008). Overexpression of AtMYB44 enhances stomatal closure to confer abiotic stress tolerance in transgenic Arabidopsis. Plant Physiology 146: 623-635.
Chen, Z., and Gallie, D.R. (2004). The ascorbic acid redox state controls guard cell signaling and stomatal movement. Plant Cell 16: 1143-1162.
Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:
735-743.
Conklin, P.L., and Barth, C. (2004). Ascorbic acid, a familiar small molecule intertwined in the response of plants to ozone, pathogens, and the onset of senescence. Plant Cell and Environment 27: 959-970.
Conklin, P.L., Pallanca, J.E., Last, R.L., and Smirnoff, N. (1997). L-ascorbic acid metabolism in the ascorbate-deficient arabidopsis mutant vtc1. Plant Physiol 115: 1277-1285.
Conklin, P.L., Norris, S.R., Wheeler, G.L., Williams, E.H., Smirnoff, N., and Last, R.L. (1999). Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis. P Natl Acad Sci USA 96: 4198-4203.
Dat, J., Vandenabeele, S., Vranova, E., Van Montagu, M., Inze, D., and Van Breusegem, F. (2000). Dual action of the active oxygen species during plant stress responses. Cell Mol Life Sci 57: 779-795.
Davletova, S., Rizhsky, L., Liang, H.J., Zhong, S.Q., Oliver, D.J., Coutu, J., Shulaev, V., Schlauch, K., and Mittler, R. (2005). Cytosolic ascorbate peroxidase 1 is a central component of the reactive oxygen gene network of Arabidopsis. Plant Cell 17: 268-281.
de Pinto, M.C., Paradiso, A., Leonetti, P., and De Gara, L. (2006). Hydrogen peroxide, nitric oxide and cytosolic ascorbate peroxidase at the crossroad between defence and cell death. Plant J 48: 784-795.
De Tullio, M.C., and Arrigoni, O. (2004). Hopes, disillusions and more hopes from vitamin C. Cell Mol Life Sci 61: 209-219.
Dubos, C., Stracke, R., Grotewold, E., Weisshaar, B., Martin, C., and Lepiniec, L.
(2010). MYB transcription factors in Arabidopsis. Trends in Plant Science 15:
573-581.
Gao, Q., and Zhang, L. (2008). Ultraviolet-B-induced oxidative stress and antioxidant
90
defense system responses in ascorbate-deficient vtc1 mutants of Arabidopsis thaliana. J Plant Physiol 165: 138-148.
Gocal, G.F.W., Sheldon, C.C., Gubler, F., Moritz, T., Bagnall, D.J., MacMillan, C.P., Li, S.F., Parish, R.W., Dennis, E.S., Weigel, D., and King, R.W. (2001).
GAMYB-like genes, flowering, and gibberellin signaling in Arabidopsis. Plant Physiology 127: 1682-1693.
Hartmann, U., Hohmann, S., Nettesheim, K., Wisman, E., Saedler, H., and Huijser, P. (2000). Molecular cloning of SVP: a negative regulator of the floral transition in Arabidopsis. Plant J 21: 351-360.
Hirai, N., Kojima, Y., Shinozaki, M., Koshimizu, K., Murofushi, N., and Takimoto, A. (1995). Accumulation of ascorbic-acid in the cotyledons of morning glory (pharbitis-nil) seedlings during the induction of flowering by low-temperature treatment and the effect of prior exposure to high-intensity light. Plant and Cell Physiology 36: 1265-1271.
Jana, S., and Choudhuri, M.A. (1982). Glycolate metabolism of 3 submersed aquatic angiosperms during aging. Aquat Bot 12: 345-354.
Johanson, U., West, J., Lister, C., Michaels, S., Amasino, R., and Dean, C. (2000).
Molecular analysis of FRIGIDA, a major determinant of natural variation in Arabidopsis flowering time. Science 290: 344-347.
Kardailsky, I., Shukla, V.K., Ahn, J.H., Dagenais, N., Christensen, S.K., Nguyen, J.T., Chory, J., Harrison, M.J., and Weigel, D. (1999). Activation tagging of the floral inducer FT. Science 286: 1962-1965.
Kobayashi, Y., Kaya, H., Goto, K., Iwabuchi, M., and Araki, T. (1999). A pair of related genes with antagonistic roles in mediating flowering signals. Science 286:
1960-1962.
Koornneef, M., Alonso-Blanco, C., Blankestijn-de Vries, H., Hanhart, C.J., and Peeters, A.J.M. (1998). Genetic interactions among late-flowering mutants of Arabidopsis. Genetics 148: 885-892.
Kotchoni, S.O., Larrimore, K.E., Mukherjee, M., Kempinski, C.F., and Barth, C.
(2009). Alterations in the endogenous ascorbic acid content affect flowering time in Arabidopsis. Plant Physiol 149: 803-815.
Koussevitzky, S., Suzuki, N., Huntington, S., Armijo, L., Sha, W., Cortes, D., Shulaev, V., and Mittler, R. (2008). Ascorbate peroxidase 1 plays a key role in the response of Arabidopsis thaliana to stress combination. J Biol Chem 283:
34197-34203.
91
Kroj, T., Journot-Catalino, N., Somssich, I.E., and Roby, D. (2006). The transcription factors WRKY11 and WRKY17 act as negative regulators of basal resistance in Arabidopsis thaliana. Plant Cell 18: 3289-3302.
Larkindale, J., Hall, J.D., Knight, M.R., and Vierling, E. (2005). Heat stress phenotypes of Arabidopsis mutants implicate multiple signaling pathways in the acquisition of thermotolerance. Plant Physiol 138: 882-897.
Lee, J.H., Yoo, S.J., Park, S.H., Hwang, I., Lee, J.S., and Ahn, J.H. (2007). Role of SVP in the control of flowering time by ambient temperature in Arabidopsis.
Genes Dev 21: 397-402.
Genes Dev 21: 397-402.