Laboratory report: Amylase activity in dormant, germinating and Seedling stages of barley
Introduction
Germination pertains to the developmental stage in plants that involves the generation of roots and shoots which serve as anatomical markers for plants growth. Germination initially starts from a dormant seed and given the optimal conditions such as presence of water, light and carbon dioxide, the primary root and shoot appears from the dormant seed. The enzyme amylase is one of the key proteins that are produced by the barley plant specifically during development. This enzyme has also served as a determinant for the quality of malt that will be produced by barley crop batches. Malt has long been employed for the production of beer (Georg-Kraemer et al., 2001). In addition, the activity of this enzyme is positively correlated with the diastatic power of the seeds from the barley species. Botanists have also determined that the weight of barley grains is positively correlated to the amount of malt that could be extracted from barley seeds (Jin-xin et al., 2006). Research studies have previously shown that amylase activity and grain weight were affected by a combination of environmental and genetic factors. In this report, we present our results from our assay that involved extraction of amylase and quantification of amylase activity in different seed stages of barley.
Materials and methods
Briefly, ten barley seeds of each developmental stage (dormant, germinating and seedling) were weighed and homogenized in 10 ml amylase extraction buffer. The solution was filtered to remove debris and the resulting volume was measured. A five-fold dilution series was established by mixing 5 ml of the barley extract with 20 ml of dilution buffer. Amylase activity was measured using a colorimetric method using a starch indicator buffer containing iodine which reacts with starch. The achromic point was determined to quantify the reaction time of amylase. Hydrolysis reaction of each development seed stage was determined using the Benedict assay.
Results
Table 1 shows the weights of ten (10) seeds representing each developmental stage (dormant, germinating and seedling) and the volume of the extract generated from crushing and filtering the seeds.
Table 1. Weight and volume of ten barley seeds at different developmental stages
Developmental stage
of barley seed
Total weight of 10
barley seeds (grams)
Volume of extract from 10 barley seeds (ml)
Germinating
1.41
9.9
Dormant
0.39
9.0
Seedling
1.92
10.1
The amount of starch from the extract was initially determined as follows:
5 mg/ml
———– = 0.714 mg/ml
7 min
The amount of starch from the barley extract was then calculated:
1.611 g
——— = 0.201 g/ml
8 ml
The dilution concentration of the barley extract was determined as follows:
0.201 g/ml
———— = 0.0403 g/ml
5
Amylase activity was then calculated as follows:
0.714 mg/ml/min
——————– = 17.73 mg/min/gram of barley
0.0403 g/ml
Our calculations thus showed that 17.73 milligrams of starch was hydrolyzed per minute, per gram of barley extract.
Discussion
The data collected from weighing ten seeds from different developmental stages of barley (germinating, dormant and seedling) showed that specific developmental stages have different weights and this also affected the amount of extract that can be collected after crushing the seeds and adding a constant volume of the extraction buffer. We have observed that the dormant seeds weighed the lightest, suggesting that dormant seeds have the least seed content. We have also observed that the seedlings weighed higher than germinating seeds, suggesting that advanced developmental stages of the seeds are correlated with an increase in the biomass of the seeds. Furthermore, the volume of the extract varied among different developmental stages. The smallest volume of extract was observed from the dormant seeds. The volume of the extract generated from the seedlings was greater than the volume of the extract collected from the germinating seeds, indicating that the content (water and other essential biological fluids) of the seedlings was substantially bigger.
The determination of amylase activity using the colorimetric method and Benedict assay served as a tool in quantifying the reaction time for the process of hydrolysis to be completed. As our calculations show, approximately 17.73 milligrams of starch could be hydrolyzed per minute by the amylase enzyme that was extract from the barley seeds. It should be noted though that the activity of amylase in a barley seed depends of the amount of amylase that is present in each seed, which is turn is dependent on the developmental stage of the seed itself. In addition, the developmental stage of a seed is dependent of the condition of the seed’s immediate environment. A barley seed will go into a dormancy stage during environmental conditions of drought or any similar condition that does not provide optimal amounts of water or humidity. The presence of water in the immediate environment of a barley seeds controls other plant biochemical activities such as nitrogen metabolism and the generation of other essential protein enzymes that could catalyze reactions that are necessary for the barley seed to grow and develop (Swanston and Molina-Cano, 2001).
This experiment has allowed us to perform a simple method in comparing the amylase activities of different developmental stages of barley seeds. We have observed that the accumulation of starch in the seed is positively correlated with amylase activity as well as the seed content of barley. The extraction assay has provided us an exposure to collect a solution that is assumed to content an essential protein that catalyzes the hydrolysis of starches in the plant. The same principle may be applied to other plants and may be used in determining the species-specific amylase activities in relation to other environmental conditions such as the presence or absence of sunlight, atmospheric gases and other essential micronutrients.
References
Georg-Kraemer JE, Mundstock EC and Cavalli-Molina S (2001): Developmental expression of amylase during barley malting. Journal of Cereal Science, 33:279-288.
Jin-xin C, Fei D, Kang W and Guo-ping Z (2006): Relationship between malt qualities and β-amylase activity and protein content as affected by timing of nitrogen fertilizer application. J Zhejiang Univ SCIENCE B. 7(1):79-84.
Swanston JS and Molina-Cano JL (2001): β-amylase activity and thermostability in two mutants derived from the malting barley cv. Triumph. Journal of Cereal Science, 33:155-161.
