Natural Variants of At HKT1 Enhance Na + Accumulation in Two Wild Populations of Arabidopsis methods
Aim. Evidence-backed execution summary for Natural Variants of At HKT1 Enhance Na + Accumulation in Two Wild Populations of Arabidopsis methods from Natural Variants of At HKT1 Enhance Na + Accumulation in Two Wild Populations of Arabidopsis.
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Biological model pending
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
General plant growth conditions.
reagent used in the protocol.
- Use
- Plants used for elemental profiling by ICP-MS analysis were grown under unstressed conditions in a controlled environment, 8 h light:16 h dark (90 µmol • m -2 • s -1 light intensity) and 19 to 22 °C [ ]. Briefly, seeds were sown onto moist soil (Sunshine Mix LB2; Carl Brehob & Son,...
Screening of the F2 population from Tsu-1 × Col-0 for survival of NaCl stress.
reagent used in the protocol.
- Use
- The evaluation of the F2 population from the Tsu-1 × Col-0 cross in response to salt stress was realized in a growth chamber under controlled environmental conditions, 8 h light:16 h dark (150 µmol • m -2 • s -1 light intensity), 21 °C:19 °C (day/night) and 60% relative hu...
Tissue Na + quantification.
reagent used in the protocol.
- Use
- Approximately 3 mg of dry weight of each plant was sampled into Pyrex tubes (16 × 100 mm) after drying at 92 °C for 20 h. After cooling, seven of approximately 100 samples from each sample set were weighed. All samples were digested with 0.7 ml of concentrated nitric acid (OmniTrace; VWR Scientific Product...
Sequencing of the At HKT1 allele from Ts-1 and Tsu-1.
reagent used in the protocol.
- Use
- provides a summary of the pairs of primers used and the size of the PCR products expected for Col-0 (based on published sequence) and obtained for Ts-1 and Tsu-1. The PCR products obtained for each one of the primer pairs were cloned using the pGEM-T Easy Vector System I according to the manufacturer's instructions...
Quantitative real-time PCR.
reagent used in the protocol.
- Use
- Plants were first analyzed by ICP-MS and further used to determine the At HKT1 transcript levels. Shoot and root from plants grown under short-day conditions (as described above in "General plant growth conditions") were separated and rinsed thoroughly with deionized water to remove any soil contaminatio...
Genetic Analysis and Mapping of the High Na + Trait
(B) Distribution of Na + accumulation in shoot tissue of F2 segregating population obtained from crossing Col-0 with Tsu-1. Presented data are distribution of Col-0 ( n = 80), Tsu-1 ( n = 16), and F2 (Tsu-1 × Col-0) ( n = 143). Na + contents were calculated for each plant as a percentage relative to Col-0 avera...
- Use
- (B) Distribution of Na + accumulation in shoot tissue of F2 segregating population obtained from crossing Col-0 with Tsu-1. Presented data are distribution of Col-0 ( n = 80), Tsu-1 ( n = 16), and F2 (Tsu-1 × Col-0) ( n = 143). Na + contents were calculated for each plant as a percentage relative to Col-0 avera...
Genetic Analysis and Mapping of the High Na + Trait
To determine the feasibility of using natural variation to identify individual loci regulating ionomic variation, we attempted to map the locus responsible for elevated Na + in Ts-1 and Tsu-1. Both accessions were crossed to Col-0, and the shoot concentrations of Na + were measured in the F1 hybrid plants ( C). The...
- Use
- To determine the feasibility of using natural variation to identify individual loci regulating ionomic variation, we attempted to map the locus responsible for elevated Na + in Ts-1 and Tsu-1. Both accessions were crossed to Col-0, and the shoot concentrations of Na + were measured in the F1 hybrid plants ( C). The...
Genetic Analysis and Mapping of the High Na + Trait
(C) Hybridization of genomic DNA from Ts-1 (blue) and Tsu-1 (red) to DNA microarray (ATH1). Data are presented as a Scaled pool hybridization difference (SPHD), the difference between the hybridization of the two pools at the SFPs, scaled so that the difference between Col-0 and the accession would be equal to 1. SF...
- Use
- (C) Hybridization of genomic DNA from Ts-1 (blue) and Tsu-1 (red) to DNA microarray (ATH1). Data are presented as a Scaled pool hybridization difference (SPHD), the difference between the hybridization of the two pools at the SFPs, scaled so that the difference between Col-0 and the accession would be equal to 1. SF...
Screening of the F2 population from Tsu-1 × Col-0 for survival of NaCl stress.
The evaluation of the F2 population from the Tsu-1 × Col-0 cross in response to salt stress was realized in a growth chamber under controlled environmental conditions, 8 h light:16 h dark (150 µmol • m -2 • s -1 light intensity), 21 °C:19 °C (day/night) and 60% relative hu...
- Use
- The evaluation of the F2 population from the Tsu-1 × Col-0 cross in response to salt stress was realized in a growth chamber under controlled environmental conditions, 8 h light:16 h dark (150 µmol • m -2 • s -1 light intensity), 21 °C:19 °C (day/night) and 60% relative hu...
DNA microarray-based BSA.
DNA microarray-based BSA was realized as previously described [, ]. Briefly, SFPs were identified between Col-0 and Ts-1 and Tsu-1 by hybridizing labeled DNA from each one of the accessions to Affymetrix ATH1 microarrays and comparing them to Col-0 hybridizations downloaded from http://www.naturalvariation.org/xam...
- Use
- DNA microarray-based BSA was realized as previously described [, ]. Briefly, SFPs were identified between Col-0 and Ts-1 and Tsu-1 by hybridizing labeled DNA from each one of the accessions to Affymetrix ATH1 microarrays and comparing them to Col-0 hybridizations downloaded from http://www.naturalvariation.org/xam...
Sequencing of the At HKT1 allele from Ts-1 and Tsu-1.
To sequence the At HKT1 gene and corresponding promoter from Ts-1 (CS1552) and Tsu-1 (CS1640), synthetic oligonucleotide primers were designed to produce overlapping amplified PCR products from 5,456 bp upstream of the At HKT1 start codon to 386 bp downstream of the At HKT1 stop codon, corresponding to positions 32,...
- Use
- To sequence the At HKT1 gene and corresponding promoter from Ts-1 (CS1552) and Tsu-1 (CS1640), synthetic oligonucleotide primers were designed to produce overlapping amplified PCR products from 5,456 bp upstream of the At HKT1 start codon to 386 bp downstream of the At HKT1 stop codon, corresponding to positions 32,...
Sequencing of the At HKT1 allele from Ts-1 and Tsu-1.
provides a summary of the pairs of primers used and the size of the PCR products expected for Col-0 (based on published sequence) and obtained for Ts-1 and Tsu-1. The PCR products obtained for each one of the primer pairs were cloned using the pGEM-T Easy Vector System I according to the manufacturer's instructions...
- Use
- provides a summary of the pairs of primers used and the size of the PCR products expected for Col-0 (based on published sequence) and obtained for Ts-1 and Tsu-1. The PCR products obtained for each one of the primer pairs were cloned using the pGEM-T Easy Vector System I according to the manufacturer's instructions...
Quantitative real-time PCR.
Plants were first analyzed by ICP-MS and further used to determine the At HKT1 transcript levels. Shoot and root from plants grown under short-day conditions (as described above in "General plant growth conditions") were separated and rinsed thoroughly with deionized water to remove any soil contaminatio...
- Use
- Plants were first analyzed by ICP-MS and further used to determine the At HKT1 transcript levels. Shoot and root from plants grown under short-day conditions (as described above in "General plant growth conditions") were separated and rinsed thoroughly with deionized water to remove any soil contaminatio...
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HKT1 Allele in Ts-1 and Tsu-1 Is Associated with Enhanced NaCl Tolerance
At HKT1 has been implicated in Arabidopsis tolerance to NaCl stress. The hkt1 null mutant is sensitive to elevated NaCl as a result of Na + overaccumulation in the shoot [ A; - ]. Under unstressed conditions, we have established that the weaker At HKT1 allele in Ts-1 and Tsu-1 is responsible for the elevated Na + content in the shoot of these two accessions, similar to that observed in the hkt1-1 null mutant. Under high NaCl concentrations (50 and 100 mM NaCl), both Ts-1 and Tsu-1 have higher levels of Na + in the shoot compared to Col-0, in all leaves at different stages of development, including the youngest ones not fully developed and localized close to the meristem (stage 1; B and C). However, this elevated shoot Na does not cause increased NaCl sensitivity in Tsu-1 ( A). Given the connection between At HKT1 expression and NaCl sensitivity, we decided to evaluate the...
HKT1 Allele in Ts-1 and Tsu-1 Is Associated with Enhanced NaCl Tolerance
(A) Relative NaCl tolerance of hkt1-1, Col-0, and Tsu-1. Five-week-old hkt1-1, Col-0, and Tsu-1 plants were treated biweekly with 100 mM NaCl. Picture was taken 6 wk after beginning of the salt treatment.
Screening of the F2 population from Tsu-1 × Col-0 for survival of NaCl stress.
The evaluation of the F2 population from the Tsu-1 × Col-0 cross in response to salt stress was realized in a growth chamber under controlled environmental conditions, 8 h light:16 h dark (150 µmol • m -2 • s -1 light intensity), 21 °C:19 °C (day/night) and 60% relative humidity. After stratification, F2 seeds as well as seeds from each one of the parents were sown onto moist soil (Scotts Potting Medium; Scotts-Sierra Horticultural Products Company, Marysville, Ohio, United States). About 2 wk after germination, seedlings were thinned to leave one plant per individual cell. Plants were bottom-watered twice per week with 0.5 × Murashige and Skoog (MS) Macro- and Micronutrients (Caisson Laboratories, Inc., Rexburg, Idaho, United States). One month after the seeds were sewn, NaCl was added to the watering solution increasingly from 50 to 10...
Na + accumulation in plants exposed to increasing concentrations of NaCl in soil.
Plants from Col-0, hkt1-1, Ts-1, and Tsu-1 used to evaluate Na + accumulation in the shoot in response to NaCl stress were grown under controlled growth conditions (as described above in "General plant growth conditions") and treated with NaCl for 2 wk. NaCl treatment was started 1 mo after the seeds were sewn. Plants were watered twice weekly without or with NaCl. The NaCl treatments included plants watered with 50 mM NaCl and plants watered once with 50 mM, once with 75 mM, and twice with 100 mM NaCl. Two weeks after the beginning of the salt treatments, plants were harvested and analyzed by ICP-MS. Na + was analyzed in leaves of different age throughout the shoot. The leaves were numbered from the meristem toward the outer leaf ring of the rosette. Four samples (indicated as Nos. 1, 2, 3, and 4 in the graph) were harvested per plant, with No. 1 containing leaves N...
Tissue Na + quantification.
Approximately 3 mg of dry weight of each plant was sampled into Pyrex tubes (16 × 100 mm) after drying at 92 °C for 20 h. After cooling, seven of approximately 100 samples from each sample set were weighed. All samples were digested with 0.7 ml of concentrated nitric acid (OmniTrace; VWR Scientific Products; http://www.vwr.com ) and diluted to 6.0 ml with 18 MΩ water. Elemental analysis was performed with an ICP-MS (Elan DRCe; PerkinElmer, http://www.perkinelmer.com ) for Li, B, Na, Mg, P, K, Ca, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Mo, and Cd. All samples were normalized to calculated weights, as determined with an iterative algorithm using the best-measured elements, the weights of the seven weighed samples, and the solution concentrations, implemented in Microsoft Excel ( http://www.microsoft.com ) [ ].
Grafting of Arabidopsis.
Seedlings to be grafted were germinated on 100 × 15-mm plates containing 0.5 × MS Macro- and Micronutrients, 0.5 × MS Vitamins (Caisson Laboratories, Inc.), 3 mg/L Benomyl [methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate; Sigma, http://www.signaaldrich.com ), 0.04 mg/L BA (6-benzylaminopurine; Sigma), 0.02 mg/L IAA (indole acetic acid; Sigma), and 12 g/L agar. Hormone treatment was found to greatly improve grafting efficiency due to at least three factors: enhanced callusing at the graft union, retardation of shoot growth which maintained contact at the graft union, and an approximately 90% reduction in adventitious root formation from the graft union. Benomyl virtually eliminated fungal contamination [ ]. Plates containing the stratified seeds were placed vertically under controlled environmental conditions (16 h light:8 h dark and 25 °C). Five-day-old seedlings...
Supporting Information
Na + contents were analyzed in leaves at four different stages of development (as described in ) across the shoot of 44-d-old Col-0 (circles), Ts-1 (triangles), Tsu-1 (squares), and hkt1-1 (diamonds) plants grown in soil under short-day conditions and treated for 17 d with (A) no NaCl addition, (B) 50 mM, or (C) 100 mM NaCl. Presented data are the mean ± SE.
Measurement outputs
What raw and processed outputs should exist?
(B) Distribution of Na + accumulation in shoot tissue of F2 segregating population obtained from crossing Col-0 with Tsu-1. Presented data are distribution of Col-0 ( n = 80), T...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
(A) Distribution of Na + accumulation in shoot tissue of F2 segregating population obtained from crossing Col-0 with Ts-1. Presented data are distribution of Col-0 ( n = 99), Ts...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
To determine the feasibility of using natural variation to identify individual loci regulating ionomic variation, we attempted to map the locus responsible for elevated Na + in...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
(C) Hybridization of genomic DNA from Ts-1 (blue) and Tsu-1 (red) to DNA microarray (ATH1). Data are presented as a Scaled pool hybridization difference (SPHD), the difference b...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
The elevated shoot Na + of Ts-1 and Tsu-1 that maps to the At HKT1 locus is associated with the loss of At HKT1 expression in roots.
from paperScoring or quantification
Quantify the primary readouts for this experiment: (B) Distribution of Na + accumulation in shoot tissue of F2 segregating population obtained from crossing Col-0 with Tsu-1. Presented data are distribution of Col-0 ( n = 80), T...; (A) Distribution of Na + accumulation in shoot tissue of F2 segregating population obtained from crossing Col-0 with Ts-1. Presented data are distribution of Col-0 ( n = 99), Ts...; To determine the feasibility of using natural variation to identify individual loci regulating ionomic variation, we attempted to map the locus responsible for elevated Na + in...; (C) Hybridization of genomic DNA from Ts-1 (blue) and Tsu-1 (red) to DNA microarray (ATH1). Data are presented as a Scaled pool hybridization difference (SPHD), the difference b....
from paperStatistical comparison
The elevated shoot Na + of Ts-1 and Tsu-1 that maps to the At HKT1 locus is associated with the loss of At HKT1 expression in roots. To address the contribution of HKT1 expressi...
from paperReporting output
Report representative outputs alongside summary comparisons for (B) Distribution of Na + accumulation in shoot tissue of F2 segregating population obtained from crossing Col-0 with Tsu-1. Presented data are distribution of Col-0 ( n = 80), T..., (A) Distribution of Na + accumulation in shoot tissue of F2 segregating population obtained from crossing Col-0 with Ts-1. Presented data are distribution of Col-0 ( n = 99), Ts..., To determine the feasibility of using natural variation to identify individual loci regulating ionomic variation, we attempted to map the locus responsible for elevated Na + in..., (C) Hybridization of genomic DNA from Ts-1 (blue) and Tsu-1 (red) to DNA microarray (ATH1). Data are presented as a Scaled pool hybridization difference (SPHD), the difference b....
inferred from protocolStructured statistical methods
The elevated shoot Na + of Ts-1 and Tsu-1 that maps to the At HKT1 locus is associated with the loss of At HKT1 expression in roots. To address the contribution of HKT1 expressi...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (7)
At HKT1 has been implicated in Arabidopsis tolerance to NaCl stress. The hkt1 null mutant is sensitive to elevated NaCl as a result of Na + overaccumulation in the shoot [ A; - ]. Under unstressed conditions, we have established that the weaker At HKT1 allele in Ts-1 and Tsu-1 is responsible for the elevated Na + content in the shoot of these two accessions, similar to that observed in the hkt1-1 null mutant. Under high NaCl concentrations (50 and 100 mM NaCl), both Ts-1 and Tsu-1 have higher levels of Na + in the shoot compared to Col-0, in all leaves at different stages of development, including the youngest ones not fully developed and localized close to the meristem (stage 1; B and C). However, this elevated shoot Na does not cause increased NaCl sensitivity in Tsu-1 ( A). Given the connection between At HKT1 expression and NaCl sensitivity, we decided to evaluate the involvement of this novel allele of At HKT1 in NaCl tolerance by screening F2 plants from a cross between Tsu-1 × Col-0 for their capacity to survive high levels of NaCl ( ). The genotyping of the F2 population from Tsu-1 × Col-0 identified 25 plants homozygous for Tsu-1- HKT1 and 50 plant...
(A) Relative NaCl tolerance of hkt1-1, Col-0, and Tsu-1. Five-week-old hkt1-1, Col-0, and Tsu-1 plants were treated biweekly with 100 mM NaCl. Picture was taken 6 wk after beginning of the salt treatment.
The evaluation of the F2 population from the Tsu-1 × Col-0 cross in response to salt stress was realized in a growth chamber under controlled environmental conditions, 8 h light:16 h dark (150 µmol • m -2 • s -1 light intensity), 21 °C:19 °C (day/night) and 60% relative humidity. After stratification, F2 seeds as well as seeds from each one of the parents were sown onto moist soil (Scotts Potting Medium; Scotts-Sierra Horticultural Products Company, Marysville, Ohio, United States). About 2 wk after germination, seedlings were thinned to leave one plant per individual cell. Plants were bottom-watered twice per week with 0.5 × Murashige and Skoog (MS) Macro- and Micronutrients (Caisson Laboratories, Inc., Rexburg, Idaho, United States). One month after the seeds were sewn, NaCl was added to the watering solution increasingly from 50 to 100 mM NaCl and maintained at that level until all the plants were recorded dead.
Plants from Col-0, hkt1-1, Ts-1, and Tsu-1 used to evaluate Na + accumulation in the shoot in response to NaCl stress were grown under controlled growth conditions (as described above in "General plant growth conditions") and treated with NaCl for 2 wk. NaCl treatment was started 1 mo after the seeds were sewn. Plants were watered twice weekly without or with NaCl. The NaCl treatments included plants watered with 50 mM NaCl and plants watered once with 50 mM, once with 75 mM, and twice with 100 mM NaCl. Two weeks after the beginning of the salt treatments, plants were harvested and analyzed by ICP-MS. Na + was analyzed in leaves of different age throughout the shoot. The leaves were numbered from the meristem toward the outer leaf ring of the rosette. Four samples (indicated as Nos. 1, 2, 3, and 4 in the graph) were harvested per plant, with No. 1 containing leaves Nos. 4 and 5 and therefore being the youngest leaves, No. 2 containing leaf No. 8, No. 3 containing leaf No. 11, and No. 4 containing leaf No. 14 and being the oldest.
Approximately 3 mg of dry weight of each plant was sampled into Pyrex tubes (16 × 100 mm) after drying at 92 °C for 20 h. After cooling, seven of approximately 100 samples from each sample set were weighed. All samples were digested with 0.7 ml of concentrated nitric acid (OmniTrace; VWR Scientific Products; http://www.vwr.com ) and diluted to 6.0 ml with 18 MΩ water. Elemental analysis was performed with an ICP-MS (Elan DRCe; PerkinElmer, http://www.perkinelmer.com ) for Li, B, Na, Mg, P, K, Ca, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Mo, and Cd. All samples were normalized to calculated weights, as determined with an iterative algorithm using the best-measured elements, the weights of the seven weighed samples, and the solution concentrations, implemented in Microsoft Excel ( http://www.microsoft.com ) [ ].
Seedlings to be grafted were germinated on 100 × 15-mm plates containing 0.5 × MS Macro- and Micronutrients, 0.5 × MS Vitamins (Caisson Laboratories, Inc.), 3 mg/L Benomyl [methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate; Sigma, http://www.signaaldrich.com ), 0.04 mg/L BA (6-benzylaminopurine; Sigma), 0.02 mg/L IAA (indole acetic acid; Sigma), and 12 g/L agar. Hormone treatment was found to greatly improve grafting efficiency due to at least three factors: enhanced callusing at the graft union, retardation of shoot growth which maintained contact at the graft union, and an approximately 90% reduction in adventitious root formation from the graft union. Benomyl virtually eliminated fungal contamination [ ]. Plates containing the stratified seeds were placed vertically under controlled environmental conditions (16 h light:8 h dark and 25 °C). Five-day-old seedlings were grafted on the plate by the 90-degree blunt end technique with a 15-degree Stab Knife (Fine Scientific Tools, North Vancouver, British Columbia, Canada) without collars [ ]. The grafted seedlings remained on the plate for an addition 5 d to allow the formation of the graft union. Successfully...
Na + contents were analyzed in leaves at four different stages of development (as described in ) across the shoot of 44-d-old Col-0 (circles), Ts-1 (triangles), Tsu-1 (squares), and hkt1-1 (diamonds) plants grown in soil under short-day conditions and treated for 17 d with (A) no NaCl addition, (B) 50 mM, or (C) 100 mM NaCl. Presented data are the mean ± SE.
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Natural Variants of At HKT1 Enhance Na + Accumulation in Two Wild Populations of Arabidopsis methods",
"description": "Evidence-backed execution summary for Natural Variants of At HKT1 Enhance Na + Accumulation in Two Wild Populations of Arabidopsis methods from Natural Variants of At HKT1 Enhance Na + Accumulation in Two Wild Populations of Arabidopsis.",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "HKT1 Allele in Ts-1 and Tsu-1 Is Associated with Enhanced NaCl Tolerance",
"text": "At HKT1 has been implicated in Arabidopsis tolerance to NaCl stress. The hkt1 null mutant is sensitive to elevated NaCl as a result of Na + overaccumulation in the shoot [ A; - ]. Under unstressed conditions, we have established that the weaker At HKT1 allele in Ts-1 and Tsu-1 is responsible for the elevated Na + content in the shoot of these two accessions, similar to that observed in the hkt1-1 null mutant. Under high NaCl concentrations (50 and 100 mM NaCl), both Ts-1 and Tsu-1 have higher levels of Na + in the shoot compared to Col-0, in all leaves at different stages of development, including the youngest ones not fully developed and localized close to the meristem (stage 1; B and C). However, this elevated shoot Na does not cause increased NaCl sensitivity in Tsu-1 ( A). Given the connection between At HKT1 expression and NaCl sensitivity, we decided to evaluate the..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "HKT1 Allele in Ts-1 and Tsu-1 Is Associated with Enhanced NaCl Tolerance",
"text": "(A) Relative NaCl tolerance of hkt1-1, Col-0, and Tsu-1. Five-week-old hkt1-1, Col-0, and Tsu-1 plants were treated biweekly with 100 mM NaCl. Picture was taken 6 wk after beginning of the salt treatment."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Screening of the F2 population from Tsu-1 × Col-0 for survival of NaCl stress.",
"text": "The evaluation of the F2 population from the Tsu-1 × Col-0 cross in response to salt stress was realized in a growth chamber under controlled environmental conditions, 8 h light:16 h dark (150 µmol • m -2 • s -1 light intensity), 21 °C:19 °C (day/night) and 60% relative humidity. After stratification, F2 seeds as well as seeds from each one of the parents were sown onto moist soil (Scotts Potting Medium; Scotts-Sierra Horticultural Products Company, Marysville, Ohio, United States). About 2 wk after germination, seedlings were thinned to leave one plant per individual cell. Plants were bottom-watered twice per week with 0.5 × Murashige and Skoog (MS) Macro- and Micronutrients (Caisson Laboratories, Inc., Rexburg, Idaho, United States). One month after the seeds were sewn, NaCl was added to the watering solution increasingly from 50 to 10..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Na + accumulation in plants exposed to increasing concentrations of NaCl in soil.",
"text": "Plants from Col-0, hkt1-1, Ts-1, and Tsu-1 used to evaluate Na + accumulation in the shoot in response to NaCl stress were grown under controlled growth conditions (as described above in \"General plant growth conditions\") and treated with NaCl for 2 wk. NaCl treatment was started 1 mo after the seeds were sewn. Plants were watered twice weekly without or with NaCl. The NaCl treatments included plants watered with 50 mM NaCl and plants watered once with 50 mM, once with 75 mM, and twice with 100 mM NaCl. Two weeks after the beginning of the salt treatments, plants were harvested and analyzed by ICP-MS. Na + was analyzed in leaves of different age throughout the shoot. The leaves were numbered from the meristem toward the outer leaf ring of the rosette. Four samples (indicated as Nos. 1, 2, 3, and 4 in the graph) were harvested per plant, with No. 1 containing leaves N..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Tissue Na + quantification.",
"text": "Approximately 3 mg of dry weight of each plant was sampled into Pyrex tubes (16 × 100 mm) after drying at 92 °C for 20 h. After cooling, seven of approximately 100 samples from each sample set were weighed. All samples were digested with 0.7 ml of concentrated nitric acid (OmniTrace; VWR Scientific Products; http://www.vwr.com ) and diluted to 6.0 ml with 18 MΩ water. Elemental analysis was performed with an ICP-MS (Elan DRCe; PerkinElmer, http://www.perkinelmer.com ) for Li, B, Na, Mg, P, K, Ca, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Mo, and Cd. All samples were normalized to calculated weights, as determined with an iterative algorithm using the best-measured elements, the weights of the seven weighed samples, and the solution concentrations, implemented in Microsoft Excel ( http://www.microsoft.com ) [ ]."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Grafting of Arabidopsis.",
"text": "Seedlings to be grafted were germinated on 100 × 15-mm plates containing 0.5 × MS Macro- and Micronutrients, 0.5 × MS Vitamins (Caisson Laboratories, Inc.), 3 mg/L Benomyl [methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate; Sigma, http://www.signaaldrich.com ), 0.04 mg/L BA (6-benzylaminopurine; Sigma), 0.02 mg/L IAA (indole acetic acid; Sigma), and 12 g/L agar. Hormone treatment was found to greatly improve grafting efficiency due to at least three factors: enhanced callusing at the graft union, retardation of shoot growth which maintained contact at the graft union, and an approximately 90% reduction in adventitious root formation from the graft union. Benomyl virtually eliminated fungal contamination [ ]. Plates containing the stratified seeds were placed vertically under controlled environmental conditions (16 h light:8 h dark and 25 °C). Five-day-old seedlings..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Supporting Information",
"text": "Na + contents were analyzed in leaves at four different stages of development (as described in ) across the shoot of 44-d-old Col-0 (circles), Ts-1 (triangles), Tsu-1 (squares), and hkt1-1 (diamonds) plants grown in soil under short-day conditions and treated for 17 d with (A) no NaCl addition, (B) 50 mM, or (C) 100 mM NaCl. Presented data are the mean ± SE."
}
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"name": "Genetic Analysis and Mapping of the High Na + Trait"
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"@type": "HowToTool",
"name": "Genetic Analysis and Mapping of the High Na + Trait"
},
{
"@type": "HowToTool",
"name": "Genetic Analysis and Mapping of the High Na + Trait"
},
{
"@type": "HowToTool",
"name": "Screening of the F2 population from Tsu-1 × Col-0 for survival of NaCl stress."
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{
"@type": "HowToTool",
"name": "DNA microarray-based BSA."
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{
"@type": "HowToTool",
"name": "Sequencing of the At HKT1 allele from Ts-1 and Tsu-1."
},
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"@type": "HowToTool",
"name": "Sequencing of the At HKT1 allele from Ts-1 and Tsu-1."
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"supply": [
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"@type": "HowToSupply",
"name": "General plant growth conditions."
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"name": "Screening of the F2 population from Tsu-1 × Col-0 for survival of NaCl stress."
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"name": "Tissue Na + quantification."
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