Forward Genetic Screen Using Transgenic Calcium Reporter Aequorin to Identify Novel Targets in Calcium Signaling

Mittal, Deepika; Mishra, Shruti; Prajapati, Ramgopal; Vadassery, Jyothilakshmi

doi:10.3791/61259

Cited by 2 publications

(6 citation statements)

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“… Rinse the mutant candidates with 25 mg/L cefotaxime solution in sterile-deionized water 2 times. Note: The use of cefotaxime reduces contamination by micro-organisms ( Mittal et al., 2020 ). Transfer the mutant candidates to 1/2 MS medium (without sucrose) with 25 mg/L cefotaxime under low light for recovery.…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

“… Discard the stimuli solution, and acquire the luminescence intensity (L rest ) at 1 s intervals about 2 min until values were within 5% of the highest discharged value, immediately after adding 1 mL of discharging buffer (2 M CaCl 2 in 20% ethanol). Note: Steps 42–45 can be adjusted depending on the type of luminometer used ( Mittal et al., 2020 , Tanaka et al., 2013 ). The total luminescence (L max ) is calculated by L rest plus L stimuli .…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

“…Note: Steps 42–45 can be adjusted depending on the type of luminometer used ( Mittal et al., 2020 , Tanaka et al., 2013 ).…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

“…This protocol aims to screen mutants with altered Ca 2+ signals using Aequorin-based luminescence imaging. Screening Ca 2+ responsive mutants with the plate reader can be conducted by following two other protocols ( Tanaka et al., 2013 , Mittal et al., 2020 ).…”

Section: Before You Beginmentioning

confidence: 99%

“… CRITICAL: A small pool allows us to regain candidate mutants with low vitality that may be lost due to growth defect, given that EMS often causes multiple random mutations that affect plant growth and fertility. Therefore, we suggest a single pedigree-based seed collection as previously described ( Mittal et al., 2020 ), although it is labor-intensive. Note: To avoid poor seed vigor or growth defect of plant materials, the EMS mutagenized pool should be generated freshly from the plants under non-stress conditions.…”

Section: Before You Beginmentioning

confidence: 99%

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Screening for Arabidopsis mutants with altered Ca2+ signal response using aequorin-based Ca2+ reporter system

et al. 2021

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Summary Environmental stimuli evoke transient increases of the cytosolic Ca 2+ level. To identify upstream components of Ca 2+ signaling, we have optimized two forward genetic screening systems based on Ca 2+ reporter aequorin. AEQ sig6 and AEQ ub plants were used for generating ethyl methanesulfonate (EMS)-mutagenized libraries. The AEQ sig6 EMS-mutagenized library was preferably used to screen the mutants with reduced Ca 2+ signal response due to its high effectiveness, while the AEQ ub EMS-mutagenized library was used for screening of the mutants with altered Ca 2+ signal response. For complete details on the use and execution of this protocol, please refer to Chen et al. (2020) and Zhu et al. (2013) .

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Section: Step-by-step Methods Detailsmentioning

confidence: 99%

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

“…Note: Steps 42–45 can be adjusted depending on the type of luminometer used ( Mittal et al., 2020 , Tanaka et al., 2013 ).…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

Section: Before You Beginmentioning

confidence: 99%

Section: Before You Beginmentioning

confidence: 99%

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Screening for Arabidopsis mutants with altered Ca2+ signal response using aequorin-based Ca2+ reporter system

et al. 2021

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High-Vigor Maize Seeds Resist Fusarium graminearum Infection through Stronger Ca2+ Signaling

Liu

Song

et al. 2022

Agriculture

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Seeds with high vigor have strong resistance to various adverse environmental conditions. However, little is known about how seed vigor affects the resistance of seeds to biotic stress. In this study, newly harvested seeds that had high vigor and seeds with low vigor, achieved via an artificially accelerated aging treatment, were used in the germination test after inoculation with Fusarium graminearum for 24 h. The results showed that high-vigor seed-related germination and seedling growth were not significantly affected by F. graminearum infection, while those related to low-vigor seeds were significantly inhibited. Analysis of transgenic maize seeds expressing the luminescent Ca2+ probe encoded by aequorin indicated that the concentration of free Ca2+ in the cytoplasm and nucleus of the embryo cells of high-vigor seeds was significantly higher than that of the low-vigor seeds. Through an experiment with Ca2+ inhibitor treatment and exogenous Ca2+ application, we further confirmed that Ca2+ played an important role in seed germination and seedling growth. Interestingly, in the presence of F. graminearum, the Ca2+ required for seed germination and seedling growth mainly came from the vacuolar calcium pool, while in the absence of F. graminearum, the required Ca2+ mainly came from the apoplastic calcium store. This study helps understand how high-vigor seeds resist disease and provides theoretical support for the wide application of high-vigor seeds in agricultural production.

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Forward Genetic Screen Using Transgenic Calcium Reporter Aequorin to Identify Novel Targets in Calcium Signaling

Cited by 2 publications

References 0 publications

Screening for Arabidopsis mutants with altered Ca2+ signal response using aequorin-based Ca2+ reporter system

Screening for Arabidopsis mutants with altered Ca2+ signal response using aequorin-based Ca2+ reporter system

High-Vigor Maize Seeds Resist Fusarium graminearum Infection through Stronger Ca2+ Signaling

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