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Ubaydullaeva Khurshida Abdullaevna, Center of Genomics and Bioinformatics of Uzbek Academy of Science, Tashkent region E-mail: [email protected] Kamburova Venera Seytumerovna, Center of Genomics and Bioinformatics of Uzbek Academy of Science, Tashkent region E-mail: [email protected] Adylova Azadahan Teshabaevna, Center of Genomics and Bioinformatics of Uzbek Academy of Science, Tashkent region Buriev Zabardast Tojibaevich, Center of Genomics and Bioinformatics of Uzbek Academy of Science, Tashkent region E-mail: [email protected]
NITRATE REDUCTASE ACTIVITY OF RNAi-BASED COTTON VARIETIES
Abstract: It has been examined nitrate reductase activity in different vegetative organs of cotton "Cocker 312" (control) and gene knock-out line, obtained by silencing phytochrome A1 gene, at different time of day and ontogenesis phases. The results of study has shown that nitrate-reductase activity in leaves of RNAi-based varieties exceeds at least by 34% the control genotypes at the late stages of cotton ontogeny. Keywords: cotton, nitrate reductase, PHYA1, RNAi.
Nitrate is the main source of nitrogen for the most higher plants. It is converted into ammonium ions by action of the enzymes nitrate reductase (NR). It is in the form of ammonium ions that is contained in amino acids, proteins, nucleotides. ATP and NADH+, generated during photosynthesis, are required for the functioning of the nitrate reductase. In turn, glucose synthesized in plants during photosynthesis, and pyruvic acid, which is the final product of carbohydrate decomposition, is the starting material for the biosynthesis of amino acids.
So the carbon and nitrogen cycles of plants are inextricably linked to each other. Usually, photosynthetic activity positively correlates with the nitrogen content in leaves, mainly because the synthesis of the photosynthetic apparatus is broken without nitrogen. And more, one of the most frequent consequences of nitrogen deficiency is changing in the amount and activity of key enzyme of photosynthesis - Rubisco [1].
The purpose of the presented work was a comparative study of nitrate reductase activities of two cotton (Gossypium hirsutum) representatives: 'Coker 312' variety (control) and "T6-1-7" line, obtained by transformation of the above cultivar using RNA interference technology. RNAi line contains the construct which suppresses expression of the phytochrome A1 gene in cotton [2].
Material and methods. Assessment of nitrogen status of cotton genotypes was determined by nitrate reductase activ-
ity (the NRA), main enzyme that reduces nitrate to ammonium - the characteristic form of nitrogen in organic substances (amino acids, nucleotides, proteins etc.) [3].
To determine the activity of nitrate reductase and the content of nitrates in plant tissues, a sample of 500 mg of leaf tissue (diameter ~ 4 mm) cut from 4 parallel leaves (each of which is collected from an individual plant) from sections of the leaf blade that do not contain large conductive veins. Samples were poured into 10 ml of a standard incubation solution [4] containing 50 mM KNO3, 100 mM potassium phosphate buffer, pH 7.5, 1% (v / v) 1-butanol, and incubated under anaerobic conditions (vacuum infiltration) for 30 min at 30 °C, in the darkness with constant stirring. After the incubation time, zinc acetate was added to aliquots of each sample (to a final concentration of 50 ^mol) [5], the samples were boiled for 2-3 minutes (for inactivation and subsequent conversion of NO2-ions to gases - N2O and NO), cooled, centrifuged at maximum RPM (Eppendorf), and then the content of nitrite (NO2) released into the incubation medium was determined using a Griss reagent [3].
The extracts evaluated on the nitrate content by reaction with salicylic acid (Cataldo et al., 1975). 0.4 ml of a freshly prepared 5% solution of salicylic acid in concentrated H2SO4 was added to 0.1 ml of the extract obtained as described above. The formation of the colored complex was continued for 20
min at room temperature. The coloration was formed in alkaline medium, so 9.5 ml of 2 N NaOH was added and measured at 410 nm on a suitable spectrophotometer. Solution in which salicylic acid was replaced with concentrated H2S04 was used as a control. Calculations were carried out according to a calibration schedule constructed according to KNO3.
Results and discussion. At the first stage, we studied the nitrate reductase activity of the vegetative organs of cot-Table 1.- Potential activity of nitrate reductase (NR)
ton. As a result, it is shown that assimilation of nitrates was carried out in all vegetative the organs of the plant irrespective of the variety. The study of the activity of the enzyme in the vegetative organs of cotton showed that the main role in the process of reduction of nitrates was assumed by the leaves. At the same time, in the roots of plants, not more than 10% of nitrates were reduced from their total amount of plants restored (Table 1). in vegetative organs of different varieties of cotton
Varieties NR, ^mol/h*g
Leaves Stems Roots
Cocker-312 15.37 ± 0.25 1.55 ± 0.08 1.19 ± 0.09
An-Bayavut-2 14.30 ± 0.21 1.57 ± 0.06 1.89 ± 0.13
^6524 17.58 ± 0.23 1.51 ± 0.07 1.76 ± 0.11
Tashkent-6 16.30 ± 0.22 1.59 ± 0.05 1.32 ± 0.10
Namangan-77 14.56 ± 0.22 1.40 ± 0.06 1.09 ± 0.08
Studies of the dynamics of the nitrate reductase activity in ontogenesis were also carried out. The results of experiments carried out on the control plants of Cocker312 were shown that the dependency diagrams of the enzyme activity on the ontogenesis phase has a dome-like character: the activity rises from the emergence phase, reaching a maximum in the budding and flowering phase, and then gradually decreases
(Table 2). This fact can be explained by the increase in the overall physiological activity of the plant from the seedling phase to the flowering phase and, consequently, the increased demand for nitrogen. The decrease in the activity of nitrate reductase to the maturation phase can be explained by the attenuation of physiological processes in the plant as a whole, and as a consequence, by a decrease in the demand for nitrogen nutrition.
Table 2.- Potential nitrate reductase activity in Coker-312 cultivar according to stages of plant development
Stage of development NR, ^mol/h*g
Leaves Stems Roots
Seedling 9.4 ± 0.15 - 0.59 ± 0.05
First real leaf 11.4 ± 0.18 0.94 ± 0.07 0.75 ± 0.07
Budding 15.37 ± 0.25 1.55 ± 0.08 1.19 ± 0.09
Flowering 13.6 ± 0.19 1.12 ± 0.09 0.84 ± 0.06
Maturation 10.6 ± 0.13 0.91 ± 0.03 0.62 ± 0.07
Figure 1. The nitrate reductase activity in Coker-312 variety depending on the time of day. Notes: on the abscissa - time after sunrise, in hours; the ordinate shows the activity of nirate reductase, in ^mol/h * g
At the same time, the rate of accumulation of nitrates and their reduction as well as depending on the time of day were investigated. According to the results of the study, a clear cir-cadian rhythm of the enzyme is traced: the minimum rate of reduction of nitrates was observed in the dark, with the onset of the day the activity of the enzyme increased, reaching its maximum in 5-6 hours after sunrise (Fig. 1).
In addition, the comparative activity of the enzyme in control and gene-knockout varieties of cotton was studied.
Table 3 shows the values of nitrate reductase activity in the leaves of the initial variety samples and their gene knockout derivatives containing the PHYA1-RNAi insertion, whose presence in the genome of the gene-knockout varieties was confirmed by PCR (results not shown) [2].
As can be seen from the presented data, in the "AN-Baya-vut-2" variety, the nitrate reductase activity during the testing varied from 11.71 ^moles (minimum value) to 17.82 ^moles (maximum), averaging 14.30 ^moles.
Table 3.- Nitrate reductase activity [^moles of NO2- /g *h) leaves of RNAi-based varieties of cotton in comparison with their original varieties
Genotypes Date of analysis* Arithmetical mean M ± m
I II III IV
An-Bayavut-2 11.71 ± 0.18 12.91 ± 0.20 17.82 ± 0.24 14.74 ± 0.22 14.30 ± 0.21
Porloq-1 18.05 ± 0.22 12.11 ± 0.20 17.82 ± 0.23 22.11 ± 0.25 17.52 ± 0.22
C-6524 14.45 ± 0.20 19.14 ± 0.23 20.39 ± 0.26 16.34 ± 0.22 17.58 ± 0.23
Porloq-2 15.31 ± 0.21 19.71 ± 0.23 21.37 ± 0.25 22.05 ± 0.27 19.61 ± 0.24
Tashkent- 6 11.37 ± 0.19 18.80 ± 0.21 21.42 ± 0.25 13.60 ± 0.23 16.30 ± 0.22
Porloq -3 27.47 ± 0.26 22.00 ± 0.22 12.51 ± 0.17 10.05 ± 0.15 18.00 ± 0.20
Namangan-77 11.99 ± 0.17 18.91 ± 0.23 13.32 ± 0.19 14.02 ± 0.21 14.56 ± 0.20
Porloq -4 15.19 ± 0.21 18.40 ± 0.23 21.53 ± 0.25 19.19 ± 0.23 18.58 ± 0.22
* I -Jule, 10, II - Jule, 15, III - Jule, 21, IV- Jule, 25
The average mean "M" of nitrate reductase activity in the Porloq-1 gene-knockout cotton variety is 17.52 ^moles, which is 22.94% higher than the original parent of the An-Bayavut-2. Similar parameters for the C-6524 variety are represented by the figures 14.45 and 20.39 ^moles, respectively, with a mean arithmetic value of "M" of 17.58 ^moles, while its gene-knockout form - Porloq-2 the indicator corresponds to 19.61 ^moles, which is 11.55% higher in comparison with the original genotype "C-6524". The average arithmetic mean "M" in Porlock-2 was the highest among all tested genotypes (Table 3). Strong differences in enzymatic activity were noted in the Tashkent-6 - Porlock-3 pair. Nitrate reductase activity in 79 day-old Porlock-3 plants was 27.47 ^mol versus 11.37 ^mol in Tashkent-6 on the same dates of the analysis. With increasing age of the plant, nitrate reductase activity in Porlock-3 gradually decreased, in Tashkent-6, on the contrary, it grew, reaching a maximum value of 21.42 ^mol in 90 day-old plants, followed by a drop to 13.60 ^mol (table, group III). Nevertheless, the average value of "M" nitrate reductase activity in the gene-knockout grade Porlock-3 was 10.43% higher than in Tashkent-6. And, finally, in the Namangan-77 variety, the minimum value of nitrate reductase activity was 11.99 ^moles, the maximum
value was 18.91 ^moles, with the value of "M" equal to 14.56 ^moles, which is 27.61% lower than the gene knockout form Porlock-4.
It is well known that the assimilation of nitrates by plants is a very complex process that depends on the species and biological characteristics of plants and is subject to significant influence of environmental external factors: plant nutrient conditions, illumination, soil pH, temperature, salt concentration in soil and nutrient medium, irrigation, i. e. [6-8]. Both in nature and in the laboratory condition they do not act on their own, but in complex. In this case, the enzymatic activity of nitrate reductase in plants is a balance between the relative rate of synthesis / degradation and the activation / inactivation of the enzyme, the factors can affect the overall activity of the enzyme by interfering with any of these processes [9].
Thus, a higher level of nitrate reductase activity in all cotton gene-knockout forms Porloq in comparison with the initial varieties (An-Bayavut-2, C-6524, Tashkent-6 and Na-mangan-77) allows to suggest that the insertion into the cotton genome PHYA1-RNAi vector induces systems involved in the transport and utilization of inorganic nitrogen fertilizers, thus increasing the overall yield of gene-knockout varieties.
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