Natural Variants of At HKT1 Enhance Na + Accumulation in Two Wild Populations of Arabidopsis methods
Show snapshot details
On this page
This experiment, in seven questions
Jump straight to the part of the recipe you need. Data and provenance labels stay close to the action they support.
Shopping and prep list
What do I need before I start?
Biological model pending
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Before you run
What should be confirmed before execution?
First confirmation
Species or subject information is missing.
Confirm before execution
Protocol does not list structured equipment.
Confirm before execution
This page shows passages quoted from the paper. Equipment, materials, times, and temperatures are hidden until the page is rebuilt from the full paper and checked by a reviewer. Check the paper before using this method.
Confirm before execution
Open the source paper before finalizing run-specific details.
Procurement checkpoint
Use source-stated vendors where present. Treat mapped products as sourcing options unless the page marks an exact source match.
Open quote workflowStep-by-step procedure
What do I do, in order?
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?
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
Review raw outputs for quality, remove unusable captures, and organize the data into a comparison-ready table or image set.
inferred from protocolScoring or quantification
Quantify the primary endpoint described in the methods before moving into any statistical comparison.
inferred from protocolStatistical comparison
Statistical method not yet structured for this page.
inferred from protocolReporting output
Report representative outputs alongside the summary comparison used to support the conclusion.
inferred from protocolSource 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": "Protocol for Natural Variants of At HKT1 Enhance Na + Accumulation in Two Wild Populations of Arabidopsis methods",
"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."
}
],
"tool": [],
"supply": [],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Natural Variants of At HKT1 Enhance Na + Accumulation in Two Wild Populations of Arabidopsis",
"datePublished": "2006",
"author": [
{
"@type": "Person",
"name": "Ana Rus"
},
{
"@type": "Person",
"name": "Ivan Baxter"
},
{
"@type": "Person",
"name": "Balasubramaniam Muthukumar"
},
{
"@type": "Person",
"name": "Jeff Gustin"
},
{
"@type": "Person",
"name": "Brett Lahner"
},
{
"@type": "Person",
"name": "Elena Yakubova"
},
{
"@type": "Person",
"name": "David E Salt"
}
],
"identifier": "10.1371/journal.pgen.0020210"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Natural Variants of At HKT1 Enhance Na + Accumulation in Two Wild Populations of Arabidopsis methods",
"item": "https://replicatescience.com/experiments/natural-variants-of-at-hkt1-enhance-na-accumulation-in-two-wild-populations-of-arabidopsis-methods-ana-rus-pmc1665649/natural-variants-of-athkt1-enhance-na-accumulation-in-two-wild-populations-of-arabidopsis-mlph342v"
}
]
}
]