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ВЕТЕРИНАРНЫЕ НАУКИ
УДК 2788
*Alikulov Z.
Associate Professor, Department of Microbiology and Biotechnology of the Eurasian National University. L.N.Gumilev E-mail: [email protected] *Shukhatova A., Undergraduate of the Eurasian National University. L.N.Gumilev E-mai:[email protected] 2Shalakhmetova G., Associate Professor, Department of Microbiology and Biotechnology of the 2The Al-Farabi Kazakh National University, Almaty
NITRATE REDUCTASE IN DETACHED EMBRYOS MAY SERVE AS A MARKER OF THE PREHARVEST TOLERANCE OF WHEAT SEEDS
Abstract
We have observed an easily determinable parameter indicative of genetic pre-harvest sprouting (PHS) tolerance - the presence of an endosperm factor, presumably ABA, capable of inhibiting nitrate reductase (NR) induction in the embryo in the presence of NO3". This finding has importance not only for the early rapid screening of PHS tolerant cultivars but may also be an important tool to determine the mechanism of NR inhibition either by genetic repression or by post-translational down regulation of NR activity. In this work we sought a simple relationship between ABA content and NR activity level that we assumed to be closely related to PHS susceptibility.
Introduction. Precocious germination of wheat grain is a serious problem in wheat production. The phenomenon of germination of physiologically mature cereal grains in the ear or panicle, usually under wet conditions shortly before harvest, is termed as pre-harvest sprouting (PHS) or vivipary. PHS occurs in many cereal crops such as wheat, barley, maize, and rice in most region of the world. PHS not only causes reduction of grain yield, but also affects the quality of grains, resulting into significant economic losses. Therefore the physiological, genetic, and environmental basis of PHS susceptibility in wheat have been a subject of intensive research during the past three decades [1]. It has been shown that mutations of genes in synthesis of the carotenoid precursors of ABA resulted in the pre-harvest sprouting, which is consequence of ABA deficiency. The phenomenon is due to the lack of ABA in the young seeds and/or their insensitivity to the dormancy-inducing hormone [2].
Materials and methods
Experiments to determine the activities of Mo-enzymes as early markers of PHS were carried out in PHS tolerant (Lutescence 70) and sensitive (Novosibirskaia 67) wheat varieties during seed germination. Wheat seeds of these varieties were obtained from the N. Baraev Cereal Research Institute at Shortandy (Kazakhstan). Preparation of seed tissue extracts and the activities of molybdoenzymes, aldehyde oxidase (AO), xanthine dehydrogenase (XDH) and nitrate reductase (NR) in the aleurone layers, endosperms and embryos were determined according to [3, 4].
Results and discussion
Mo-enzyme activities in the parts of dry wheat seeds. The activities of NR XDH and AO were determined in different parts of dry dormant seeds - the embryo, endosperm and aleurone layer. The embryo and aleurone layer extracts showed XDH and AO activities while these activities were not detected in the endosperm (Table 1). Dry seeds were incubated in distilled for 24 h, after which only the embryo showed no NR activity. NR activity developed in seed embryos only after incubation in the presence of KNO3 and its levels in embryos of both wheat varieties were nearly the same (not shown). Early results showed that NR-antibodies did not cross-react with protein of extracts of wheat seed embryos, endosperm and aleurone layer [4].
Международный научный журнал «СИМВОЛ НАУКИ»_№4/2015_ISSN 2410-700X
Table I
Activities of AO, XDH and NR in different seed parts of PHS-tolerant cultivar Lutescence 70
Seed parts AO1 XDH2 NR3
Embryo 75.7 ± 6.3 1.5 ± 0.2 0.0
Aleurone layer 55.3 ± 4.5 1.2 ± 0.3 0.0
Endosperm 0.0 0.0 0.0
*nmo1 phenantridone mg-1 protein min-1; 2цто1 NADH mg-1 hour-1; 3цто NO2- mg-1 hour-1
Mo-enzymes in developing and mature seeds. The activities of NR XDH and AO in developing seeds were determined at five days intervals starting from 10th day after pollination (DPA) (Table II). Water content of the seed attained a peak at 30 DPA and then started declining to its lowest level a maturity. A linear increase in fresh as well as dry matter of the seed was observed between 10 and 30 DPA (not shown). XDH activity in the embryo and aleurone layer in maturing seeds of both varieties remained at a steady level. A slow increasing activity of AO reached a maximum at 40 DPA (Table II). Low AO activity was detected in the embryo and aleurone layer of seeds until the age of 30 DPA. After this age the activity of the enzyme in the embryo increased steeply reaching a maximum at 40 DPA, the enzyme activity in the aleurone layer increased slightly. Thereafter, AO activity in the embryo fell significantly at full maturity of the seeds, while AO activity in the aleurone layer remained at a steady level.
Table II
Changes in AO, XDH and NR activities in seed embryo during seed development and maturation
DPA 10 20 30 40 50 60 FM
AO 10.2±1.7 11.8±2.1 18.3±2.4 67.4±8.7 65.7±9.3 66.4±7.4 66.8±7.4
XDH 1.6±0.2 1.6±0.2 1.7±0.3 1.8±0.2 1.9±0.3 1.9±0.2 1.8±0.3
NR 0.0 0.0 0.0 0.3±0.02 2.3±0.1 4.7±0.5 5.1±0.7
NR activity in germinating seeds of PHS-tolerant and sensitive wheat cultivars. The following experiments were carried out to study the activities of NR in pre-harvest sprouting tolerant and sensitive wheat varieties during their germination. The seeds were separated into aleurone layer, endosperm and embryo. These parts of dry seeds showed no detectable NR activity. Seeds were incubated in distilled water for 24-30 h, then separated and into their parts to determine NR activity in each of the tissues. NR activity appeared in seed embryo only after incubating them in the presence of KNO3 and its levels varied among wheat varieties (Table III). Seeds were incubated in nitrate and at appropriate times embryos were removed from the seeds to estimate the enzyme activity. In the present set of experiments embryos were first excised and then incubated in nitrate. Study of NR induction in the excised embryos showed a significant level of the NR activity as early as 6 h of germination. After this, the enzyme activity increased and showed a peak at 12 h of incubation followed by a decrease in the level of the enzyme activity which came to the minimum activity after 30 h of incubation (Table III). It is interesting to note that the embryos of ungerminated wheat seeds of both varieties could not synthesize NR in the presence of 60 mM KNO3 after 12 h induction. Since wheat seeds germinate in the absence of nitrate and without NR synthesis, seed germination is not depended on nitrate assimilation in the embryo.
Table III
NR activity of attached and detached seed embryos of PHS-tolerant Lutescence 70 and PHS-sensitive
Novosibirskaya 67 wheat varieties
Wheat varieties Type of embryo Incubation time, h
0 6 12 18 24 30
PHS-tolerant Attached 0.0 0.0 0.0 0.0 0.0 0.0
Detached 0.0 2.7±0.2 4.3±0.7 3.2±0.5 2.5±0.4 1.8±0.2
PHS-sensitive Attached 0.0 0.5±0.1 1.2±0.1 2.4±0.3 1.9±0.3 1.3±0.2
Detached 0.0 3.2±0.4 5.2±0.5 4.8±0.6 4.2±0.5 3.6±0.6
Since in PHS-tolerant seeds the presence of the endosperm delayed NR induction, attempts were made to study effects of cross-combinations of PHS-tolerant and PHS-sensitive endosperm extracts on the induction of NR in their detached embryos. Results of these experiments are shown in Table IV.
Table IV
Effects of cross-combinations of fresh endosperm extracts of PHS-tolerant and sensitive on NR-induction in their detached embryos in after 12 h induction presence of 60 mM KNO3
Combinations of endosperm extracts and detached embryos in KNO3 NR activity in detached embryos
Detached embryo of PHS-tolerant seed in 4.6 ± 0.9
Endosperm of PHS-tolerant seed + its detached embryo 0.5 ± 0.1
Detached embryo of PHS-sensitive seed 5.5 ± 0.7
Endosperm of PHS-sensitive seed + its detached embryo 2.9 ± 0.6
Endosperm of PHS-tolerant seed + detached embryo of PHS-sensitive seed 1.9 ± 0.3
Endosperm of PHS-sensitive seed + detached embryo of PHS- tolerant seed 0.8 ± 0.1
The endosperm of PHS-tolerant wheat seeds contains a factor inhibiting NR induction by nitrate in embryo, while this factor is not present in PSH-sensitive cultivars. This inhibitory factor present in the endosperm gets gradually destroyed with time (not shown). Seed sensitivity of different wheat varieties to PHS depends on the levels of ABA in their endosperm and embryo, i.e. PHS-tolerant wheat seeds contain higher concentrations of ABA [5]. On the basis of these facts we proposed that ABA regulates the activity or synthesis of NR in wheat embryo. Detached embryos of PHS-tolerant seeds were incubated for 12 h in the presence of different ABA concentrations plus 60 mM KNO3. Increasing concentrations of ABA increase the inhibition of NR activity in detached wheat embryo (Table V).
Table V
Time course decrease of NR activity in PHS-tolerant embryo by increasing ABA concentrations
(NR activity in nmoles of nitrite/embryo/h)
ABA concentrations Control 10 nM 100 nM 1.0 ^M
NR activity 4.8 ± 0.8 4.3 ±0.8 3.5 ± 0.3 1.3 ± 0.2
Since ABA content in both seed types is nearly the same the endosperm of PHS-sensitive seeds may contain higher concentrations of ABA inactivating enzymes than the PHS-tolerant seeds, and this is one of reasons of PHS-sensitivity of wheat seeds. These observations are significant in view of the fact that NR is induced in wheat seed embryo linked to the seed tolerance to PHS. The inhibition of NR in detached embryos following 4-6 h of imbibition by endosperm extract may serve as an early marker of the tolerance of wheat seeds to PHS.
In the embryos present in the intact seed there was no NR activity until 24 h of incubation. It appears that the presence of endosperm in the intact seed inhibited the induction of NR in the embryos during the (first 24 h) germination of the PHS-tolerant wheat seeds but not in the embryos of the PHS-sensitive wheat seeds. Thus, only the endosperm of PHS-tolerant seed appeared to inhibit NR induction. Other enzymes of nitrate assimilation pathway, such as nitrite reductase, glutamine synthase and glutamate dehydrogenase were also found to be present in in situ embryos during the initial stages of germination in wheat (not shown). Thus, it appears that among the key enzymes of nitrate assimilation only NR shows a long lag period in its development that seems to be exerted by the endosperm of PHS-sensitive seeds for NR. The level of the inhibitory endosperm factor of PHS-sensitive wheat seeds appears to be lower in PHS-tolerant seeds. The inhibitory factor gradually disappears with seed aging, similarly to the fate of the dormancy controlling ABA, the level of which determines the PHS sensitivity of the seeds, suggesting that ABA may regulate the synthesis of NR in wheat embryos and perhaps in other plant tissues [6]. These observations are significant in view of the fact that NR induction in wheat seed embryos correlates with their level of PHS tolerance. Thus, the 4-6 h inhibition by the endosperm extract of NR in detached embryos may serve as a marker of the PHS tolerance of wheat seeds, and may be used as an early parameter for fast genetic screening.
Literature cited
1. Fang J. and Chu Ch. Abscisic acid and the pre-harvest sprouting in cereals. Plant Signal Behav. 2008. 3(12): 1046-1048.
2. Gerjets T.; Scholefield D.; Foulkes M.J.; Lenton J.R.; Holdsworth M.J. An analysis of dormancy, ABA responsiveness, after-ripening and pre-harvest sprouting in hexaploid wheat (Triticum aestivum L.) caryopses. Journal of Experimental Botany, v.61, p.597-607, 2010.
3. Terao M., Kurosaki M., Saltini G., Demontis S., Marini M., Salmona M., Garattini E. 2000. Cloning of the cDNA for two aldehyde oxidase and xanthine oxidoreductase. J.Biol.Chem. V.275 (39): 30690-30700.
4. Alikulov Z. and Schieman. 1985. Presence of active molybdenum cofactor in dry seeds of wheat and barley. Plant Sci. 40: 161-165.
5. Kawakami N, Miyake Y. and N. Kzuhiko. 1997. ABA insensitivity and low ABA levels during seed development of non-dormant wheat mutants. J.Exp. Botany. 48(312): 1415-1421.
6. Finch-Savage W.E.; Cadman C.S.C.; Toorop P.E.; Lynn J.R.; Hilhorst H.W.M. Seed dormancy release in Arabidopsis Cvi by dry after-ripening, low temperature, nitrate and light shows common quantitative patterns of gene expression directed by environmentally specific sensing. Plant Journal, v.51, p.60-78, 2007.
© Z. Alikulov, A. Shukhatova, G. Shalakhmetova, 2015
УДК 2788
Alikulov Z.
Associate Professor, Department of Microbiology and Biotechnology of the Eurasian National University. L.N.Gumilev E-mail: [email protected] Talapova Zh., Dyussembayev K.
Undergraduate of the Eurasian National University. L.N.Gumilev E-mail: [email protected]
ROLE OF ANIMAL MOLYBDOENZYMES IN DETOXIFICATION OF XENOBIOTICS
Abstract
In animals xanthine oxidase (XO) and aldehyde oxidase (AO) are closely related enzymes with similar molecular properties but differ somewhat in substrate specifity; they catalyze the oxidation of a wide range of heterocyclic compounds containing nitrogen atoms. Unlike membrane-bound cytochrome P450 monooxygenases, XO and AO are cytosolic and stable during the oxidative stress (lipid peroxidation). The review considers that the activities of these enzymes can be regulated by administration with molybdenum (activation) and its deficiency may cause oncological diseases.
Xenobiotic-transforming enzymes. Most the attention to date in metabolism of drugs and foreign compounds has been focused on the microsomal cytochrome P450 (CYP) enzyme family or monooxygenase system. This membrane bound system plays an important role in the oxidation of aromatic carbocyclic compounds in animals and human. However, the presence of the one or more nitrogen atoms in the aromatic rings makes heterocyclic compounds also susceptible to oxidation via a second group of enzymes known as the "molybdenum hydroxylases". These cytosolic enzymes, which include xanthine oxidase aldehyde oxidase (AO, EC 1.2.3.1) and (XO, EC 1.2.3.2) form a closely related group with similar molecular properties but differ somewhat in substrate specificity [1].
These enzymes catalyze both oxidation and reduction of a broad range of drugs and other xenobiotics indicating the importance of these enzymes in drug oxidation, detoxification and activation. Xanthine oxidoreductase (XOR) appears in two interconvertible forms xanthine dehydrogenase (XDH), and xanthine oxidase (XO). Xanthine oxidoreductase catalyzes the hydroxylation of hypoxanthine to xanthine and of xanthine to urate. XDH reduces NAD+, but XO reduces molecular oxygen at the flavin center. Molybdenum-containing hydroxylases catalyze the hydroxylation of carbon centers using oxygen derived ultimately from water, rather than O2, as the source of the oxygen atom incorporated into the product, and do not require an external source of reducing equivalents [1].
The relative importance of these two groups of oxidative enzymes is illustrated by comparing the in vitro oxidation of several bicyclic ring system. Naphtalene is oxidized via the CYP P450 system to an unstable epoxide intermediate which ultimately gives rise to a mixture of 1-naphtol and 2-naphtol. However, naphthalene is not a substrate for the molybdenum hydroxylases. Quinoline, 1-azanaphtalene, reacts not only with the CYP P450 system but also with aldehyde oxidase to give a number of mono- and dihydroquinolines with rabbit or rat liver fractions.
