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Urea Induced Urease Activity Is Coupled to the Synthesis of Primary Monoamine: a Possible Mechanism of Enhancing Fertilization of Mango Flower

Md. Shahidul Haque*
Department of Biochemistry and Molecular Biology, Laboratory of Protein and Enzyme Research, University of Rajshahi, Rajshahi-6205, Bangladesh

Uttam Kumar Roy*
Department of Biochemistry and Molecular Biology, Laboratory of Protein and Enzyme Research, University of Rajshahi, Rajshahi-6205, Bangladesh

Farzana Ashrafi Neela
Department of Botany, University of Rajshahi, Rajshahi-6205, Bangladesh

Md. Monirul Islam
Department of Biochemistry and Molecular Biology, Laboratory of Protein and Enzyme Research, University of Rajshahi, Rajshahi-6205, Bangladesh

Md. Faisal Kabir
Department of Biochemistry and Molecular Biology, Laboratory of Protein and Enzyme Research, University of Rajshahi, Rajshahi-6205, Bangladesh

Abstract/ Executive Summary:

Mango is a delicious and nutritious fruit however its production is retarded by different ways although the mechanism is not clarified. In this study, we analyzed mango flower and examined the effect of urea on urease activity in flower extract. The flower had 12.0 g of protein, 1.21 g of lipid, 2.65 g of sugar, 0.01 g of reducing sugar, 4.01 g of starch, non reducing sugar 2.64 g in 100 g of flower and a moisture content is 77.15%. The higher urease activity in response to 100 mM urea was found in flower extract rather than 50 and 200 mM concentrations. The Km and Vmax were 62.5 mM and 0.04 µmole/mg of protein/min for the above doses respectively. Moreover, 100 mM urea was found to cause a higher synthesis of protein in the flower. Primary monoamine has been recognized to be the fertilization of flower and was found to increase whenever the flower extract was treated with 100 mM urea. Therefore, the results demonstrate that urea is involved in enhancing urease activity which is coupled to the synthesis of protein and the overall bioprocess might be linked to the higher synthesis of primary amine augmenting the higher fertilization of mango flower.

Keywords: Mango flower, Urease, Uptake of NH4 + , Primary monoamine, Fertilization of mango flower

Location:

Start Date:

End Date:

Program Area: Crop-Soil-Water Management

Commodity: Fertilizer, Mango

Objectives:

The current investigation has been designed to clarify the role of urea on the synthesis of primary monoamine in the mango flower and may induce fertilization in this plant.

Methodology:

Plant material Mango flower (Magnifera indica)(Langra) was collected from the mango garden located to the northern side of the University Campus during February-March. The flowers were quickly stored at –80 ^oC refrigerator. About 5–6 g of the flower were homogenized with mortar and pestle with 10 mM phosphate buffer (pH 7.0) and centrifuged at 6000 rpm for 10 min. After centrifugation, the supernatant was collected and used as crude extract while the sediment was discarded. Analysis of mango flower For determination of water soluble protein content from the flower extract, 5–6 g of mango flower were used and analyzed protein following the method of Lowry et al. (1951). Lipid content in flower was determined by the conventional method as described in Laboratory Manual in Biochemistry (Jayaraman, 1981) where 2 g of mango flower were used. Total sugar and reducing sugar content of mango flower was determined colorimetrically as described by Jayaraman (1981). Five to six g of flower were used for this assay. Non reducing sugar or sucrose content was calculated from the following formula: % sucrose or non reducing sugar = (% of total sugar – % reducing sugar) The starch content of the mango flower was determined by the anthrone method accordingly as described by Jayaraman (1981) where approximately 5 g of mango flower were used and the moisture content was determined by the conventional procedure in which 2 g of the flower were used for this determination. Assay of urease activity 5.0 g of mango flower was placed into a mortar with pestle and were homogenized with 30 ml of 0.2 M phosphate buffer (pH 7.0) and centrifuged at 6000 rpm for 10 min. The supernatant was collected and used as crude extract. The urease activity in crude extract of flower was assayed by the method as described by Jayaraman (1981). For assay of urease activity, 0.2 or 0.4 ml of the crude extract were used. To examine the effect of urea on urease activity, 50, 100 and 200 mM of urea were used as a substrate of the enzyme. The enzyme activity was expressed as µmole/min/mg of protein. Assay of protein content in flower extract Flowers (5 g) were homogenized with 10 mM phosphate buffer (pH 7.0) (30 mL) and were centrifuged at 6000 rpm for 10 min. The supernatant from flower homogenate were used as crude extract for assay of protein. 400 µL flower extract, 3.1 mL of phosphate buffer (pH 7.0) and 0.5 mL of 100 mM urea were taken in a test tube and was incubated with 55 oC for 15 min. After incubation, 1 mL 1N H2SO4 was added to the test tube to stop the reaction. For control, 400 µL flower extract, 3.6 mL phosphate buffer (pH 7.0), 1 mL of 1N H2SO4 were mixed in a test tube and was incubated with 55 oC for 15 min. After incubation, the above mixture of the above sample and control was determined by the procedure of Lowry et al. (1951). Briefly, alkaline solution was prepared by mixing 50 ml of alkaline Na₂CO₃ solution (2% Na₂CO₃ in 0.1N NaOH) and 1.0 mL of copper-sodium potassium tartarate solution (1 g sodium potassium tartarate and 0.5 g CuSO4. taken to the test tube and made up to 1 mL with distilled water. For blank, 1 mL water was used in place of tissue extract. 5 mL alkaline solution was added to each tube and mixed well. The tubes were allowed to stand for 10 min at room temperature and 0.5 mL of diluted FCR (Commercial FCR was diluted with an equal volume of water) was added and mixed well. After 30 min, the absorbance was taken at 650 nm against the blank. The protein content in flower extract was calculated from the standard graph of bovine albumin (1 mg/mL) and is expressed as g/100 g of flower weight. Test of primary monoamine 400 µL samples (5 g of flower in 30 mL solution) in a test tube was taken, mixed well with 0.5 mL urea (100 mM) and incubated at 55 ^oC for 15 min. After incubation, 1.0 mL diluted HCl and 4 drops of 10% NaNO₂ were added and cooled. In another test tube, a few drops of alkaline β-napthanol were taken and the mixture of the previous test tube was added slowly to the alkaline β-napthanol. An orange deep color was appeared showing the synthesis of primary monoamine in the flower sample. Control tube was similarly used for identification of primary amine where no urea was used and 1.5 mL diluted HCl were used and the color pigmentation was different from the urea induced sample tube.

Source of Information: Journal of Agricultural Technology 2014 Vol. 10(6):1537-1552

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Achievement/Findings:

Although different doses of urea were used in this study, however, the results reveal that 100 mM concentration plays the critical role on enhancing urease activity in mango flower extract. Urea induced urease activity was found to be co-related to the synthesis of protein. Because, the amino nitrogen derived from urea during catalysis of urease is metabolized to synthesize protein in cells of plants. The increase activity of urease shows the higher uptake of urea in the flower because higher concentration of NH4 + is formed in response to urea. Primary monoamine has been believed to be involved in fertilization of mango flower and other protective functions. The higher color pigmentation was demonstrated in response to 100 mM urea treatment during the assay showing the higher synthesis of primary monoamine in flower induced by urea thereby causing fertilization and fruiting.

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