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UDK 577.152.087:581.2 doi: 10.15407/biotech9.01.103
CHLOROPLAST STRUCTURAL AND FUNCTIONAL CHANGES AS BIOMARKERS OF HEAVY METAL
CONTAMINATION
M. V. Vodka, N. O. Bilyavs'ka
Kholodny Botany Institute of the National Academy of Sciences of Ukraine, Kyiv
E-mail: marinavodka@yandex.ru
Received 28.12.2015
The aim was to confirm the hypothesis of possibility to use the chloroplast structural and functional changes in higher plants as biomarkers to assess heavy metal contamination.
Chloroplast ultra-structural changes of Pisum sativum L were detected using the transmission electron microscopy. This work deals with studies of chloroplast structure responses to a high content of copper (80 pM) and zinc (200 pM). Data on changes in the structure of chloroplasts in particular, heterogeneity in the grain thylakoid packing, increase of interthylakoid gaps and thickness of chloroplast grain thylakoids in comparison with controls were obtained. The results of studies on structural and functional chloroplasts changes offer challenges for their use as markers for an early diagnostics of abiotic stress effects and in biotechnological studies to produce novel advanced varieties of crops resistant to stress.
Key words: Pisum sativum L., chloroplast changes, heavy metal contamination, biomarkers.
Industrial emissions are a powerful factor that affects the vegetation cover development. Air, water and soil pollution by abnormal concentrations of chemicals has a direct influence on the plant photosynthetic apparatus. Effects of heavy metals (HM) excess concentrations on plants result in anatomical and morphological changes, physiological and biochemical disturbances. Excess of heavy metals negatively impacts cell organelles, changing their structure and characteristics.
In many regions of Ukraine environmental pollution arouses a strong interest in studies of heavy metal as a stress factor and finding mechanisms for organism protection against their toxic effects. Methods of bioindication are becoming increasingly important for the environmental conditions monitoring. Thus, for example, plant indicators are used to assess mechanical and acid composition of soils, their fertility, moisture content and salinity.
Based on the literature data, algae (Prasinocladus marinus, Phaeodactylum tricornutum) are known to be formerly used as indicators to assess a degree and pattern of contamination with heavy metals, such
experiments using plants have not yet been conducted.Indication methods of heavy metals using biological objects are today very urgent since they have a low cost and because of an increasing man-made environmental pollution. Industrial emissions are a powerful factor that affects the vegetation cover development. Abnormal concentration chemical pollution has a direct impact on the plant photosynthetic apparatus.
According to Reimers classification, metals are referred as heavy when their density is higher than 8 g/cm3, among them are — Pb, Cu, Zn, Ni, Cd, Co, Sb, Sn, Bi, Hg [1]. Excessive heavy metals concentrations in plants result in anatomical and morphological changes, in physiological and biochemical processes disturbances. Redundancy of heavy metals has a negative influence on organelle of cells by changing their structure and properties.
Trace amounts of some heavy metals (Me2+) are required for plant growth and development.
Copper and zinc ions are in particular involved in the photosynthetic apparatus formation: plastocianine electrons
transporter contains copper while zinc is a part of plastocyanin, and zinc — superoxide dismutase and carbonic anhydrase. However, high concentrations of these metals in water or soil disturb many plant physiological processes; interrupt transportation functions, and various plant organs show destructive processes associated with an oxidative stress
[2], there are also observed ultra-structural changes in the chloroplast membrane system
[3]. Since plants are able to accumulate metal ions in cell envelopes and vacuoles of various tissue and organ cells, a substantial quantity of Me2+ is excluded from an active metabolism and their concentration in various cell compartments may differ by several orders.
Thereby, the interpretation of physiological and structural changes in plant leaf organelles developing in conditions of an excessive Me2+ content in soil requires additional studies because the observed effects may be caused either by an increasing Me2+ concentration in the leaf or by dehydration and/or oxidative damages of the plant as a whole.
Most of metals enter the cell as cations (for example, Zn2+, Cu2+) as a result of an ordinary diffusion, and also via special transport systems [2]: CPx-type ATPases transporting essential and non-essential metals along the plasmatic cell membrane; cation/H+ antiporters; transport proteins of the NRAMP families (natural resistance associated macrophage proteins) [4] and CDF (cation diffusion facilitors) involved in the transportation of ions of Zn. Zn2+, Co2+, Mn2+ h Cd2+, and also the ZIP family proteins (Zinc-regulated transporters, Iron-regulated transporter-like Proteins) transporting ions of Fe2+, Mn2+, Cd2+ h Zn2+ [4]. These proteinstransporters were found in vacuole membranes and in chloroplast enclosures [2-6]. The higher HM content causes a sharp increase in plasmolemma permeability and, consequently, ion misbalance, leads to the loss of turgor and inhibition of the cell metabolism [2].
Mechanisms of cell protection against a HM abundance have been studied partially and include HM adsorption by internal and external enterosorbents, vacuolar compartmentation, activation of transporters exporting HM from cells, induction of proteins-chaperones [7]. Today, the mechanisms of HM effects on the plant structural components where photosynthesis occurs, namely on the chloroplasts membrane system, i.e. on the photosynthetic apparatus, remain studied insufficiently. The chloroplast internal membrane contains grana thylakoids
and stroma thylakoids. Near the other components of the chloroplast membrane there is an important enzyme of their functioning, carbonic anhydrase (CA). The enzyme CA plays an important role in photosynthetic plant activities and also contributes to C02 transport facilitation and reduction of diffuse resistance to intracellular C02 transfer. It is known that carbonic anhydrase function blocking results from action of HM ions particularly, Zn2+ and Cu2+. Also proceeding from that, it can be assumed that carbonic anhydrase activity reduction may be a direct or indirect cause of photosynthesis disturbances. The aim of our studies was to estimate HM effects on the photosynthetic apparatus in pea seedling leaves.
14-day leaves of pea seedlings were used in our experiments. The leaf blades of Pisum sativum L. were soaked at 22 °C and lighting of 15 pM quanta/( m2xs) for 2.5 days in Petri dishes in versions: in distillates, 250 pmM CuSO4 or 400 pmM ZnCl2. From leaves exposed to CA inhibitor solutions there were cut out 2-3 cm long segments. The preliminary fixation was done in conditions of sample vacuum infiltration in 1% glutaraldehyde on 0,1 M phosphate buffer (pH 7,0) at the room temperature. Samples fixation was carried out with 2,5% glutaraldehyde on 0,1 M cacodylate buffer, pH 7.6 for 4 hours at 4 °C. After washing in the same buffer (2 times for 20 min), the material post-fixation was conducted in solution of 1% OsO4 on 0,1 M cacodylate buffer, pH 7,6, for one night at 4 °C.
A dehydration was done by sample washing in ethyl alcohol at increasing concentrations and in acetone. Then the material was soaked in epoxide resins and acetone mixture, poured in Epon-Araldite mix resin and transferred to thermostat for polymerization at 60 °C. Ultra-thin cell sections were obtained using ultramicrotome LKB-IV (LKB, Sweden). Sections were stained with uranyl acetate and potassium permanganate mixture (1:1) for 15 min in darkness. Ultra-thin sections were studied and filmed using transmission electron microscope JEM-1300 (JEOL, Japan). Preparation sections images were filmed by film for electron microscopy EB19H (AGFA, Belgium). After processing to analyze chloroplasts pictures and their segments morphometrically, the negatives were scanned. Pictures were made using the computer software Adobe Photoshop 7.0 and Corel Photo-Paint 11. 30-39 grana thylakoids enlarged x10 000, or x15 000, and x100 000
Thylakoid thickness and width of interthylakoid gaps
Variant Control 80 pM Cu2+ 200 pM Zn2+
Thylakoid thickness, nm 4.7 ± 0.55 5.21 ± 0.55* 8.46 ± 0.86*
Width of interthylakoid gaps, nm 3.12 ± 0.50 3.43 ± 0.23 ** 4.37 ± 0.69**
Note. * — P < 0.05 relative to control (80 pM Cu2+); ** — P < 0.05 relative to control (200 pM Zn2+).
were analyzed for each experiment version. The thylakoid dimensions on section pictures were measured using the free software UTHSCSA ImageTool 3.0. Statistic methods of data processing were also applied. The reliability of average values difference between test and control versions was evaluated by means of t-Student criterion. The difference was regarded statistically reliable at Р < 0,05.
Table shows data on calculation of thylakoid thickness and width of interthylakoid gaps.
The picture (Fig., a) presents chloroplast segments from control version showing clear-cut individual grana, plastoglobules, and thylakoids of stroma. The significantly enlarged picture presents an intact granum that consists of 42 thylakoids at the terminal segments of which there are attached stroma thylakoids (Fig., b). Under exposure to Cu2+ (Fig., c, d), with a preserved granum general structure and uniform thylakoid packing in grana, the thylakoid of grana thickness exceeded that of control by 11%, while interthylakoid gap thickness — by 10%. Following the chloroplast treatment with Zn2+ (Fig., e, f) there were observed some heterogeneity in grana thylakoid packing, granum structure changes that resulted in increase of thylakoid gaps by 14%, grana thylakoid thickness also increased by 18% as compared to control.
Chloroplast ultrastructure disturbances involving HM are one of the important causes of plant pigment content reduction and decrease in photosynthesis intensity [8]. Chloroplast membranes were found to contain proteins-transporters Zn/Cd that is an evidence of HM possible occurrence in these organelles [6] and, consequently, their direct effect on the chloroplast ultrastructure organization [3, 9, 10]. The photosystem II (PS II) is the most sensitive to metal ions [11]. Variation in the response center proteins structure is assumed to be the reason why PS II activity decreases when heavy metals occur in high concentrations [12]. There are available literature data indicating that heavy metals have a direct impact on electros transport in
_
¡00 run
Fragments of chloroplasts:
a, b — control (HC x30 000, x100 000); c, d — Cu2+ (x15 000, x100 000); e, f — Zn2+ (x 15 000, x 100 000); G — grana;
GT — grana thylakoids; PG — plastoglobules; ST — stroma thylakoids
photochemical reactions [11]. High metals concentrations reduce CO2 assimilation intensity both as a result of a direct action of their ions on some photosynthetic reactions and due to an indirect influence on other physiological processes. Insignificant changes in indices of thickness increase of thylakoids and interthylakoid gaps as compared to the spinach results we obtained previously [13, 14] may be related to the fact that heavy metal ions do not accumulate in chloroplasts despite the fact that their membranes were found to have Cd/Zn transporting ATPase, indicating that metals are probably delivered to these organelles [6]. Chloroplasts are able to accumulate heavy metals in protoplasts i.e. when plant cells have no cell wall [15].
Thus, heavy metals can affect photosynthesis that causes a reduction in photosynthetic pigments content and in carbonic anhydrase activity, which breaks the ultrastructure. The results obtained are important for understanding the mechanisms of heavy metals toxic effects, estimation of plant potential to adapt to unfavorable factors of the environment and may be used in solving the problems of the theory of plant resistance to extreme factors action.
Results of studies on chloroplast structural and functional changes may be used as markers in early diagnostics of abiotic stress effects, namely heavy metals, and also in biotechnological researches to produce novel, advanced technology crops resistant to stresses.
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СТРУКТУРНО-ФУНКЦЮНАЛЬШ ЗМ1НИ
ХЛОРОПЛАСТ1В ЯК Б1ОМАРКЕРИ ЗАБРУДНЕННЯ ВАЖКИМИ МЕТАЛАМИ
М. В. Водка Н. А. БЫявська
1нститут ботанши iM. М. Г. Холодного НАН Укра'ни, Ки'!в
E-mail: marinavodka@yandex.ru
Метою роботи було тдтвердження гшо-тези щодо можливостi використання струк-турно-функцiональних змiн хлоропластiв вищих рослин як бiомаркерiв для ощнки за-бруднення важкими металами.
Ультраструктурнi змiни хлоропластiв Pisum sativum L. реестрували методом транс-мiсiйноi електронно'! мiкроскопii. У робота дослщжено реакцiю ультраструктури хло-ропласйв на пiдвищений вмiст мiдi (80 цМ) i цинку (200 цМ). Отримано данi про змiни у структурi хлоропластiв, зокрема неоднорщ-шсть упаковки гран тилакоiдiв, зб^ьшен-ня мiжтилакоiдних промiжкiв i товщини гран тилаковдв хлоропластiв порiвняно з контролем. Результати дослщжень стосов-но структурно-функщональних змiн хло-ропластiв можна застосовувати як маркери за ранньо'! дiагностики впливу абiотичних стресiв, зокрема важких металiв, а також у бютехнолопчних дослiдженнях при ство-реннi нових стшких до стресiв високотех-нолопчних сортiв сiльськогосподарських культур.
Ключовi слова: Pisum sativum L., змши хлоропласт1в, забруднення важкими метала-ми, б1омаркери.
СТРУКТУРНО-ФУНКЦИОНАЛЬНЫЕ ИЗМЕНЕНИЯ ХЛОРОПЛАСТОВ КАК БИОМАРКЕРЫ ЗАГРЯЗНЕНИЯ ТЯЖЕЛЫМИ МЕТАЛЛАМИ
М. В. Водка, Н. А. Белявская
Институт ботаники им. Н. Г. Холодного НАН Украины, Киев
E-mail: marinavodka@yandex.ru
Целью работы было подтверждение гипотезы о возможности использования структурно-функциональных изменений хлоро-пластов высших растений в качестве биомаркеров для оценки загрязнения тяжелыми металлами.
Ультраструктурные изменения хлоро-пластов Pisum sativum L. регистрировали методом трансмиссионной электронной микроскопии. В работе исследована реакция ультраструктуры хлоропластов на повышенное содержание меди (80 цМ) и цинка (200 цМ). Получены данные об изменениях в структуре хлоропластов, в частности неоднородность упаковки гран тилакоидов, увеличение межтилакоидных промежутков и толщины гран тилакоидов хлоропластов по сравнению с контролем. Результаты исследований о структурно-функциональных изменениях хлоропластов можно применять в качестве маркеров при ранней диагностике влияния абиотических стрессов, в частности тяжелых металлов, а также в биотехнологических исследованиях при создании новых устойчивых к стрессам высокотехнологичных сортов сельскохозяйственных культур.
Ключевые слова: Pisum sativum L., изменения хлоропластов, загрязнение тяжелыми металлами, биомаркеры.
