Download Drought Tolerance Assays

Experimental procedures
The measurement of the root cell length
The roots of wild-type and mutant plants were photographed, and the length of the
root cells in the elongation zone was measured using Image J Microsoft.
Drought Tolerance Assays
Drought tolerance assays were performed for both seedlings and 5-week-old plants.
For seedlings, an3 mutant and wild type seeds were separately grown at high density
in soil in 2×2-inch pots. When seedlings were 15 day old, watering was withheld for
19 d before rewatering. To assay drought tolerance more vigorously at later
developmental stages, an3 mutant plant was grown with wild type in the same pot
under long-day conditions in the greenhouse. When plants were 1 week old, watering
was withheld for 10 days.
Physiological Analysis
Relative SWC indicates the soil water relative to the soil water at day 0 of
withholding water and is the average of three pots. Relative soil water content (SWC)
[Relative SWC was calculated as (final fresh weight –dry weight)/(initial weight –
dry weight) ×100], daily water loss, leaf relative water content (RWC) [Leaf RWC
was determined as (FW －DW)/(TW－DW)×100; fresh weight (FW); dry weight
(DW); leaf turgid weight (TW); leaf dry weight (DW) ], integrated water use
efficiency (WUE) (biomass/water use) and leaf gas exchange of fully expanded leaves
were determined using a portable photosynthesis system (LI-COR LI-6400) in the
morning (9:00 to 11:00AM) under natural sunlight (22 ± 2°C) on the same plants
mentioned above before stomatal observation, as has been described in our previous
reported (Yu et al., 2008). Five measurements were made for each plant, and five
plants were used for the wild type and the mutant.
Integrated WUE was determined as final shoot dry weight divided via total water
loss over a period of 6 weeks. Individual pots were covered with plastic wrap with a
central hole through which four to five seeds for each genotype were sown. One week
after germination, pots were thinned to one seedling per pot. Individual pots were
weighed before and after irrigating with water every 5 to 6 day to assay moisture loss.
Water loss from control pots (without plants) was subtracted from treatment pots.
When the experiment is ended, shoot dry weight was calculated .
Stomatal Density, Index and Aperture Analysis
To assay stomatal density, leaf blades of the same age and from the same relative
position were sampled from rosette stage plants of the wild type and mutant grown on
the same short-day conditions. A leaf blade surface imprint method was used. That is,
a drop of crazy glue was applied to a glass slide, and the abaxial side of a sampled
leaf was pressed on the glue for ~30 s. The abaxial epidermis of the leaf blade was
removed and the imprint on the glass slide was observed under a Hirox
three-dimensional video microscope. For statistical analysis of stomatal density, five
leaves were sampled for each plant and five plants were sampled for the wild type and
the mutant.
The stomatal density (stomatal number per area), stomatal index (stomata number
to total epidermal cells that includes stomata, stomatal precursor cells and pavement
cells), number of stomatal precursor cells and pavement cell density (pavement cells
per area) were determined from a leaf area of ~ 0.007 mm². Stomatal precursor cells
were recognized at the meristemoid or the GMC stage. The stomatal aperture was
measured as previously described by Song et al (2005), in details, stomata were
opened via exposing plants for 10-12 h to light and high humidity, and leaf blades
were incubated for 1.5-2.0 h in stomatal-opening solution supplemented with 60 mM
KCl, 15 mM CaCl2, and 15 mM Mes, pH 6.2. Stomatal apertures were measured
1.5-2.0 h after adding 3mM ABA. Data represent means ± SD (n = ~100 stomata).
Subsequently, the epidermis was placed onto a slide and photographed under a Hirox
three-dimensional video microscope. These experiments were repeated at least three
times with similar results.
Measurement of Anthocyanin
For anthocyanin extraction, the relative sample were placed in 500 mL of 1% HCl in
methanol (v/v) and then incubated overnight in the dark at 4 °C with gentle shaking.
After this process, 300 mL of water and 300 mL of chloroform were added and mixed
to the extract. After centrifugation at 12,000 rpm for 2 min, the absorbance of the
supernatant was measured at 530 and 657 nm, and the concentration of anthocyanin
was determined by using A530- 0.25A657 (Rabino and Mancinelli, 1986).These
experiments were repeated at least two times with similar results.
Quantitative PCR
When the seedlings grew up to 2 weeks, they were collected. Total RNA was
extracted from at least 10 independent seedlings of every lines using the TRIZOL
reagent (Invitrogen), as has been described by Yu et al (2008). cDNA was synthesized
using oligo-(dT)18 primer and ReverTra Ace M-MLV RTase (Toyobo, Osaka, Japan)
according to the manufacture’s recommendation. SYBR green was used to monitor
the kinetics of PCR product in real-time RT-PCR, as has been described by Yu et al
(2008). For analyzing the expression of YDA, MPK6, SDD1 and TMM in Col-0, an3-4
and pGEM-35Spro-AN3/an3-4 seedlings, primers F-5'-GCA GCA AGA TCG GTC
GCG GA-3' and R-5'- ACC GGG TCT CAG GTC GAG GG-3' were used for YDA;
and primers F-5'-ACG GGA CTC ACA CCG CCT CA -3' and R-5'- TCA AGC GTG
CCT GCG CCA AT -3' were used for SDD1; and primers F-5'-GCA CGG AGC CTG
ACG AGC AA -3' and R-5'- GGC GCG GCC TAA ACA TCG GT -3' were used for
TMM; and primers F-5'-ACG CAT TGG CCC ATC CAT ACC T -3' and R-5'-GCA
AGC GCC TCG CGG TAG AT -3' were used for MPK6. For analyzing AN3
expression in Col-0, sdd1, yda and tmm seedlings; primers F-5'-GCC TCA GCC ACC
AAG TGT GCA T-3' and R-5'-ACC GCC ACC ACC ACT TCC CA-3' were used.
For identifying E1728 an3-4 ∆ N-YDA and YDA, SQ-RT-PCR is performed, and
primers has been described (Kang et al., 2009). These experiments were repeated at
least two times with similar results. The specificity of the primers for the target genes
verified by using OLIGE.6 software. UBQ5 (At3g62250) was used as a control.
Real-time PCR was performed using the Rotor Gene RG 3000 thermocycler (Corbett
Research, San Francisco, CA, USA), and the data were collected and analysed using
the Rotor Gene 6.0 software (Corbett Research). The gene expression data were
calculated by using standard curve methods.
Plasmid Constructs
For AN3 and YDA promoter analysis, promoter–GUS constructs of At5g28640 and
At1g63700 were created by inserting about 2.0 Kb promoter fragments, respectively,
into pCB308R, as previously described by Lei et al (2007). Used primers are P1-ggg
gac aag ttt gta caa aaa agc agg ct TTT GTA AGC GTT TCA GAA TCC T and P2ggg gac cac tttg tac aag aaa gct ggg t TAA CTA TTG AAG ATG TGT ATC TC for
AN3, Used primers are P1-ggg gac aag ttt gta caa aaa agc agg ct GCA TCT AGA
ATT ACT AAA TCA, P2- ggg gac cac ttt gta caa gaa agc tgg gt CTC TCC TTT GAC
CAC TTC AGA G for YDA. These amplified fragments were inserted into pCB308R,
as previously described by Lei et al (2007).
For obtaining pCB2004-35Spro-AN3 plasmid, used primers were F-ggg gac aag
ttt gta caa aaa agc agg ct ATG CAA CAG CAC CTG ATG CAG AT and R--ggg gac
cac tttg tac aag aaa gct ggg t TCA ATT CCC ATC ATC TGA TGA TTT C. For
obtaining pMD111-YDApro-GFP plasmid (containing two TCTC motif), used
primers were F-ggg gac aag ttt gta caa aaa agc agg ct aat cga aat aat taa tta ag and
R-ggg gac cac tttg tac aag aaa gct ggg t gag atc ata ctt ggt tga cc. For obtaining
pMD111-YDApro-GFP plasmid (without two TCTC motif), used primers were F-ggg
gac aag ttt gta caa aaa agc agg ct gtt tgt gtg tga tta ttc cat tac and R-ggg gac cac tttg
tac aag aaa gct ggg t aat act tcc aat tca tta ggt gtt.
Confocal Laser Scanning Microscope for GFP Imaging
Observation of fluorescent light in the abaxial epidermis of 1-week-old transgenic
plants harboring pMD111-YDApro-GFP was performed. An Olympus IX-70
microscope (http://www.olympus-global.com/) was used to detect GFP expression
using λ = 488 nm and λem = 510 nm. The sections were photographed under a
confocal laser scanning microscope.
GUS Staining
Using a mix buffer (1 mM X-gluc, 60 mM NaPO4 buffer, 0.4 mM of
K3Fe(CN)6/K4Fe(CN)6 and 0.1% (v/v) Triton X-100), samples (Transgenic plants
harboring and expressing pCB308R-AN3pro-GUS or pCB308R-YDApro-GUS) were
stained, and then incubated at 37°C for 8 h. After GUS staining, chlorophyll was
removed using 30, 50, 70, 90 and 100% ethanol about 30 min for every process. GUS
staining was performed as previously described by Lei et al (2007).
ChIP Assay
The transgenic lines overexpressing pGEM-35Spro-AN3-3XGFP in an3-4 mutants
were used in this assay. ChIP was performed on 2-week-old seedlings growing on MS
medium as described previously (Yoo et al., 2010). GFP and HA tag-specific
monoclonal antibody were used for ChIP analysis. The ChIP DNA products were
analyzed via PCR using three pairs of primers that were synthesized to amplify
~300-bp DNA fragments in the promoter region of YDA in ChIP analysis. The primer
sequences were P1-tgt gtc act aac tca ctt cac; P2-gaa aac cct aag tag aac aac; P3-gct
ttc gat ttg att cca ttt caa; P4- tca atg tga tct tca acc ta; P5- tac aaa gat taa cgc acc aaa
gg; and P6-tca aaa gca atc gaa gaa tcc aa. These experiments were repeated at least
three times with similar results
References
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