THE INFLUENCE OF ABSCISIC AND INDOLE-ACETIC ACIDS ON THE ACTIVITY ASCORBATEPEROXIDASE OF COTTON UNDER SALINITY

ABSTRACT

This article examines the interaction of phytohormones with the antioxidant system by studying the exogenous effects of abscisic and indole-acetic acids Porlok-4 cotton variety under salinity conditions. Comparative studies of interactions of abscisic and indole-acetic acids in the activation of ascorbateperoxidase in roots of biotechnological cultivar Porlok-4 demonstrated that the speed of formation of responses depended on duration of the salt stress effect and concentration of NaCl in the medium. Increase in the APX activity under exogenous effect of phytohormones speaks of starting the systemic protection from negative effects of reactive oxygen spicies forming under high salt concentrations not entirely forclose the participation in transduction of signals causing expression of protective proteins.

АННОТАЦИЯ

 В данной статье рассматривается взаимодействие фитогормонов с антиоксидантной системой путем изучения экзогенных эффектов абсцизовой и индолилуксусной кислот сорта хлопчатника Порлок-4 в условиях засоления. Сравнительные исследования взаимодействия абсцизовой и индолилуксусной кислот при активации аскорбатпероксидазы в корнях биотехнологического сорта Порлок-4 показали, что скорость формирования ответных реакций зависит от длительности воздействия солевого стресса и концентрации NaCl в среде. Повышение активности АПО при экзогенном воздействии фитогормонов свидетельствует о запуске системной защиты от негативного воздействия активных форм кислорода, образующихся при высоких концентрациях соли, не полностью исключая их участие в передаче сигналов, вызывающих экспрессию защитных белков.

Keywords: salinity, phytohormone, abscisic acid, indole-acetic acid, ascorbateperoxidase, cotton.

Ключевые слова: засоление, фитогормон, абсцизовая кислота, индолилуксусная кислота, аскорбатпероксидаза, хлопчатника.

INTRODUCTION. Salinity, drought and low temperature are the common forms of abiotic stress encountered by land plants. To cope with these adverse environmental factors, plants execute several physiological and metabolic responses. Among abiotic stresses, high salinity is the most severe environmental stress, which impairs crop production on at least 20 % of irrigated land worldwide. In addition, increased salinity of arable land is expected to have devastating global effects, resulting in up to 50 % land loss by the middle of 21st century. All these forms of abiotic stresses result in both general and specific effects on plant growth and development, reduced productivity and significant crop losses [1].

Phytohormones play a crucial role in the growth and development of plants by regulating many processes. Phytohormones may enhance stress tolerance and minimize the yield loss of plants caused by abiotic stress [2]. Plant growth and development are regulated by internal signals and by external environmental conditions. One important regulator that coordinates growth and development with responses to the environment is the sesquiterpenoid hormone abscisic acid (ABA) [3]. ABA is considered as a stress phytohormone playing a key role in the plant stability to the effects of adverse abiotic and biotic factors. In particular, its effect applies to practically all processes of cell metabolism. ABA plays an extremely significant role in plant response to the dehydration, salinization and effects of temperature. Various studies have determined the roles of ABA and indole-acetic acid (IAA) in plants under stress conditions [4].

Thus, this stage of our work aimed at studying effects of phytohormones, to name, ABA and IAA on antioxidant responses of biotechnological cotton variety Porlok-4 under short-and long-term exposure to various concentrations of NaCl.

Materials and methods

Cotton plants of gene-knockout cultivar G. hirsutum Porlok-4 with suppression of the PHYA1 gene were also used, which shortens the growing season of plants [5,6]. Seeds of the gene-knockout cotton cultivar, such as Porlok-4, was provided by the Center of Genomics and Bioinformatics, Uzbekistan Academy of Sciences. Seeds of the cotton cultivar under study were denuded in concentrated sulfuric acid, washed under cold running water for 15 minutes and kept in the distilled water for 12 hours [7]. The swollen seeds wrapped in the paper rolls were germinated in a dark wet chamber for 7 days at 27°C.

To get the enzymatic extract a sample of tissue (500 mg) previously frozen in the liquid nitrogen was ground in a cold porcelain mortar with addition of appropriate extraction buffer (0.1 M Na-phosphate buffer pH 7.0 containing 20 mM EDTA, 2 mM PMSF, 1% triton X, 150 mM PVP) in 1:10 ratio. The homogenate was centrifuged at 8000 g for 15 min at temperature not higher than 4°C. The APX was assayed according to Nakano and Asada [8]. Lowry’s method was used for protein measurement [9].

We used concentrations of NaCl as low, 172 mM and high, 862 mM limits of salt stress to identify stress-adaptive effects of phytohormones. The 7-day-old seedlings of cotton cultivars were separately placed in the vessels containing indole-acetic acid (IAA) and abscisic acid (ABA) in the concentration of 10^-7 M, those containing respective salt solution in respective concentrations separately and jointly with the phytohormones with 1-hour and 24-hour exposure. The control seeds were germinated in water.

Results and Discussion

Same as other antioxidant enzymes and low molecular weight antioxidants, APX participates to the maintaining cell redox balance and necessary level of the reductive elements, to name glutathione and ascorbate. APX splits Н2О2 with necessary level of ascorbate.

Comparative study on interaction of phytohormones in the APX activation in the roots of Porlok-4 seedlings demonstrated that under effect of 172 mM NaCl, the APX activity in 24 hours was 1.5 times lower than the one after 1-hour exposure (Fig.1).

In roots of seedlings placed in the ABA solution and under combined effect of 172 mM NaCl + ABA within the first hours after their effect starting, the APX activity was found to increase by 2.4 and 1.2 times as compared to the control, respectively. Some differences in responses of the APX activity to the combined effect of 862 mM NaCl + ABA were found; within the first hours after their effects starting, the enzyme activity could be seen to reduce by 2.4 times, after 24- hour exposure it was found to increase by 3.3 times.

Figure 1. Integrated effects of  ABA (a), IAA (b) and salt stress (1hour and 24 hour) on the activity of ascorbateperoxidase in roots of the 7-day seedlings of Porlok-4 cotton cultivar

The activity of APX under effect of IAA and combined effect IAA with 172 mM and 862 mM NaCl after 1-hour exposure was of ambiguous character; thus, 172 mM NaCl + IAA > IAA > 862 mM NaCl + IAA where the enzyme activity was lower than the control one by 1 > 1.3 > 1.6 times, respectively. After 24 hours of exposure the APX activity decreased; thus, IAA > 172 mM NaCl > 862 mM NaCl + IAA where the enzyme activity was lower than the control one by 1.8 > 2.6 > 2.8 times, respectively.

APX splits Н2О2 with presence of necessary level of ascorbate; consequently, under short salt stress the hydrogen peroxide spike suppressed the APX activity, and additional synthesis of antioxidant compounds is known to require time. Combined effects of 862 mM NaCl and ABA under 24-hour exposure promoted activation of the enzyme slowing the hydrogen peroxide spike down and decreasing the oxidation processes. IAA is a phytohormone of growth; both in its presence and in combinations under prolonged salt stress, the APX activity increases to be the evidence for stimulation of growth processes in cotton, respite high concentrations of salts. In some variants of experiment, under combined effects of IAA and ABA with salinization, the APX activity decreased. The reduction in the APX activity under salt stress could be associated with its high specificity to ascorbate quickly losing activity with its absence. Probably, effects of ABA and IAA on the cotton seedlings are determined by the quantities of ascorbate in the salt stress.

Thus, our findings demonstrated a specific sequence in starting of protective mechanisms under salt stress. At the second stage (24 hour), efficacy of antioxidant systems increased due to increased activity of APX. Thus, successive starting of protective mechanisms with participation of ABA in response to increments of salt stress prevents oxidative stress in the cotton roots.

References:

1. Aryadeep R., Saikat P., Supratim B. Cross-talk between abscisic acid-dependent and abscisic acid-independent pathways during abiotic stress // Plant Cell Rep (2013) 32:985–1006 DOI 10.1007/s00299-013-1414-5
2. Liming X., Jian-Kang Z. Regulation of Abscisic Acid Biosynthesis // Plant Physiology, September 2003, Vol. 133, pp. 29–36.
3. Ilias I, Ouzounidou G, Giannakoula A, Papdopoulou P (2007) Effect of gibberellic acid and prohexadione-calcium on growth, chlorophyll fluorescence and quality of okra plant. Biol Plant 51:575–578.
4. Amzallag GN, Lener HR, Poljakoff-Mayber A (1990) Exogenous ABA as a modulator of the response of Sorghum to high salinity. J Exp Bot 541:1529–1534.
5. Pat. NAP 20130014, Cotton cultivar Porloq-1, Abdurakhmonov, I. Y., Buriev, Z. T., Abdukarimov, A., Ubaydullaeva, K. A., Makamov, A. H., Shapulatov, U. M., Darmonov, M. M., Turaev, O. Y., Ruziboev, H. S., Publ. 15.11.2013.
6. Pat. NAP 20130017, Cotton cultivar Porloq-4. Abdurakhmonov, I. Y., Buriev, Z. T., Abdukarimov, A., Ubaydullaeva, K. A., Makamov, A. H., Shapulatov, U. M., Darmonov, M. M., Turaev, O. Y., Ruziboev, H. S., Publ. 15.11.2013.
7. Babaeva, D. T., Esanov, R. S., Akhunov, A. A., Gafurov, M. B., Khashimova, N. R. & Matchanov A. D. (2020). Biological activity of the supramolecular complex of glycyrrhizic and salicylic acids. Chemistry of Natural Compounds, 56:278-281. https://doi.org/10.1007/s10600-020-03006-1
8. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with Folin phenol reagent. Journal of Biological Chemistry, 3(1): 265-275.
9. Nakano, Y. and Asada, K. 1981. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiology, 22:867-880