International Journal of Current Research and Review (IJCRR)

Full Html

IJCRR - Vol 11 Issue 06, March

Pages: 16-21

Date of Publication: 30-Mar-2019

Print Article   Download XML  Download PDF

Alcohol Dehydrogenase (ADh) Enzyme is a Potent Biochemical Marker for Submergence Tolerance in Rice (Oryza sativa L.) During Seedling Stage of Growth

Author: Biswajit Pradhan, Moushree Sarkar, Sabyasachi Kundagrami

Category: Life Sciences

Abstract:Aim: The incorporation of sub 1 gene in high yielding rice mega genotypes indicate that higher amount of Adh enzyme synthesis is controlled by the genetic factors. Adh enzyme will be the one of the vital factor for underwater respiration process of survival. Hence, rice genotypes with high amount of alcohol dehydrogenase enzyme synthesis during anoxic condition in compare with tolerant checks could be good promise for early introduction to the submergence prone area.
Methodology: Fresh and disease free seeds of fifty diverse rice genotypes including tolerant and susceptible checks were taken and germinated. 0.1 gm cut five days old rice seedlings of various submergence dose of day 1, 2, 3, 4 and 5 along with control were evaluated separately in three replications for Adh enzyme estimation by standard protocols with spectrophotometer.
Results: The peak time of Adh enzyme synthesis was during 72 hours to 96 hour of submergence period. Among paddy genotypes Mahananda along with Sabita and Purnendu performed well in compare with tolerant check paddy genotypes.
Conclusion: As it is controlled by genetic factors, so estimation of Adh enzyme activity under submerged stage could be the potent biochemical marker for the screening for paddy genotypes for seedling stage of crop growth during flash flooding condition.

Keywords: Alcohol dehydrogenase enzyme, Rice, Submergence, Biochemical marker


Full Text:


Under flash flooding, few characters were identified as playing a key role in submergence tolerance in rice, the most critical are: maintenance of slow elongation of stem, maintenance of high carbohydrate concentration, retention of high chlorophyll percentages, optimum rates of alcoholic fermentation and energy conservation by maintaining low elongation growth rates during submergence (Jackson and Ram, 2003). Submergence tolerance is a metabolic adaptation in response to anaerobiosis that enables cell to maintain their integrity so that the plant survives hypoxia/anoxia without major damage (Sarkar et al., 2006). Rapid increase of water due to various reasons is very much detrimental for crop establishment where direct sowing is practices (Kawano et al., 2009). Tolerance to submergence stress during various stage of crop growth is also important aspect of submergence breeding (Ito et al., 1999; Mohanty et al., 2000). Tolerance to submergence stress during seedling stage of crop growth is very important for quick regeneration and initiation of new leaves after submergence (Setter et al., 1997). Alcoholic fermentation is the alternative metabolic process that plants seems to get adapted under submerged or oxygen free atmosphere (Green Way and Setter, 1996). Under anaerobic conditions aerobic respiration shift to a less efficient anaerobic fermentation to provide energy for survival (Green Way and Setter, 1996).  Alcohol dehydrogenase (Adh) involves in ethanol fermentation pathway that is responsible for the reduction of toxic acetylaldehyde to ethanol, resulting in continuous regeneration of nicotinamide adenine dinucleotide (NAD) in the cytoplasm (Chung and Ferl, 1999). Hence, induction of Adh can enhance survival of plants under flooded conditions (Johnson et al., 1994). Changes in enzyme activity levels have been noted within a day under hypoxic conditions and may occur more quickly under anoxic conditions (Keeley and Franz, 1979). Variation of Adh activity was increased from aerial to submerged condition (Chan and Burton, 1992). Adh activity under submerged condition reaches to an optimum level which provide sufficient energy level for better survival. Estimation of Adh activity could be used as a tool to differentiate tolerant and susceptible level.

So an attempt was done to estimate the alcohol dehydrogenase enzyme activity in paddy genotypes which could provide an aid for bio chemical marker for submergence tolerance. Hence an attempt was made to study the alcohol dehydrogenase enzyme activity of fifty diverse paddy genotypes for tolerance under complete submergence condition during seedling stage of growth.


Estimation of alcohol dehydrogenase enzyme: The Adh enzyme estimation was done by the modification of process of Tong, W.F. et al. 1989. The fresh and disease free seeds of fifty diverse rice genotypes were taken and germinated. Five days old rice seedlings was transferred to the test tube flooded with deionized water along with control. 0.1 gm cutted shoots of five days old rice seedlings of submergence dose of day 1, 2, 3, 4 and 5 along with control in separate set up with three replications were homogenated in a pre chilled mortar with 1.0 ml of 10 mM Tris-Hcl buffer solution (pH-7.6), containing 0.5mM zinc chloride, 0.5g polyvinylpyrolidone. The homogenate was centrifuged at 5600 x g for 20 mins, at -40C and the supernatant serve as a crude extract for assay of Adh activities. Adh activity was determined by modifying the method of Bonnichsen and Brink (1955). The assay mixture contained 0.1 M glycine- NaOH buffer (pH-9.0), 75mM ethanol and 0.26 mM NAD+. After the addition of enzyme solution to the mixture the initial rate of NAD+ reduction was measure at 340nm with spectrophotometer. One unit of Adh activity was defined as the amount which catalysed 1.0µ mole of NAD+ per min. The calculation of Adh activity was based on manuals of Worthington and Worthington (2011), Units/mL = (A340/min x Cuvette volume x Enzyme dilution) / (6.22 x Sample volume).

This experiment was carried out under laboratory condition at the Dept. of Genetics and Plant Breeding, Calcutta University during 2015-2016.


Table 1 represents an account of estimated value of alcohol dehydrogenase enzyme units/gm/min of fifty diverse rice genotypes of five days aerial condition and five successive days of submergence during seedling stage of growth. The mean value ranges from 42.59 – 98.41 Adh units/gm/min. It was IR64 sub1 showed maximum activities of Adh enzyme. In compare with tolerant checks, rice genotypes like Mahananda, Sabita and Purnendu performed well. From chart 1, 2 and 3, it was revealed that gradually alcohol dehydrogenase synthesis took place during submergence period than the aerial condition. During seedling stage of rice genotypes, the enzyme synthesis reached its peak around 3-4 days of submergence. After that the synthesis of Adh enzyme falls rapidly during 5th day of submergence.


The interesting feature is that rice genotypes like Mahananda, Sabita and Purnendu showed Adh units above from the FR43B tolerant check genotypes and below the other tolerant check genotypes. But these rice genotypes performed appreciably well in compare with all tolerant check genotypes. Among three well performed rice genotypes Mahananda is best followed by Sabita and Purnendu. The exhaustion of reserve food materials of the nascent rice seedlings was unable to maintain the Adh activity so long period. It may be the age which determines the reserve food matters and Adh activity during extreme anaerobiosis stress condition. During early seedling stage of growth these genotypes may be considered to be a good promise for cultivation of paddy for submerged prone areas where direct sowing is practiced.


Flash flood and heavy rain during just grown rice seedling stage specially for direct seed sowing condition is very much fatal to the crop. As the rapid increase of water causes total inundation of the nascent rice seedlings, survival during seedling stage is very much crucial for regeneration after submergence. The well established sub 1 incorporated rice mega varieties like IR64 sub 1 and Swarna sub 1 showed commendable Adh enzyme activity in the experiment. In compare with the sub 1 introgressed rice varieties, genotype like Mahananda, Sabita and Purnendu performed at par during seedling stage of growth. So these rice genotypes could be a good promise for early selection to be introduced into the submerged prone area where flash flood may occur. So screening of Adh activity could a good tool for differentiating submergence tolerant and susceptible rice genotypes during seedling stage of growth.


Authors acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. The authors are also grateful to authors / editors / publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.




  1. Bonnichsen, R.K. and Brink, N.G. (1955). Liver alcohol dehydrogenase. Methods Enzymology. 1: 495-500

  2. Chan, JWY. and Burton, RS. (1992). Variation in alcohol dehydrogenase activity and flood tolerance in white clover, Trifolium repens. Evolution. 46(3): 721-734

  3. Chung, H. J. and Ferl, R.J. (1999). Arabidopsis alcohol dehydrogenase expression in both shoots and roots is conditioned by root growth environment. Plant Physiology. 121: 429–436

  4. Greenway, H. and Setter, T.L. (1996). Is there anaerobic metabolism in submerged rice plants? a view point. In: Singh VP et al (ed) Physiology of stress tolerance in rice: Proceedings of the international conference on stress physiology of rice, IRRI. 11-30

  5. Ito, O., Ella, E. and Kawano, N. (1999). Physiological basis of submergence tolerance in rainfed lowland rice ecosystem. Field Crops Research. 64: 75–90

  6. Jackson, M. B. and Ram, P. C. (2003). Physiological and molecular basis of susceptibility and tolerance of rice plants to complete submergence. Ann. Bot. 91: 227–241

  7. Johnson, J. R., Cobb, B.G. And Drew, M.C. (1994). Hypoxic induction of anoxia tolerance in roots of Adh1 null Zea mays. Plant Physiology. 105: 61–67

  8. Kawano, N.; Ito, O. & Sakagami, J.I. (2009). Morphological and physiological responses of rice seedlings to complete submergence (flash flooding). Annals of Botany. 103(2):  161–169

  9. Keeley, J. E. and Franz, E.H. (1979). Alcoholic fermentation in swamp and upland populations of Nyssa sylvatica: temporal changes in adaptive strategy. American Naturalist. 113: 587–592

  10. Mohanty, H. K., Mallik, S. and Grover, A. (2000). Prospects of improving flooding tolerance in lowland rice varieties by conventional breeding and genetic engineering. Curr. Sci. 78: 132–137

  11. Sarkar, R.K., Reddy, J.N., Sharma, S.G. and Ismail, A.M. (2006). Physiological basis of submergence tolerance in rice and implications for crop improvement. Current Science. 91: 899–906

  12. Setter, T.L., Ellis, M., Laureles, E.V., Ella, E.S., Senadhira, D., Mishra, S.B., Sarkarung, S. and Datta, S. (1997). Physiology and Genetics of Submergence Tolerance in Rice. Annals of Botany. 79: 67-77

  13. Tong, W.F., Yu, J.J. and Lin, S.W. (1989). Flooding-mediated Induction of Alcohol Dehydrogenase in Rice Seedlings: Involvement of New Synthesis of Enzyme Molecules in the Induction. Biol Normal.24: 1-18

  14. Worthington, K. and Worthington, V. (2011). Worthington Enzyme Manual. Worthington Biochemical Corporation.