ISSN 1563-034Х; еISSN 2617-7358 Экология сериясы. №2 (67). 2021 https://bulletin-ecology.kaznu.kz
IRSTI 34.31.27 34.31.17 https://doi.org/10.26577/EJE.2021.v67.i2.08
U. Erezhetova1* , G.M. Sultangalyeva1 , N.V. Terletskaya1 , M.S. Kurmanbayeva1 , M.H. Hoffmann2
1Al-Farabi Kazakh National University, Kazakhstan, Almaty
2Martin Luther University Halle-Wittenberg, Germany, Halle
*e-mail: [email protected]
ANATOMICAL PARAMETERS OF THE FLAG LEAF OF ALLOPLASMIC LINES AND THEIR PARENTAL FORMS
IN DROUGHT CONDITIONS
Anatomical parameters of the flag leaf as criteria for drought tolerance of two alloplasmic lines (interspecific hybrids) and their parental forms − T. aestivum L. (cultivar of Mironovskaya-808) and T.
dicoccum wheat were presented. The parameters studied were the thickness of the abaxial and adaxial epidermis, the thickness of the mesophyll and central vein, and the diameter of the central conducting bundle of the flag leaf. Different degrees of negative impact of sudden drought-stress and permanent water deficit on the anatomical structure of the flag leaf were shown. At the stage of flag leaf formation, anatomical parameters, for example an increase or preservation of the size of the protective and mechan- ical leaf tissues and the thickness of the mesophyll under stress can serve as criteria for the selection of drought-resistant forms of wheat. It revealed that among the studied genetic lineages the alloline D-d-05 showed the best adaptability and the most stable anatomical parameters of the flag leaf under conditions of moisture deficiency. The different degrees of drought tolerance of the studied lines indicate that the combination of the nucleus and cytoplasm of the parental forms can contribute to both an increase and a decrease in the level of drought tolerance in interspecific hybrids, which indicates the need to continue research with additional molecular genetic analysis.
Key words: wheat, allolines, flag-list, anatomy, drought tolerance.
У. Ережетова1*, Г.М. Султангалиева1, Н.В. Терлецкая1, М.С. Курманбаева1, М.Х. Хоффман2
1 Әл-Фараби атындағы Қазақ ұлттық университеті, Қазақстан, Алматы қ.
2Мартина Лютер атындағы Галле-Виттенберг университеті, Германия, Галле қ.
*e-mail: [email protected]
Құрғақшылық жағдайындағы аллоплазмалық сызықтардың жалауша жапырақтарының және олардың ата-аналық формаларының
анатомиялық параметрлері
Мақалада жалаушаның анатомиялық параметрлерін зерттеу нәтижелері екі аллоплазмалық сызықтың (түр аралық будандар) және олардың ата-аналық формаларының – T. aestivum L.
(Мироновская-808сорты) және T. dicoccum бидайының құрғақшылыққа төзімділігі критерийлері ретінде ұсынылған. Абаксиальды және адаксиальды эпидермистің қалыңдығы, мезофилл мен орталық тамырдың қалыңдығы, жалауша жапырағының орталық өткізгіш шоғырының диаметрі зерттелді. Зерттелген формалардың жалауша жапырағының анатомиялық құрылымына кенеттен болатын құрғақшылық-стресс және тұрақты су тапшылығының теріс әсерінің әр түрлі дәрежелері көрсетілген. Қорғаныс және механикалық жапырақ ұлпаларының мөлшерін және мезофилл қалыңдығын жоғарылату немесе сақтау сияқты анатомиялық параметрлер стресс кезінде бидайдың құрғақшылыққа төзімді түрлерін таңдау критерийі бола алады деп атап өтілген. D-d-05 аллолиниясы зерттелген түрлердің арасында әр түрлі ылғалдылық тапшылығы жағдайында ең жақсы бейімделгіштік көрсететіндігі және жалауша жапырақтарының тұрақты анатомиялық параметрлерімен сипатталатындығы анықталды. Зерттелетін сызықтардың әр түрлі құрғақшылыққа төзімділігі ядро мен цитоплазманың ата-аналық формаларының бірігуі түр аралық будандардағы құрғақшылыққа төзімділік деңгейінің өсуіне де, төмендеуіне де ықпал етуі мүмкін екенін көрсетеді, бұл молекулалық-генетикалық талдауды қатыстыра отырып зерттеуді жалғастыру қажеттілігін көрсетеді.
Түйін сөздер: бидай, аллолиниялар, жалауша-жапырақ, анатомия, құрғақшылыққа төзімділік.
79 U. Erezhetova et al.
У. Ережетова1*, Г.М. Султангалиева1, Н.В. Терлецкая1, М.С. Курманбаева1, М.Х. Хоффман2
1Казахский национальный университет имени аль-Фараби, Казахстан, г. Алматы
2Галле-Виттенбергский университет имени Мартина Лютера, Германия, г. Галле
*e-mail: [email protected]
Анатомические параметры флагового листа аллоплазматических линий и их родительских форм в засушливых условиях
В статье приведены результаты изучения анатомических параметров флагового листа как критериев засухоустойчивости двух аллоплазматических линий (межвидовых гибридов) и их родительских форм − видов T. aestivum L. (сорт Мироновская-808) и T. dicoccum пшеницы.
В процессе исследования изучали толщину абаксиального и адаксиального эпидермиса, толщину мезофилла и центральной жилки, а также диаметр центрального проводящего пучка флагового листа. Показана различная степень негативного воздействия внезапной засухи- стресса и перманентного водного дефицита на анатомическую структуру флагового листа изучаемых форм. Отмечено, что такие анатомические параметры, как увеличение или сохранение неизменными при стрессе размеров защитных и механических тканей листа и толщины мезофилла, могут служить критериями отбора засухоустойчивых форм пшеницы на стадии флагового листа. Выявлено, что среди изучаемых форм аллолиния D-d-05 характеризуется наилучшей возможностью к адаптации и наиболее стабильными анатомическими параметрами флагового листа в условиях недостатка влаги различной степени. Различная степень засухоустойчивости изучаемых линий свидетельствует о том, что сочетание ядра и цитоплазмы родительских форм может способствовать как повышению, так и понижению уровня засухоустойчивости у межвидовых гибридов, что говорит о необходимости продолжения исследований с привлечением молекулярно-генетического анализа.
Ключевые слова: пшеница, аллолинии, флаг-лист, анатомия, засухоустойчивость.
Introduction
The main direction in wheat breeding is to increase the overall potential of grain productivity.
At the same time, breeding to increase productivity is one of the most difficult tasks, because plant productivity is a complex trait controlled by a complex genetic system that closely interacts with many parameters of the external environment.
Approximately up to 90-95% of dry weight of the crop is created by photosynthesis of leaves [1].
The ability of leaves to absorb photosynthetically active radiation is in direct proportion to the size of the assimilating surface of the plant leaves and their longevity [2].
Leaf area is frequently used as a proxy for yield and productivity although green stems may contribute too. It revealed a positive relation between the yield and the leaf area of different strength; from strong (r = 0.95-0.98) to medium (r = 0.45-0.56) [3, 4].
The leafiness (the number of leafs) of wheat plants attracted much attention of researchers. It is thought that drought-resistant genotypes during the period of grain formation had increased but not maximum indicators of leafiness [5]. At this time of the development, the assimilating surface is formed mainly by the two upper leaves, their sizes are closely correlated with the elements of grain yield [6]. A positive correlation was found between the
length of the flag leaf and the length and weight of the ear, the width of the leaf with the total number of spikelets, the number of productive spikelets, and the productivity of an ear. An increase of the size of the flag leaf led to an increase of productivity due to an increase in the grain content of the ear, however, the grain size decreased [7]. Many studies including our previous research deal with changes of the photosynthetic apparatus of wheat in relation to an increased level of ploidy and the transition from wild to cultural forms and also with changes in the leaf anatomy [8], in the photochemical and photophosphorylating activity of photosynthetic pigments [9, 10] as well as features of the anatomical organization of the wheat leaf [11]. It was shown that the structural features of the leaf at different phases of its development are of great importance for the formation of resistance to unfavorable factors – extreme temperatures and damages induced by diseases and pests. So, the analysis of anatomy and morphology of the leaf blade in different varieties and species revealed the possibility for selecting more resistant forms based on characteristics like the thickness of the leaf blade, the thickness of the cell walls of the epidermis and sclerenchyma, and the presence of pubescence [12].
Nevertheless, the cellular and tissue levels of organization of the wheat photosynthetic apparatus are the least studied currently [13]. Alloplasmic
Anatomical parameters of the flag leaf of alloplasmic lines and their parental forms in drought conditions
lines represent a unique tool for studying the effect of genome substitution on the adaptive potential of plants under stress conditions.
The aim of this study is to reveal changes of anatomical parameters of the tissues of the flag leaf of winter wheat in relation to drought. To understand the reasons for the different resistance of varieties and lines of winter wheat to unfavorable drought conditions, we compared the anatomical and morphological structure of flag leaves of alloplasmic lines of wheat and their parental forms in the heading phase under different moisture backgrounds.
Materials and research methods
Species T. aestivum (Mironovskaya-808 cultivar) and T. dicoccum were taken as material for research, which were studied by us earlier and showed high resistance to osmotic and salt stress [14], as well as two interspecific hybrids (alloplasmic lines) obtained from crosses of these species with subsequent backcrossing (up to 5 years) and long- term selection (F11). These allolines were created by Professor N.A. Khailenko.
The plants were grown on the field plot of the RSE “IBBR” KN MES RK in two different drought conditions – with optimal irrigation (up to 60% of full moisture capacity) and with minimal watering (up to 30% of full moisture capacity, i.e. in conditions of permanent water deficit (“water deficit” variant).
Some of the plants grown in optimal irrigation were subjected to severe drought for 5 days at the stage of flag leaf formation by completely stopping irrigation (“drought stress” variant). The thermostat-weight method was used to measure soil moisture [15].
The calculation of the field soil moisture is carried out according to the formula: W= (a x 100) : b (%), where (W) − the field soil moisture, in% of the dry soil mass; (a) − the mass of water in the soil sample, g; (b) − dry soil mass, g. When processing the data, the plant characteristics was determined from 1 m2 from each variant. Leaf samples for anatomical studies were taken from each treatment of the experiment with at least three biological replicates and at least three plants in each replicates.
Plant material was preserved for anatomical studies according to the Strasburger-Flemming method. The fixation was carried out in 70% ethanol.
The fixed material was preserved in a mixture of ethanol: glycerol: water in a 1: 1: 1 ratio. Anatomical preparations were prepared using a microtome with a TOS-2 freezing unit. The sections were placed in glycerin and balsam in accordance with the methods of Barykina [16]. Microphotography of anatomical
sections was performed using a microscope with an MC 300 CAMV400 / 1.3M camera (Austria).
Statistical analysis was performed using the program R (R Core Team. 2014. R: A language and environment for statistical computing, version 3.1.0. website: www.r-project.org [accessed 16 June 2014].). TukeyHSD tests as implemented in R were calculated to reveal significant pairwise ANOVA tests that were graphically presented as boxplots.
Results of research
Figure 1 shows the data for the thickness of the adaxial (upper) epidermis of the flag leaf. The maximum value observed is in control conditions in T. dicoccum. The stressful conditions of a sudden intense drought caused a thickening of the adaxial epidermis in Mironovskaya-808; in T. dicoccum and in alloline D-40-05 there was noted an insignificant decrease in the value of this parameter. But under conditions of prolonged moderate water deficit, the indicator values decreased significantly in all forms except the D-d-05 alloline. In alloline D-d-05, the thickness of the adaxial epidermis under stress conditions remained at the level of control values.
Figure 1 − Thickness of the adaxial epidermis of the flag- leaf of allolines and parental forms. The x-axis of the boxplot
represents the studied lines and treatments. Within each line, the significantly different comparisons (p < 0.05) between the
treatments were connected by lines
81 U. Erezhetova et al.
The line D-40-05 were characterized by the greatest thickness of the abaxial (lower) epidermis of the flag leaf under control conditions. Under conditions of severe stress caused by a sudden drought, thickening of the abaxial epidermis compared to the control can be noted in all studied forms, except for D-40-05; In M-808 and D-d- 05 there thickening was significant. And under conditions of growth with a moderate water deficit, all forms showed a decrease in the value of this parameter. Only for line D-d-05 and M-808 it remained equal to the control indicator (Fig. 2).
Figure 2 − The thickness of the abaxial epidermis of the flag-leaf of allolines and parental forms.
For further explanations, see Fig. 1
The thickness of the mesophyll in the studied forms also differed in both control and stress conditions (Fig. 3). But if a sudden stress-drought caused a sharp thickening of the mesophyll in cultivar of Mironovskaya-808, line D-d-05, in line D-40-05 it decreased. Under conditions of constant water deficit, a decrease in the thickness of the mesophyll was observed in all studied forms, but the decrease in the line D-d-05 was minimal in relation to the control.
According to the data presented in Fig. 4, severe sudden drought stress caused a significant thickening of the central vein of the flag leaf of all studied forms, and constant water deficit caused a decrease in the values of this parameter. In relation to the control value, the decrease was smaller for the D-d-05 alloline.
Figure 3 − The thickness of the mesophyll of the flag-sheet of allolines and parental forms.
For further explanations, see Fig. 1
Figure 4 − Thickness of the central vein of the flag-leaf of allolines and parental forms.
For further explanations, see Fig. 1
Anatomical parameters of the flag leaf of alloplasmic lines and their parental forms in drought conditions
The diameter of the central conducting bundle under severe drought-stress increased significantly in all studied forms, except for the M-808 (Fig. 5).
The constant water deficit caused a decrease in the values of the diameter of the central conductive bundle, but in the D-d-05 alloline, this decrease in relation to the control was less than in other studied forms but still significant.
Figure 5 − Diameter of the central conducting bundle of the flag-sheet of allolines and parental forms.
For further explanations, see Fig. 1
The discussion of the results
Leaf structure is a reliable diagnostic feature for assessing the relationship between the plant and the environment. The dynamics of changes in the leaf apparatus of plants under stressful conditions reflects, firstly, the degree of influence of stress on the aboveground part of plants, and secondly, the degree of consistency of the work of the entire plant organism [17]. At the same time, as a rule, an increase or decrease in the values of one or another anatomical sign depends on the susceptibility to stress.
Since the growth of the first leaves occurs in spring, when the soil usually has the necessary amount of moisture, and the growing conditions during the formation of the upper leaves can change to a very wide extent, the size and anatomical
characteristics of the upper tiers leaves (sub-flag and flag) vary much more than those of the lower ones.
Reduction of the area of the leaf blade is an essential and most common feature that arises in the process of evolutionary adaptation to arid environmental conditions.
From the point of view of resistance to stress, both the absolute values of the studied parameters and the relative (stress / control) values for each studied form were important for us.
The epidermis covered with numerous trichomes contribute to the survival of plants in conditions of extreme temperatures, low or high humidity and are elements of the supporting skeleton of the leaf.
Thickening of the epidermis, as well as the thickness of the cuticle layers, can play a positive role in conditions of limited water consumption [18, 19].
We consider that especially important is to focus on changes in the thickness of the abaxial (lower) epidermis of the flag leaf of the studied forms under stress conditions. As the lower epidermis of the leaf has a greater number of stomata than the upper one, carrying out the main gas exchange and transpiration, its thickening during drought may be an important adaptive feature. Our measurements of the thickness of both adaxial and abaxial epidermis in the studied forms under control and experimental conditions of stress of different intensity and duration give an opportunity to characterize the Dd-05 line as more stable, and the D-40-05 line as more responsive to the action of sudden stress caused by drought, and to the action of moderate water deficit for a long time.
The mesophyll of cereals can be characterized as a loose, cellular, isolateral-palisade. This structure of the mesophyll combines signs of resistance to unfavorable environmental conditions and the possibility of saturated metabolism [20]. Wheat mesophyll is heterogeneous in the composition of the cell population. The shape of the cell size changes depending on external conditions, which suggests the presence of an appropriate adaptation mechanism, directed at optimizing the structural and functional organization of the assimilation apparatus of the leaf [21, 22].
An increase in the thickness of the mesophyll under stress conditions can serve as one of the indicators of stress resistance. The cells remain relatively large, they are less densely packed, due to which a greater volume of the inner assimilation surface of the leaf is preserved. This is typical in our experiment for the D-d-05 line as well as parental forms M-808 and T. dicoccum.
Veins serve as conductors of water and distributors of assimilates, in addition, they
83 U. Erezhetova et al.
mechanically strengthen the leaf [23]. Therefore, thickening of the central vein under severe stress can be considered as an adaptation mechanism.
And the minimum decrease in this parameter under conditions of constant water deficit in relation to the control in the D-d-05 line indicates its greater drought tolerance, compared to other forms.
A decrease in the size of the main conducting bundle during drought is directly related to a decrease in the size of the xylem region, which is directly responsible for the ability of plants to absorb water and conduct nutrients, changing the diameter of the vessel [21]. Drought-resistant forms of wheat under conditions of water and temperature stresses form a more powerful conducting system of leaves [8, 24-26]. Judging by the data presented, line D-d- 05 in terms of the diameter of the central conducting bundle can also be considered the most drought- resistant of the presented forms.
Conclusion
Thus, experiment dates showed the different degrees of drought tolerance of the studied lines. that indicate that the combination of the nucleus and cytoplasm of the parental forms can contribute to both an increase and a decrease in the level of drought tolerance in interspecific
hybrids. We noted that such anatomical parameters as an increase or preservation of the size of the protective and mechanical leaf tissues and the thickness of the mesophyll under stress can serve as criteria for the selection of drought- resistant forms of wheat at the stage of the flag leaf. Among the studied forms, the D-d-05 line can be considered the most drought-resistant both under conditions of sudden severe drought-stress and under conditions of constant water deficit during the growing season, based on the studied anatomical parameters of the flag leaf.
Conflict of interests
All authors have read and are familiar with the content of the article and have no conflicts of interest.
Funding
The work was carried out within the framework of research under the project SP AR05131734
“Physiological and molecular genetic features of the functioning of the photosynthetic apparatus of alloplasmic lines of wheat obtained as a result of interspecific crosses, in connection with their drought resistance”, 2018-2020 years.
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