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Section 1. Biology
Nenko N. I., Kisileva G.K., Ulyanovskaya E. V., Karavaeva A. V., Federal state budgetary scientific institution "North-Caucasian federal scientific centre of horticulture, viniculture and winemaking", Krasnodar, Russia01 E-mail: [email protected] Yablonskay E. K.,
Federal State Budget Educational Institution of Higher Education "IT Trubilin Kuban State University of Agriculture", Krasnodar, Russia
WINTER RESISTANCE OF THE APPLE-TREE VARIETIES IN THE SOUTH OF RUSSIA
Abstract
Objective: revealing the apple-tree varieties, promising for growing in the south of Russia.
Methods: In winter period in normal conditions and in model experiment, in frost penetration (25 ° C) the content of bound water was determined in the shoots of apple-tree by a weight method, that of starch - by a spectral method, phenolcarbonic acids, malondialdehyde - by a capillary electrophoresis method on Kapel 105 M tool, anaerooxydase activity - by a spectral method, anaerooxydase isoenzyme composition in water-salt-soluble protein fraction - by a vertical electrophoresis method. When making the anatomical preparations, the methods of conventional botanical microengineering were used.
Results: the most significant indicators, characterizing the varieties' resistance to the low-temperature stress, determining the frost tolerance of apple-tree varieties were studied. The domestic varieties (Soyuz, Rodnichok), as well as Erli Mac variety ofAmerican selection were revealed to store up more starch, making them more resistant to the low temperatures. The correlation dependence between the content of bound water and that of osmoprotectors (saccharose, proline), phenolcarbonic acids (chlorogenic and caffeic), pigments (anthocyans and chalcones) in the apple-tree shoots was established. The parameters such as anaerooxydase activity, and phenolcarbonic acids, malo-ndialdehyde contents were used to reveal the apple-tree varieties' resistance to the low-temperature stress. The electrophoretic spectrum of the proteins' water-salt-soluble fraction with anaerooxydase activity was studied for the apple-tree varieties. A model experiment determined the proteins, having
the molecular weight of 50 and 60 kDa and exhibiting anaerooxydase activity to appear in protein spectrum, as the shoots of the apple-tree varieties under study are exposed to the cold stress.
Scientific novelty
The physiological-biochemical and morphoanatomic criteria were established for the frost-tolerance of the apple-tree varieties of home (Prikubanskoye, Rassvet, Fortuna, Soyuz, Rodnichok), Polish (Ligol) and American (Idared, Erli Mac and Dayton) selection.
The proteins, appearing in the shoots of the apple-tree varieties under study when exposed to the low-temperature stress, were revealed to have the molecular weight of 50 and 60 kDa and exhibit anaerooxydase activity.
Practical implications
Soyuz, Rodnichok and Erli Mac varieties were established to be tolerant to the low temperatures effect and promising for selection process and growing in the south of Russia.
Keywords: apple-tree; variety; frost tolerance; oxidative stress, protein spectrums; anaerooxydase.
Introduction
The issues of the apple-tree plants higher resistance to abiotic and biotic stress-factors of environment were high on the list in solution of the high-productive fruit coenoses engineering problem. They became mostly dramatic in the recent years in the terms of the local changes in climate [1, 2].
The exposure to the natural stressors brings to the sharp decrease in productivity and fruits quality, and more often than not to the death of plants of the fruit crops. If affected by the stress factors, the intensive varieties may realize only 15-30 percent of potential productivity [1].
That is why only the varieties, combining the high quality with adaptedness to conditions of the region, may be successfully grown on a rather wide scale basis [3, 4]. The natural and climatic conditions ofthe south of Russia favour the cultivation of the fruit crops, but obtaining the steadily high yields is restricted by the impact of unfavourable environmental factors, such as winter frosts, especially after the long-time warm weather, and summer droughts. Therefore the varieties, both locally selected, and introduced ones, adapting to the cultivation region conditions take on particular significance. In this context the importance of selection, as a basic method to improve the commercial range of apple-tree culture grows [3, 5].
In this regard it is particularly topical not only to single out the promising genotypes, but to determine the expressivity of genetic systems, in other words the ability to realize the potentialities of genotype in the changing conditions of environment. The study of the physiological-biochemical criteria of adaptability will permit to find out the varieties resistant to abiotic factors of winter period in the south of Russia.
The objective of work is to study the physiological-biochemical and morphoanatomic parameters of resistance of the apple-tree varieties different in ecological-geographical origin to the stress winter period conditions in the south of Russia.
Methods of investigations
The investigations were carried out on the basis of Pilot production farm "Centralnoye", Federal State Budgetary Scientific Institution North Caucasian Federal Scientific Centre for Horticulture, Viticulture and Wine making, Krasnodar. The physiological-biochemical regularities of the apple-tree adaptation were studied on the varieties as follows:
- Idared (USA), Ligol (Poland), Prikubanskoye (Russia, NCRRIH&V) planted in 2010 on CK 4 parent stock with 0.9 x 4.5 planting system;
- Rassvet (2n = 2x), Fortuna (2n = 2x), Soyuz (2n = 3x), Rodnichok (2n = 3x) (Russia,
NCRRIH&V) planted in 2000 on M 9 parent stock with 2 x 5 planting system;
- Erli Mac (2n = 2x) (USA), Dayton (2n = 2x) (USA) planted in 1998 on M 9 parent stock with 2 x 5 planting system.
To estimate the apple-tree plants frost tolerance in the shoots both in the usual conditions, and under the low-temperature stress in the model experiment at the temperature of minus 25 ° C, they determined water status with the use of methodology [6], content of starch [7], phenolcarbonic acids, malondi-aldehyde - by the capillary electrophoresis method on Kapel 105 M tool [8], activity of anaerooxydase -by a spectral method [9], isoenzyme composition of anaerooxydase in the water-salt-soluble protein fraction - by a vertical electrophoresis method [10]. When making anatomic preparations, the methods of conventional botanical microengineering were used [11].
The measurement data were processed with the use of conventional methods of variation statistics [12].
Results of investigations and discussion
As distinguished from winter period of 20142015, 2015-2016, the 2016-2017 winter was cold-
er, the minimum air temperature lowered down to minus 17-16-14 °C, respectively, the maximum temperature reached 12-22 °C, the temperature drop made 15-35 °C. The increase in the maximum air temperature from December of 2014 (12 °C) through December of 2017 (16 ° C) by 4 ° C and minimum air temperature from - 9 up to - 4 °C, characterizes climate warming. The high air temperature and the large depth of rainfall in November of 2016 promoted the later entrance of the apple-trees of the varieties under study into the state of winter dormancy. It was a reason for the apple-tree plant to be out of the state of true dormancy. This fact may become a reason of their lower resistance to the damaging factors of winter period.
The starch content in the shoots of plants is among the winter resistance indices of the appletree varieties (Fig.1). The decrease in temperature involves an intensive hydrolysis of starch in the fruit trees, as a result of which the starch turns into the fats and osmotically active compounds, first of all into the different sugars, being the protective agents (they diminish the processes of proteic substances denaturation, when freezing, and stabilize the structure of protoplasm) [13].
B 2016
H 2017
Figure 1. The dynamics of starch content in the shoots of apple-tree varieties in 2015-2017 December
During the winter period in December of 2015- Prikubanskoye varieties in 2015, in 2016 - by Idared, 2017 the lesser accumulation of starch in the zone of Ligol, Prikubanskoye varieties and in 2017 - by Idared, fine-cellular shoot core was shown by Idared, Ligol, Rassvet, Fortuna, Rodnichok varieties (Fig. 2).
Transversal section of shoot Longitudinal section of bud Starch grains
Figure 2. Morphoanatomic structure of the one-year shoots and the buds of Idared apple-tree variety in December of 2015
Thus, depending on the weather conditions, the different varieties accumulate starch in the shoots variously, that may stem from their photosynthetic activity under the conditions of the previous drought of summer period [14].
The histochemical and morphoanatomic research of the one-year shoots of the apple-tree varieties under study showed them to be in the state of induced dormancy for the analyzed period of 2015-2017 in December, that is confirmed by the method of the cut branches sprouting (in all variants of experiment all the buds began to grow). The morphoanatomic examinations of the fruit buds on the annual increment showed them to be in the state of induced winter dormancy. The fruit buds are at the Vr stage of organogenesis - the formation of ar-chesporial tissue in anthers (Fig. 2).
The intravital anatomic investigations for the tissues of the one-year shoots and the buds of the apple-tree varieties under study, conducted in February of 2016, showed them to be in the state of induced dormancy. The transversal sections of shoot in the core zone show the multiple plasmodesmata (intercellular cytoplasmic cords between the cells), proving the activity of metabolic processes in the plant cells. In Soyuz and Rodnichok triploid varieties the starch content in the zone offine-cellular shoot core significantly lowered in response to its hydrolysis,
and made 4.4 and 4.3 points respectively. The hydrolysis of starch was noted to be delayed in diploid varieties, its quantity varied from 4.4 to 4.6 points. For example, the starch content of Rassvet, Erli Mac, Idared, Prikubanskoye diploid varieties in the zone of the fine-cellular shoot core made 4.6 points, that in Fortuna variety - 4.5 points, characterizing it as a winter-hardy one, in Dayton and Ligol diploid varieties - 4.4 points (medium winter-hardy).
Thus, in February conditions of 2016 Soyuz and Rodnichok triploid varieties, proved themselves to be highly winter-hardy according to the physiological-biochemical and histochemical data.
The most important biochemical markers of the plants frost-tolerance are the content indicators of such osmoprotectors as proline and saccharose (15-17).
The study for dynamics of the change in bound water content and such stress-protectors, as proline and saccharose, in December of 2014-2016, as exemplified by Idared, Ligol, Prikubanskoye varieties, showed the resistance of the Idared apple-tree plants be determined by saccharose content to the greater extent (K .= 0.89), and by proline - in Prikubanskoye variety (K .= 1.0). As for Ligol variety, both proline and saccharose participate in the maintenance of water economy, but the relation between the bound water content and these osmoprotectors
is not significant (K , = 0.55 and 0.61). At the time
c v correl '
of freezing through the shoots in a model experiment in December of 2014-2016 the relationship between the change in the content of bound water and that of saccharose, proline, phenolcarbonic acids was established. The maximum coefficient of correlation was noted between the content of bound
water and that of proline (Korel = 0.97), anthocyans (0.92), chalcones (Kgnel = 1.0), which is due to inhibition of the free-radical processes under oxidative stress, characterizing the change in permeability of the cell membranes [18]. The anaerooxydase activity of the studied apple-tree varieties varies in the range from 12.02 to 28.15 sec-1(Fig. 3).
® before freezing B after freezing
Figure 3. Anaerooxydase activity in the shoots of apple-tree in December of 2017
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Figure 4. The phenolcarbonic acids content in the shoots of appletree in December of 2016 before and after freezing
In this case the lesser anaerooxydase activity before in Rassvet, Fortuna, Dayton varieties and greater - in
the shoots are frozen through is noted for Rassvet, Prikubanskoye, Soyuz, Rodnichok, Erli Mac varieties,
Rodnichok and Erli Mac varieties and the higher that agrees with the higher content ofphenolcarbonic
one for Soyuz, Prikubanskoye, Fortuna and Dayton acids before the exposure of Rassvet, Fortuna, Dayton
varieties, and after freezing the shoots the lesser one - varieties to the cold stress. (Fig. 4), and lesser one - of
Prikubanskoye, Rodnichok, Soyuz, Erli Mac varieties, to characterize the specificity of variety and it may involve the differences in the spectrums of proteins, featuring the anaerooxydase activity.
To characterize the degree of oxidative stress development and functioning of the plants'
0,3
75 0,25
antioxidant defence in the apple-tree shoots, the content of malondialdehyde was determined [19-21]. It appears in organism, when the active forms of oxygen degrade the polyunsaturated fatty acids and serves as one of important indicators for degree of the stress-factor damaging action (Fig. 5.)
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I after freezing
Figure 5. Malondialdehyde content of the apple-tree shoots in December of 2016 before and after freezing
before freezing after freezing
1 - Prikubanskoye, 2 - Soyuz , 3 - Rodnichok, 4 - Fortuna , 5 - Rassvet Figure 6. The electropherogram of the water-salt-soluble fraction of the proteins featuring the anaerooxydase activity of the apple-tree varieties before and after freezing the shoots in December of 2016
The malondialdehyde content of Rassvet, Fortuna, Dayton varieties both before and after freezing was established to be higher, as compared with other apple-tree varieties under study. To characterize the genetic systems' expressivity of the studied apple-tree varieties adaptation to the low temperatures in the state of true dormancy, the cold stress proteins, showing the anaerooxydase activity, were studied (Fig. 6).
The protein complex with anaerooxydase activity of the apple-tree varieties under study was established to be represented by the proteins of 250, 150, 100, 90, 75, 60, 50 kDa molecular weight. Pri-kubanskoye, Soyuz, Rodnichok varieties differ from other studied apple-tree varieties by the presence of protein of 90 kDa molecular weight and larger proteins content with molecular weight of 100 kDa, that can be ascribed to their ecological-geographical origin. The proteins with 60 kDa molecular weight are abundant in Rodnichok, Rassvet varieties, as distinguished from other varieties under test.
When freezing through the shoots of apple-tree in a model experiment, Prikubanskoye, Soyuz and Rodnichok varieties show the rise in the content of proteins with molecular weight of 50 kDa. Thus, one may suppose, that the proteins of cold stress in the studied varieties, when exposed to the low-
temperature stress, are represented by the proteins with molecular weight of 50 and 60 kDa, featuring the anaerooxydase activity.
Conclusions
The most significant parameters, characterizing the apple-tree varieties' resistance to the low-temperature stress, determining their frost tolerance, were studied. The domestic varieties (Soyuz, Rodnichok), as well as Erli Mac variety ofAmerican selection were revealed to store up more starch, making them more tolerant to the low-temperatures effect. The correlation dependence between the content of bound water and that of osmoprotectors (saccharose, proline), phenolcarbonic acids (chlorogenic, caffeic) pigments (anthocyans and chalcones) in the shoots of apple-tree was established. The parameters such as anaerooxydase activity and phenolcarbonic acids, malondialdehyde content were used to reveal the apple-tree varieties' resistance to the low-temperature stress. The apple-tree varieties' electropho-retic spectrum was studied for the water-salt-soluble fraction of the proteins, exhibiting anaerooxydase activity. It was established in a model experiment, that the proteins, having the molecular weight of 50 and 60 kDa and exhibiting anaerooxydase activity, appear in a protein spectrum on exposure of the studied apple-tree varieties to the cold stress.
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