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Diss Factsheets

Administrative data

additional ecotoxicological information
Type of information:
experimental study
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Not a guideline study. This study has been published in generally accepted and reviewed scientific paper.

Data source

Reference Type:
review article or handbook
Glycine betaine involvement in freezing tolerance and water stress in Arabidopsis thaliana
Weibing, X., Rajashekar, C.B
Bibliographic source:
Envronmental and Experimental botany 46 (2001) 21-28

Materials and methods

Test guideline
other: The materials and methods are described in the reviewed paper.
All experiments were conducted with four replicates.
Principles of method if other than guideline:
Levels of endogenous glycine betaine in the leaves were measured in response to cold acclimation, water stress and exogenous ABA application in Arabidopsis thaliana. According to this study, the methodology was the following:

Growing conditions

Plants were grown in greenhouse (23/18 C) day/night temperatures under natural day light in pots (15 cm) in sterile medium of peat, perlite and soil (2:2:1 v/v). Plants were irritated every 2 days and fertilized weekly (N-P-K 10-10-20). Irritation water contained 250 ppm nitrogen.

Cold acclimation was studied in cold chambers set for 4/2 C (day/night) temperatures 4 weeks. Plants were watered when needed to keep the soil near field capacity.

Plants were also grown under axenic conditions to test the effects of glycine betaine on freezing tolerance. Plant seeds were sterlized with 2.62 % sodium hypoclorite for 10 min and planted in a Magenta vessel. Plants were grown at 25/18 C day/night temperatures with a 16-h photoperiod in the growth chamber.

Exogenous substance addition

10 mM of anhydrous glycine betaine solution with 0.0.2 % Tween-20 was applied into plants grown 3 weeks in greenhouses. Addition was done by foliar spray twice a day for 3 consecutive days. Leaf were sampled for freezing tolerance and leaf glycine betaine levels evaluation. Plants grown in axenic conditions, glycine betaine solution (10 mM) was added into the medium through sterile filter. Plants were grown for 4 weeks and leaves were sampled to evaluate the freezing tolerance.

Exogenous abscisic acid

Mixed isomers of ABA were dissolved in small volume of 0.1N KOH and diluted to 1mM with distilled water. 3 weeks grown plants were treated with ABA. ABA solution with 0.02 % Tween-20 was sprayed three times a day for 3 consecutive days. Leaves were sampled each day to evaluate freezing tolerance and endogenous glycine betaine levels.


Freezing tolerance of leaf samples were determined after freezer pretreatment (-1 and -2 C). The plant injuiry was evaluated using the electrolyte leakage test and conductivity of the leachate was measured. Leaf samples were killed and the total elctrolytes were released from the tissue. The final conductivity measurement was done after holding the samples for 10 h at 22 C.

Water stress and leaf water potential of the plants were determined using thermo-couple psychrometer with a dew point microvoltmeter. The leaf samples in psychometer chamber were equilibrated at 25 C for 5 h. The water potential was measured in the dew point mode with a cooling time of 20s.

Endogenous glycine betaine was determined from harvested plants. Before analysing the glycine betaine concentrations of the leaf samples, collected samples were frozen in liquid nitrogen. The leaf samples were pretreated by thawing, and extracts of the samples were dried in nitrogen stream in a desiccator, and diluted up to 1 ml with D2O. Glycine betaine was quantified using NMR spectrometer. All NMR spectra were measured at 30 C using a 5mm triple-esonance inverse detection probe. The peak intenties were measured digitally using NMR spectrometer's integration sofware.

Leaf dry weight was determined after drying the samples at 80 C for 4 days.

GLP compliance:
Type of study / information:
This study provides additional information on the substance abiotic behaviour and occurance in nature (plants). The accumulation of the substance in plants and the water stress leading to increase in the freezing tolerance were studied. This additional information is provided in the registration process for data waiving for ecotoxicity of the substance.

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:

Results and discussion

Any other information on results incl. tables

According to this study, the freezing tolerance of Arabidopsis plant leaves increased in cold acclimation treatment. The freezing tolerance reached the maximum after 1 week treatment. Also the leaves became thicker and dark green compared to the unhardened control plants. Also endogenous glycine betaine content in the leaves increased during this cold acclimation treatment. The glycine betaine content was shown to be higher in the cold-hardened plants compared to the unhardened plants. In this study, the results showed that maximun increase in freezing tolerance of leaves occured at 10 mM application. Higher concentrations of glycine betaine caused damage to leaves.

When exogenous glycine betaine was applied into the test plant, the maximum freezing tolerance of Arabidopsis was increased. The glycine betaine content in plant leaves was measured during the experiment. This exogenous glycine betaine application showed that glycine betaine was rapidly taken up by the leaves, and the concentration was increased nearly 30 umolg-1 dw in 1 day. The results showed that the exogenous glycine betaine application increased the freezing tolerance of the plants grown in greenhouse as well as in axenic conditions.

The freezing tolerance was increased as well when ABA was applied to Arabidopsis plants. Endogenous glycine betaine content was also increased in this application. Also the water stress of these plants were studied. The induction of water stress in plants resulted in a significant increase in the freezing tolerance of the leaves. The increase in freezing tolerance by water stress was smaller than that induced by the cold acclimation treatment.

Applicant's summary and conclusion

The key conclusions of this study shows that betaine is known to accumulate in a wide range of plants typically when plants are introduced to salt and drought stress. The results showed that cold acclimation and ABA treatments lead to the endogenous glycine betaine accumulation in test plant (Arabidopsis thaliana). The results also showed that glycine betaine accumulates in large quantities to test plants during water stress. Glycine betaine content is increased in Arabidopsis thaliana and other plants (i.e. barley, wheat, Puma rye) and improves the freezing tolerance of the plants during cold acclimation and water stress.