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Ubaydullaeva Khurshida Abdullaevna, Center of Genomics and Bioinformatics of Uzbek Academy of Science, Tashkent region E-mail: hurshida_70@mail.ru Kamburova Venera Seytumerovna, Center of Genomics and Bioinformatics of Uzbek Academy of Science, Tashkent region E-mail: venera_k75@mail.ru Nematov Sherzod, Tashkent State Technical University named afer Islam Karimov, Tashkent, Uzbekistan E-mail: shnematov@gmail.com 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: zabar75@yahoo.com
STUDY OF CARBON AND NITRATE EXCHANGE IN COTTON GENOTYPES OBTAINED BY "PHYA1-RNAi" TECHNOLOGY
Abstract: The study of photosynthetic and nitrate reductase activities of cotton "Cocker 312" (control) and its "T6-1-7" line, obtained by silencing phytochrome A1 gene, using gene-knockout technology, has shown the positive correlation between the tested parameters in both genotypes. At the same time, higher potential of photosynthetic apparatus was established in gene knock-out plants. The nitrate-reductase activity in leaves exceeds at least 34% the control genotypes at the late stages of cotton ontogeny.
Keywords: cotton, photosynthesis, PHYA1, RNA interference (RNAi).
RNA interference (RNAi) is an "innovative technology" nitrate reductase activities of two cotton (Gossypium hirthat helps to determine the functions of agricultural genes, sutum) representatives: "Coker 312" variety (control) and and thus solve the problems of cotton growing by creating a "T6-1-7" line, obtained by transformation of the above culti-"biotechnological" cotton varieties with suppression of un- var using RNA interference technology. RNAi line contains wanted genes, but improved expression of the desired trait the construct which suppresses expression of the phyto-(s). Currently, RNAi is used for functional studies of many chrome A1 gene in cotton [7].
agronomically, biologically and physiologically significant Material and methods. Determination the photosyn-
cotton genes associated with the development of cotton fiber thetic activity of cotton genotype accessions was carried (Gossypium spp.); early maturation and flowering; increased out by method, based on the measurement of parameters of yield; fertility and embryogenesis; resistance to viral, fungal fluorescence of chlorophyll at excitation by pulse-amplitude-diseases and insects; resistance to various abiotic stresses; and modulated light by means of - MINI-PAM PAM- fluorim-improving the quality of seeds and oil [1, 2]. eter Watlz (Germany). The fluorimeter has such advantages
However, detailed molecular intracellular mechanisms as the informativity, expressiveness, preservation nativity of responsible for the realization of RNA interference effects the sample after measurement and high sensitivity [8]. Pho-are rarely established [3-7]. At the same time, photosynthe- tosynthetic efficacy was estimated by parameter "YIELD" sis is one of the main physiological processes whose activ- (the quantum yield of photochemical processes in photo-ity affects the plants yield. In this context, the purpose of system II) which was calculated by formula: this work was a comparative study of photosynthetic and YIELD = (F'M - F)/F'M),
where, F - is the fluorescence intensity at a given time, and F'M - maximum intensity, which most fully characterizes the efficiency of the primary, light phase of photosynthesis [8, 9].
Assessment of nitrogen status of cotton genotypes was determined by nitrate reductase activity (the NRA), main enzyme that reduces nitrate to ammonium - the characteristic form of nitrogen in organic substances (amino acids, nucleotides, proteins etc.) [10].
Results and discussion. The analysis of the photosynthet-ic activity of the studied genotypes at different stages of vegetative growth has shown that, since 70-75 day of vegetation (with emergence and developing of reproductive organs) the average "YIELD" value of the gene-knockout cotton plants was reliably high (2.5%) related control genotypes, that testifies of higher assimilation (acceptor) activity of their leaves.
The highest value of the "YIELD" parameter at both genotypes has fallen on July 22-25 (approximately 97-100
day plants), followed by the expected plant ageing related to regression of this parameter. Moreover, how it shown in Fig. 1, in the bearing stage of cotton plants the "YIELD" value of gene-knockout line was 12% lower, than in Cocker 312 control genotypes (Fig. 1).
Accordance to the physiological life cycle of cotton plants the decrease of leaves photosynthetic activity at the later stages of ontogeny is followed by catabolic processes activation. As a result it must be followed by an outflow of synthesized nutrients from leaves to the developing fruit. The understating revealed by us (concerning control) "YIELD" values in gene - knockout lines at late stages of ontogenesis can be result of earlier shift at them acceptor - donor relationship towards the developing fruit. Not accidentally that the "T6-1-7" line, as well as others PHYA1-RNAi cotton genotypes, are differ both high productivity and also early maturity.
Figure 1. The dynamics of alteration 'YIELD" parameter of "T6-1-7" line (■) and "Coker312" (I) during tested period
PAM-fluorimetry is one of advanced opportunity to determine not only the maximum quantum yield of photosynthesis but also effective quantum yield of photosynthesis (determined after the adaptation of plants to darkness) which allows to calculate the depth of the fall of the quantum yield of photosynthesis caused by the phenomenon of "photo-inhibition" (Fig. 2).
Above parameters are permissive to discuss how effective solar power utilized by plants. It is considered that the basis of "photo-inhibition" has decreased the photo-chemical activity of chloroplasts - initially without significant degradation of pigments and membrane proteins, and after prolonged action of high intensity light, especially in the stress conditions (temperature, lack of water, etc.). Photo-inhibition is followed by photooxidation of pigments, destruction of chloroplasts structures and other irreversible changes [9, 11].
Comparisons of the "YIELD" parameter have shown that during all of daily testing period "T6-1-7" line has reliable higher "YIELD" value than "Cocker-312" - the control genotype (Fig. 2). It means that the gene-knockout cotton plants are more resistance to photo-inhibition and therefore "the effective quantum yield of photosystem II" of these plants is higher in comparison to the genotype "Coker 312".
It is pertinent to note that one of the noticeable phe-notypic characteristics of "T6-1-7" line, as well as other PHYA1-RNAi cotton genotypes (set of Porlock cultivars), is saturated pigmentation of the leaves - "anthocyanin suntan", which appears not only in closer to the autumn (senescence), but is present in the leaves of these genotypes throughout the entire physiologically active period of plants [7]. Anthocyanins were known as a substance par-
ticipating in oxidation-reduction reactions long time ago. According A. E. Solovchenko and M. N. Merzlyak (2008) the presence of both anthocyanins and carotenoids in high concentrations in certain tissues and cell compartments is capable to prevent damage of chlorophyll and other poten-
tial photosensitizers [12]. Probably, the high content of pigments in leaves of the gene-knockout cotton genotype makes them more resistant to photo-inhibition, providing thereby a rather high potential of theirs photosynthetic apparatus in comparison with initial genotype.
Figure 2. Daily change curves of cotton line T6-1-7 (■) and cultivar "Coker-312" (■) 'YIELD" parameters The investigation of nitrate reductase activity of tested activity - 98th plant growing day. The enzyme value corre-
cotton genotypes has shown a definite correlation between there leaves nitrogen status and photosynthetic activity. The higher value of nitrate reductase activity was determined approximately at the same date, as the peak of photosynthetic
sponded to 2090.3 nmol nitrite ions (NO2-), produced by "Coker-312" leaves tissue during 1 hour of incubation time (per mg of protein) and 2032.6 [nmol NO2- (mg protein, hour) -1] - produced by "T6-1-7" genotypes (Table 1).
Table 1.- Age -related changes of nitrate reductase activity in leaves of "Coker 312" cotton cultivar u "T6-17" RNAi line, [nmol NO2- (mg protein * hour)-1]
Genotypes Age of plants, days
86 98 116
Coker-312 1371 2090.3 1767.86
T6-17 1472 2032.6 2677.08
With the age of plants, nitrate-reducing activity of the leaves, as well as their photosynthetic activity, was decreased, amounting in "Coker 312" enzyme value of 1767.86 [nmol NO2 - (mg protein, hour) -1] and in "T6-1_7" gene-knockout genotype- 2677.08 [nmol NO2(mg protein, hour) -1] for (table 1). Comparison among themselves above two tested genotypes in this parameter has shown that at late stages of ontogenesis, nitrate reductase activity of leaves of T6-17 line significantly (for 34%) exceeds ones of control genotypes.
Although cotton fiber almost does not contain nitrogen, there is a lot of nitrogen in cottonseeds. Thus, according to
Tarp (1960), seeds of one bale of raw cotton contain about 16 kg of nitrogen [13], the mobilization of which in seeds is ensured by the coordinated functionalization of nitrate transportation and nitrate reducing systems.
Thus, increasing productivity of cotton PHYA1-RNAi lines, observed in this work, apparently, is integrated expression of the developed/elongated root system, saturated pigmentation, effective use of light quant and high nitrate reductase activity of their leaves at the bearing stage of cotton plant.
References:
1. Abdurakhmonov I. Yu. RNA Interference - A Hallmark of Cellular Function and Gene Manipulation // In RNA interference. In-Tech.- 2016 a.- P. 1-18.
2. Abdurakhmonov I. Yu., et al. RNA interference for functional genomics and improvement of cotton (Gossypium spp.) // Frontiers in Plant Science. 7.- 2016 b.- 202 p.
3. Hao J., et al. GbTCP, a cotton TCP transcription factor, confers fiber elongation and root hair development by a complex regulating system // J Exp Bot. 63.- 2012.- P. 6267-81.
4. Tan J., et al. A genetic and metabolic analysis revealed that cotton fiber cell development was retarded by flavonoid narin-genin // Plant Physiol. 162.- 2013.- P. 86-95.
5. Gao X., et al. Cotton GhBAK1 mediates Verticillium wilt resistance and cell death // J Integr Plant Biol. 55.- 2013.-P. 586-96.
6. Zhang X., et al. Genome-wide identification of mitogen-activated protein kinase gene family in Gossypium raimondii and the function of their corresponding orthologs in tetraploid cultivated cotton // BMC Plant Biol. 14.- 2014.- 345 p.
7. Abdurakhmonov I. Y., et al. Phytochrome RNAi enhances major fibre quality and agronomic traits of the cotton Gossypium hirsutum L. // Nat. Commun. 5.- 2014.- 3062 p.
8. Khabibullaev P. K., et al. Evalution of the effects of drought on cotton plants using characteristics of chlorophyll fluorescence // Proceedings of Biological Sciences - MAIK. 392.- 2003.- P. 442-444.
9. Valikhanov K. M., et al. Monitoring of photoinhibition plant leaves using prompt and delayed fluorescence // Indian Journal of Plant Physiology. 8.- 2003.- P. 144-148.
10. Hageman R. H., Hucklesby D. P. Nitrate reductase from higher plants // Methods Enzymol. 23.- 1971.- P. 491-503.
11. Valikhanov K. M., et al. Kinetics of photoinhibition and delayed fluorescence in the plant photosynthetic system // Report ofAcad. Sci.- M. 38 (6).- 2002.- P. 331-334.
12. Solovchenko A. E., Merzlyak M. N. Screening of visible and UV radiation as a mechanism of photoprotection in plants // Physiology of plants. 55 (6).- 2008.- P. 803-822.
13. Tharp W. H. The cotton plant. How it grows and why its growth varies. US Department of Agriculture. Agriculture Handbook - No. 178.- 1960.
