May 29, 2025
Measuring Glycine Betaine concentrations in Typha/Spartina
- Suzanne M Thomas1,
- Zoe Cardon1,
- Ilana Stein1
- 1Marine Biological Laboratory
Protocol Citation: Suzanne M Thomas, Zoe Cardon, Ilana Stein 2025. Measuring Glycine Betaine concentrations in Typha/Spartina. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g75rr9lwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 25, 2025
Last Modified: May 29, 2025
Protocol Integer ID: 125031
Keywords: saltmarsh, glycine betaine, plant stress, sediment, glycine betaine content, spartina gycine betaine, measuring glycine, betaine concentrations in typha, microbes in salt marsh, betaine concentration, roots of typha angustifolia, salinity, many tidal marsh plant species, salt marsh, important aspect of salt tolerance, different salinity, typha angustifolia, compatible osmolyte that many plant, salt tolerance, marine sediment, compatible osmolyte, glycine betaine concentrations in typha, measuring glycine betaine concentration
Funders Acknowledgements:
Simons Foundation
Grant ID: 1247132
DOE
Grant ID: DE-SC002470
Abstract
Gycine betaine (GB) is a compatible osmolyte that many plants produce in response to various types of
stress, particularly drought and salinity (Ashraf & Foolad, 2007; Jarin et al., 2024). GB accumulation is an important aspect of salt tolerance for many tidal marsh plant species (Adrian-Romero et al., 1998), as well as being a substrate for methanogenesis by microbes in salt marsh and marine sediments (Boysen et al., 2022; Bueno De Mesquita et al). We measured glycine betaine content in both the leaves and roots of Typha angustifolia grown under different salinities, using the method of Grieve and Grattan (1983), as modified by Valdez-Bustos et al (2016). We made a further modification, using a plate reader instead of a spectrophotometer, which makes it possible to analyze samples more quickly.
Image Attribution
All images taken by Suzanne Thomas
Materials
Betaine, anhydrous Spectrum Chemical MFG CorpCatalog #B1572
Sulfuric acidFisher ScientificCatalog #A300C-212
1,2-dichloroethaneThermo Fisher ScientificCatalog #032462.K2
potassium iodideThermo Fisher ScientificCatalog #A12704.18 Iodine flakesFisher ScientificCatalog #137-100
Equipment:
Equipment
MM200
NAME
Grinding mill
TYPE
Retsch
BRAND
MM200
SKU
Equipment
Synergy H1
NAME
Plate Reader
TYPE
BioTek
BRAND
H1M
SKU
Equipment
96 well plate
NAME
acrylic UV transparent plate
TYPE
Corning
BRAND
3635
SKU
Equipment
block heater
NAME
module block heater
TYPE
American Scientific
BRAND
2025-1
SKU
Equipment
Neoprene
NAME
lab gloves
TYPE
MicroFlex
BRAND
NPG-888
SKU
Equipment
Eppendorf 5415R Refrigerated Centrifuge
NAME
Refrigerated Centrifuge
TYPE
Eppendorf
BRAND
EP-5415R
SKU
Equipment
Eppendorf tubes
NAME
2mL clear
TYPE
USA Scientifc
BRAND
1620-2700
SKU
Equipment
Eppendorf tubes
NAME
2mL amber
TYPE
Eppendorf
BRAND
022363379
SKU
Troubleshooting
Safety warnings
1,2-dichloroethane (also known as ethylene dichloride) is toxic, corrosive, and carcinogenic. It is also highly volatile. This method calls for using undiluted 1,2-dichloroethane. It is imperative to use neoprene gloves, safety glasses, lab coat, and work in a fume hood at all times.
Before start
Iodine is light sensitive. Excess iodine will be removed in Step #20, but iodine will also be incorporated into the betaine-iodide crystals. All steps where the iodine reagent is involved (Steps 14-27) were done under low light. The graph below shows how quickly iodine reacts with light. A black cover was used to protect the samples from light as they were being pipetted into the 96 well plate to read the absorbance.
Repeated absorbance readings on samples (plate removed from plate reader between readings- volatile dichlorethane could damage the plate reader). The iodine reacts with light and quickly changes the absorbance.
96 well plate lid covered with black duct tape. This helps to decrease the reaction of iodine with light while samples are being pipetted to read absorbance in the plate reader.
It is critical to remove the iodine reagent when it is in excess. In the photo below, I ran samples in quadruplicate, intentionally leaving a small drop of iodine solution in the vial with the betaine iodide crystals. This shows that it is critical to remove the iodine reagent in Step #20.
96 well plate showing samples run in quadruplicate, where the 4th sample of each set was intentionally contaminated with a small drop of iodine solution from Step # 20.
Collect and prepare plant material
12h 10m
snip ~20cm of a leaf (we are currently looking at glycine betaine in saltmarsh and brackish marsh plants (Sporobolus and Typha)) and place in a labeled paper bag
Place sample + bag in a 60ºC drying oven overnight or longer
12h
Grind sample into a homogenized powder
Retsch mill grinder and stainless steel grinding vessels
10m
Store ground sample in a closed vial
Make Reagents and Standards
4h 45m
2N Sulfuric Acid
Add 28 mL 36 Molarity (M) H2SO4 (sulfuric acid) to ~300mL deionized water in a 500mL volumetric flask. Swirl to mix. When cool, fill volumetric flask to 500mL. Invert to mix 10x. Transfer to a glass bottle for storage.
Safety information
- Work in a fume hood when handling full-strength sulfuric acid.
- Use nitrile or latex gloves and remove gloves immediately if you come into contact with any concentrated acid.
- Wear eye protection.
- Wear a lab coat.
1h
Potassium Iodide - Iodine Solution
1.57 g I2 (iodine flakes/chunks) + 2 g KI (potassium iodide) + 10 mL deionized water in a dark bottle. Swirl from time to time to mix. It will take some time for the KI-I2 to dissolve.
Safety information
Use appropriate PPE when handling these chemicals- lab coat, safety glasses, latex or nitrile gloves.
2h
Prepare 1,2-dichloroethane (also known as ethylene dichloride)
Pour 1,2-dichloroethane into a smaller glass bottle (50 or 100mL) as a working reagent (full strength)
Safety information
This chemical is very hazardous! It is toxic, corrosive, carcinogenic, and flammable.
You must:
- Work in a fume hood
- Wear special lab gloves- neoprene
- Wear safety glasses and a lab coat
- Always use and store this chemical in a fume hood. It is very volatile
15m
Make Standard Stock Solution (200mg/mL)
4 g betaine + 20 mL 2N H2SO4
From this solution, I made standards of 0, 1, 2, 5, 10, 20, 50, 100, 200, and 500 µg/mL
1h 30m
Extract Glycine Betaine from Ground Plant Material (timing estimates for ~15 samples)
2d 19h 45m
Weigh out ~0.02 g ground material and place in a 2mL Eppendorf tube (can be clear)
30m
Add 1.5mL 2N H2SO4 to tube with ground plant material
Safety information
You should be wearing gloves and safety glasses
10m
Vortex closed tube to mix
Heat samples to 60 °C for 10 minutes in a heating block (or similar)
10m
Centrifuge sample tubes at ~14,000 rpm, 22°C (our centrifuge only goes to 13,200rpm and that has been fine) for 10-25 minutes at room temperature. The centrifugation time depends on how much plant material is in the vial and how dense it is.
25m
Transfer125 µL sample supernatant to an amber 2mL Eppendorf tube and add 50 µL cold KI-I2 solution
Note
The iodine solution is said to be light sensitive, so I do this step with the overhead lab lights off and a small light on to see by.
20m
Cap tube and place mixture on ice immediately. Ice should be kept in a cooler with a lid to keep the samples dark.
Place cooler with ice and samples in a refrigerator overnight. Samples should remain at 0-4ºC for 16 hrs.
16h
Cool Centrifuge to 0ºC. Ours take 30-60 minutes to cool down.
Note
It is crucial that everything stays cold during these next steps! The betaine iodide complexes that form can dissolve at temps > 0ºC.
1h
Centrifuge samples at 0ºC and ~14,000 rpm for 30 minutes.
30m
Remove samples from centrifuge and return to ice.
5m
Remove supernatant from sample tube. Only the crystals of betaine iodide should remain. Do a quick visual inspection to ensure that all liquid iodine reagent has been removed. Supernatant should be disposed of in a labeled hazardous waste container.
Note
Again working with the overhead lab lights off and a small light on to see by.
15m
Once the supernatant has been removed, the sample tube may be placed in a holder at room temperature.
Move samples to a fume hood and change gloves to neoprene gloves. Double check that you have your safety glasses on.
Note
Continue to work in low light, but dichloroethane is very nasty, so we want to make sure we can see well. Our lab has two banks of lights. We work with our fume hood light off, but with one bank of lab lights on.
10m
Add 1.4 mL 1,2- dichloroethane to sample vial with crystals. Cap.
Safety information
Collect pipette tips in a glass beaker to evaporate in the fume hood. Then dispose of as hazardous waste when dry.
10m
Let crystals dissolve for 48 hrs. I agitate the vial holder several times over this period to encourage dissolution. We place an opaque plastic box over the samples to ensure darkness.
Safety information
These vials need to stay in the fume hood for this step! The dichloroethane is very volatile.
2d
Determine Concentration of Glycine Betaine in Samples on a Plate Reader
1d 0h 22m 30s
Agitate samples in holder to mix. Prepare plate reader and 96 well plate. We have found that reading one row of 12 samples at a time works well for us, as long as each well in the row is covered as it is filled. The plate reader should be set to read absorbance at 290nm (and 365nm, although we're not sure this wavelength is necessary).
Note
This step can also be done on a spectrophotometer in a fume hood. You will have to use a removable glass/quartz cuvette (as opposed to pump tubing and a sipper unit. The dichlorethane would dissolve pump tubing). Being in a fume hood is critical to working with 1,2-dichloroethane.
96 well plate lid covered with black duct tape. When 100µL of sample is pipetted into a well, the plate cover is repositioned to cover the well and block out most of the light. The 96 well plate itself is clear, so some light gets in through the bottom plate.
Note
I am again working with only half the lab lights on and am in the fume hood. It is crucial to cover each well after it is filled with sample. The iodine that is released from the betaine iodide crystal is very light sensitive. I use a 96 well plate lid covered in black duct tape to cover each well as I fill a row. Then I place the covered 96 well plate on the plate reader arm and remove the cover as the plate is being pulled into the plate reader.
10m
Add 100µL of sample to a well and read a row of 12 samples at a time in the plate reader at 290nm
Safety information
Working in a fume hood, move quickly (light sensitive extract) but carefully with these samples. The dichloroethane is toxic, corrosive, carcinogenic, and volatile. Do NOT leave the plate in the plate reader for any longer than is necessary. The fumes will degrade the instrument.
All pipette tips should be ejected into a glass beaker. Once dry (overnight), dispose of as hazardous waste.
2m 30s
Repeat until all samples have been read.
10m
Allow samples to evaporate from the wells. Yes, you are still in a fume hood! The well will have been distorted and stained from the sample. The plates are single-use for this experiment.
1d
Protocol references
Adrian-Romero M, Wilson SJ, Blunden G, Yang M-H, Carabot-Cuervo A, Bashir AK. (1998). Betaines in coastal plants. Biochemical Systematics and Ecology 26: 535–543.
Ashraf M, Foolad MR. 2007. Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environmental and Experimental Botany 59: 206–216.
Boysen AK, Durham BP, Kumler W, Key RS, Heal KR, Carlson LT, Groussman RD, Armbrust EV, Ingalls AE. 2022. Glycine betaine uptake and metabolism in marine microbial communities. Environmental Microbiology 24: 2380–2403.
Bueno De Mesquita CP, Wu D, Tringe SG. 2023. Methyl-Based
Methanogenesis: an Ecological and Genomic Review. Microbiology and Molecular
Biology Reviews 87: e00024-22.
Citation
LINK
Jarin A, Ghosh UK, Hossain MdS, Mahmud A, Khan MdAR. 2024. Glycine betaine in plant responses and tolerance to abiotic stresses. Discover Agriculture 2: 127.
https://doi.org/10.1007/s44279-024-00152-w
Citation
LINK
Citation
LINK