Short-term heating causes thylakoid restructuring in pea chloroplasts and modifies spectral properties of pigment-protein complexes

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Short-term heating causes thylakoid restructuring in pea chloroplasts and modifies spectral properties of pigment-protein complexes

V.V. Shevchenko, O.Yu. Bondarenko, D.Yu. Kornyeyev

institute of Plant Physiology and Genetics, National Academy of Sciences of Ukraine

31/17 Vasylkivska St., Kyiv, 03022, Ukraine

2Texas Tech University, Lubbock, TX 79409, USA

The effect of short-term heating on ultrastructure of the pea chloroplasts and the spectral characteristics of the subchloroplast fragments has been investigated. The size of chloroplasts decreased at heating for 5 min in the darkness at either 25, 35 or 45 °C. The phenomenon was caused mainly by a decrease of the length of long axis of chloroplast. The extent of the reduction was less after the heating at 45 °C in the presence of light. Detailed analysis has been conducted for heating at 45 °C because more prominent changes in circular dichroism (CD) spectra have been observed indicating the greater effect of heating. A decrease in у-bands of the spectrum has indicated a disturbance in long range ordering in arrangement of membrane macrodomains. The quantity of grana with an increased number of thy- lakoids (more than 6) became greater after the heating of isolated chloroplasts. Low temperature fluorescence spectra of chloroplasts showed a decrease in intensity of both bands of the spectrum. The spectra of subchloroplast fragments corresponding to the grana particles revealed an increase in intensity of both bands. An increase of the short-wavelength band belonging to emission of PSII, indicates an increase of quantity of PSII in grana particles originated from heated chloroplasts. An increase of the long-wavelength band belonging to PSI emission, was caused by an increasing of the size of antenna, that was detected by the rise of intensity in excitation spectrum of fluorescence detected at 735 nm. A broadening of short-wavelength band which was greater after the heating in the presence of the illumination was evoked by a loosening of the PSII complex antenna. An analysis of the spectral characteristics of the fragments of the granal thylakoids margin region provided additional data about the changes in the grana organization caused by heating. The obtained data are in line with the hypothesis that links changes in the organization of the thylakoid membranes to the interaction between PSII and the «mobile antenna» represented by light-harvesting complex II (LHCII). It has also been demonstrated that illumination mitigates the effect of short-term heating.

Key words: Pisum sativum L., short-term heating, chloroplasts ultrastructure.

Досліджено вплив короткочасного прогріву на ультраструктуру хлоропластів гороху та спектральні характеристики субхлоропластних фрагментів. Розмір хлоропластів зменшувався при нагріванні протягом 5 хв у темряві за 25, 35 або 45 °C. Явище було спричинене переважно зменшенням довжини довгої осі хлоропласта. Ступінь змен-шення знижувався після нагрівання за 45 °С на світлі. Детальний аналіз був проведений для нагрівання за 45 °C, оскільки спостерігались помітніші зміни в спектрах кругового дихроїзму (CD), що свідчить про більший ефект нагрівання. Зменшення ^-смуг спектра свідчить про порушення великомасштабного впорядкування в розміщенні мембранних макродоменів. Кількість гран зі збільшеною кількістю тила- коїдів (понад 6) зросла після нагрівання ізольованих хлоропластів. Низькотемпературні спектри флуоресценції хлоропластів показали зменшення інтенсивності обох смуг спектра. Спектри субхлоропластних фрагментів, що відповідають частинкам грани, виявили збільшення інтенсивності обох смуг. Збільшення інтенсивності короткохвильової смуги, що належить до випромінювання PSII, свідчить про зростання кількості PSII у частинках грани, отриманих із прогрітих хлоропластів. Підвищення інтенсивності довгохвильової смуги, що належить до випромінювання PSI, спричинене збільшенням розміру антени, яке було встановлено за зміною інтенсивності спектра збудження флуоресценції, детектованого за 735 нм. Після нагрівання при наявності світла спостерігалось розширення короткохвильової смуги, яке було викликане зменшенням антени комплексу PSII. Аналіз спектральних характеристик фрагментів крайової ділянки гранальних тилакоїдів дав змогу отримати додаткові дані про зміни в організації грани, спричинені нагріванням. Отримані дані узгоджуються з гіпотезою, яка пов'язує зміни в організації тилакоїдних мембран із взаємодією між PSII та «мобільною антеною», представленою світлозбиральним комплексом II (LHCII). Також було продемонстровано, що освітлення пом'якшує ефект короткочасного прогріву.

Ключові слова: Pisum sativum L., короткочасний прогрів, ультраструктура хлоропластів.

The progress in our understanding of the chloroplast response to the shortterm heating relies on the accumulation of the data on the heat-induced changes in various characteristics of photosynthetic apparatus, and subsequent generation of the hypotheses regarding the mechanisms behind these changes [1, 2, 3]. Several important experimental reviews have been published by now. It was reported partial destacking of grana thylakoids in bean chloroplasts. Changes in the spatial orientation of grana and in their number per chloroplast have been described for winter wheat plants [4, 5]. Number of thylakoids per grana can also be modified upon heat treatment. The chiral LHCII macrodomains in intact thylakoid membranes have been shown to gradually disassemble after heating at temperatures above 40 °C [6]. Short-term heating also led to alterations in the antenna complexes of both photosystem I (PSI) and photosystem II (PSII) [7, 8]. Interestingly, illumination during heat treatment has been shown to have protective effect on the functional activity of PSII and PSI [9, 10, 11].

In this study, we provide additional experimental evidence of the modifications of the pea chloroplasts ultrastructure after short-term heating and test the hypothesis that designates the light-harvesting complex II (LHCII) as a key player controlling restructuring the chloroplast membranes during the heat stress. Several techniques (electron microscopy, circular dichroism spectra, and low-temperature spectroscopy) were applied to obtain the information related to the different hierarchical organization levels of the organelles performing photosynthetic function in higher plants.

Material and methods

thylakoid restructuring pea chloroplast

Plant material. The pea (Pisum sativum L.) plants were grown outdoors or in greenhouse for two weeks. The leaves of two upper layers were used. The chloroplasts with partially broken sheath were isolated as described earlier [12]. The isolated chloroplasts were suspended in the medium containing 10 mM Tricine buffer (pH 7.5), 0.4 M sucrose, 10 mM NaCl, and 5 mM MgCl2. Subchloroplast fragments were obtained by solubilization of the chloroplasts with 0.3 % digitonin, and the subsequent differential centrifugation according to the previously published procedure [13]. The particles sedimented at 1000 g and 40000 g after 20000 g were used for the measurements. Chlorophyll concentration (Chl) was estimated spectrophotomet- rically in 80 % acetone extracts [13] and the yield of the fractions was calculated as the ratio of the total Chl in the particles to the total Chl in chloroplast suspension taken for fragmentation. Chl a/b ratio was calculated as described [13].

Temperature treatment. The whole leaves were placed into a transparent flexible polyethylene bag and submerged in the water warmed up to a designated temperature. While being under water, the leaves were in the darkness or under illumination with the white light (260 gmol/(m2 ¦ s)). The suspension of isolated chloroplasts was let to settle down in a flat-bottomed flask to form a thin layer. Then the flask was placed in the water at certain temperature (25, 35 or 45 °C) and incubated either in the darkness or under the white light (260 gmol/(m2 ¦ s)) for 5 min. The control sample was kept at 5 °C in the darkness. Chlorophyll content in the chloro- plast suspension used for measurements of low temperature fluorescence spectra and CD spectra was 30 mg/ml and 10 gg/ml, respectively.

Electron microscopy. The preparation of the samples for the electron microscopy was as following: the control and heated leaves were placed into the agarose blocks and fixed with 3 % glutaraldehyde solution in cacodylate buffer. An additional fixation was performed in 1 % osmium tetroxide solution prepared in the same buffer. Then agarose blocks were embedded into Epon--Araldite mixtures and polymerized. Ultrathin sections of fixed material were obtained with an UMTP-6M microtome (Slmi, RF) and stained with lead citrate. Digital images were acquired with a JEM 1230 electron microscope (JEOL, Japan). About 30 images of chloroplasts were selected in a random manner from the pictures of the leaves ultrathin sections for further measurements of the chloroplasts spatial characteristics. The measurements were performed using MapInfo software modified for the analysis of the microscopic images. To obtain the frequency diagram of the thylakoids number per grana, about 80 grana images from the ultrathin sections of the chloroplasts have been processed.

Spectral measurements. Circular dichroism spectra were measured with Jobin Yvon CD6 dichrograph. The measuring cuvette was placed into a thermostated sample holder. The length of optical path within the cuvette was 1 cm. The distance between the sample and a photomultiplier was 5 cm. CD spectra were recorded with a sampling step of 1 nm, the integration time of 0.3 s, and the band-pass length of 2 nm.

Low-temperature fluorescence spectra were measured using air-dried thin films of chloroplasts deposited on a glass disc. The films were prepared as described earlier [14] and placed into an optical cryostat filled with vapours of liquid nitrogen. The fluorescence spectra were measured as described earlier [13] using modified lab-made setup. The films were highly transparent and their low temperature fluorescence spectra proved to be identical to those of corresponding to frozen buffer-glycerol suspension [13]. Light absorption by the samples in a peak of the red band did not exceed 15 %.

Fluorescence was excited with LED light at 450 nm and was recorded at wavelengths from 650 to 800 nm with a sampling step of 0.5 nm. Bandpass of monochromator was 1 nm. Intensity of the actinic light which passed through the sample was monitored using an additional optical channel at the time of recording the fluorescence spectrum of the sample. These data were used for the normalization of the fluorescence spectra to the spectrum of the control sample, to remove small variations in the light absorbance of various samples.

Excitation spectra of fluorescence were obtained at the detection wavelength of 735 nm for the same samples as for the measurement of the fluorescence spectra as described earlier [13]. The samples were illuminated with the monochromatic light in the interval 630--720 nm using an excitation monochromator with the band-pass of 2 nm and a sampling step of 0.5 nm. The fluorescence at 735 nm was detected with band-pass equal to 4 nm using an emission monochromator.

The means were calculated from five replicates. The tables and the figures show the arithmetic mean and standard error of the mean. The obtained data were processed by generally accepted methods of variation statistics.

Results and discussion

The analysis of the electron microscopy images implies that the heat- induced changes in the lengths of the long and short axes of pea chloro- plasts varied depending on the mode of treatment. The cross section of the chloroplasts decreased after heating in the darkness with the increase of the temperature maintained during the treatment (Fig. 1). After the short-term heat treatment, the length of the long axis decreased whereas the length of the short axis increased. The higher was the temperature the greater were these changes. The effect of the heating at 45 °C was more pronounced for the leaves kept in the darkness in comparison to the illuminated ones.

Fig. 1. Changes (experiment/control) of cross section of chloroplast (1), lengths of the long (2), and the short (3) axes

The observed alterations in lengths of the axes mainly resulted from changes in the grana organization. The most prominent change was in the number of the thylakoids per stack. The typical image of the heat-treated grana is shown on Fig. 2, b. The percentage of the grana consisting of the large quantity of the thylakoids was noticeably higher in case of heating in the darkness (Fig. 3) when compared to samples heated under illumination. The majority of the grana from the control samples had small number of thylakoids (7 or less). The relative amount of the small grana decreased after heating whereas the percentage of the large grana (8 or more thylakoids per stack) increased. Quite a few grana containing 17--23 thylakoids were observed in the heated samples but none was detected in control ones.

Fig 2. Electron microscopy images of typical configurations of grana in control chloroplasts (a), chloroplasts which are heated at 45 °C in the darkness (b), and at light (c)

Fig. 3. Changes in a distribution of granas containing various number of thylakoids in the control sample (1), in the heated ones in the darkness (2), and in the heated ones at light (3)

CD signals originate from molecular systems of different complexity, such as granal thylakoid membranes or lamellar aggregates of LHCII. PSII and LHCII in the granum forms chirally organized macrodomains (e.g., the so-called psi-type (y) aggregates), large arrays with diameters of several hundred nanometers and long-range chiral order. These chiral macrodomains have also been shown to be capable of undergoing termo-induced reversible structural changes. In chirally organized macroarrays, with sizes commensurate with the wavelength of the visible light, very intense and «anomalously shaped» so-called y-type CD bands are given rise; y-type bands are exhibited by intact granal thylakoid membranes, in particular by LHCII-containing macrodomains, as well as by lamellar aggregates of LHCII.

Short-term heat treatment induced changes in CD spectra of the pea chloroplasts manifested mainly in the decreased intensity of both y-bands (Fig. 4, Table 1). The most prominent changes occurred after heating at 45 °C. Therefore, this temperature was chosen for further studies performed with subchloroplast particles isolated from chloroplasts subjected to heating in the presence or in the absence of the illumination.

Fig. 4. CD spectra of chloroplasts of control sample (1), and of heated in the darkness at 25, 35, 45 °С (2, 3, 4, respectively)

TABLE 1. Changes in ф-bands of CD spectra of chloroplasts, heated at various modes of-treatment