Acta Botanica 1-2017 - za web.indd ACTA BOT. CROAT. 76 (1), 2017 107 Acta Bot. Croat. 76 (1), 107–110, 2017 CODEN: ABCRA 25 DOI: 10.1515/botcro-2016-0043 ISSN 0365-0588 eISSN 1847-8476 Short communication Temperature-dependent chlorophyll accumulation and photosystem II assembly during etioplast to chloroplast transition in sunfl ower cotyledons Hrvoje Lepeduš1*, Mario Jakopec2, Jasenka Antunović Dunić3, Goran Krizmanić4, Sanida Osmanović5, Vera Cesar3 1 Faculty of Humanities and Social Sciences, Josip Juraj Strossmayer University of Osijek, Lorenza Jägera 9, 31000 Osijek, Croatia 2 Vodovod Osijek, Poljski put 1, 31000 Osijek, Croatia 3 Department of Biology, Josip Juraj Strossmayer University of Osijek, Ulica cara Hadrijana 8/A, 31000 Osijek, Croatia 4 Agricultural Institute Osijek, Južno predgrađe 17, 31000, Osijek, Croatia 5 Faculty of Natural Sciences, University of Tuzla, Univerzitetska 4, 75000 Tuzla, Bosnia and Herzegovina Abstract – Despite numerous data dealing with the biogenesis of photosynthetic membranes connected with specifi c functional alterations in higher plants this is still an insuffi ciently understood topic and is one of the most promising areas of research in plant biochemistry. The main goal of our study was to detect the impact of different temperatures on chlorophyll biosynthesis and the maximum quantum yield of PSII (Fv/Fm). There- fore, we investigated the greening processes in etiolated sunfl ower cotyledons (Helianthus annuus L.) grown at different temperatures (10, 20 and 30 °C) during 24 h. The dynamics of chlorophyll a and b (Chl a and Chl b) accumulation as well as photosystem II (PSII) effi ciency were observed. We also evaluated combined ef- fects of different temperatures (20 and 30 °C) and short-term application of increased irradiation (800 μmol m–2 s–1) on effective quantum yield of PSII (ΔF/F’m) and non photochemical quenching (NPQ) in cotyledons with fully developed PSII. Our results showed reduced chlorophyll accumulation and the arrest of PSII as- sembly at 10 °C in comparison with 20 and 30 °C. Further, the increased irradiance induced equal down regu- lation of effective quantum yield of PSII at 20 and 30 °C, with signifi cantly higher capability of heat dissipa- tion at 30 °C. Keywords: chlorophyll, chloroplast biogenesis, photosynthesis, photosystem II, thylakoid membranes * Corresponding author, e-mail: hlepedus@yahoo.com Introduction Thylakoid membranes in chloroplasts of higher plants are arranged in a highly fl exible way so as to conduct effi - cient photosynthesis (Tikkanen and Aro 2014). They com- prise several multiprotein complexes responsible for key photochemical processes such as light harvesting, trapping of excitons and electron transport to its fi nal acceptor (NADP+). A lot of studies have been undertaken with the aim of elucidating the de novo development of thylakoid membranes with special emphasis on the precise order of multiprotein complex assembly as well as on their interplay with photosynthetic pigments and their precursors (Ru- dowska et al. 2012). Still, some key processes have re- mained insuffi ciently understood and thus became much- discussed topics in scientifi c debates (Pudelski et al. 2009). However, it is well known that the key step in the transition of etioplast to chloroplast is regulated by catalytic activity of protochlorophyllide oxidoreductase A (POR A). The en- zyme was found in etioplast prolamellar bodies and was shown to catalyze the reduction of protochlorophyllide to chlorophyllide in angiosperm plants upon exposure to light (Reinbothe et al. 2010). One of the key regulatory compo- nents of the thylakoid electron-transport chain is chloro- plast water-plastoquinone oxidoreductase, known as photo- system II (PSII), which is divided into three functionally distinct parts: antennae, reaction centre (RC) and oxygen evolving complex (OEC) (Barber et al. 1997). The effi cien- cy of PSII can be quantifi ed thanks to the fl uorescence fea- ture of chlorophyll molecules. The methodology enables determination of the maximum quantum yield of PSII (Fv/ Fm) refl ecting the light-harvesting effi ciency of PSII and is widely used to evaluate its general functionality (Schreiber et al. 1995). LEPEDUŠ H., JAKOPEC M., ANTUNOVIĆ DUNIĆ J., KRIZMANIĆ G., OSMANOVIĆ S., CESAR V. 108 ACTA BOT. CROAT. 76 (1), 2017 The aim of our study was to investigate the dynamics of chlorophyll a and b accumulation as well as PSII effi ciency during greening process in etiolated sunfl ower cotyledons exposed to the different temperatures (10, 20, 30 °C). Since it is generally well known that low temperatures inhibit en- zymatic activity, we hypothesized that the lowest tempera- ture would result in slower accumulation of chlorophylls in developing chloroplasts. However, we aimed to place par- ticular emphasis on the way this would be refl ecteed in the Fv/Fm and Chl a/b ratio, the important parameter that infl u- ences PSII stability (Sakuraba et al. 2010). Also, we en- deavored to evaluate the effects of greening temperature and short-term application of increased irradiation on the effective quantum yield of PSII (ΔF/F’m) and its capacity for non photochemical quenching (NPQ) as very important protective process against photoinhibition. Materials and methods Sunfl ower (Helianthus annuus L.) seedlings were ger- minated in the darkness at room temperature for 7 days. Etiolated cotyledons were then removed from the dark- grown plants and subjected to greening by exposure to 100 μmol m–2 s–1 of white light (Osram Powerstar HQI BT 400 W/D E40), for 3, 6, 12 and 24 h. Three separate experi- ments were done by changing the temperature during greening process: 10, 20 and 30 °C. Chlorophylls a and b were quantifi ed spectrophotometrically, and concentrations were calculated according to Lichtenthaler (1987). In vivo chlorophyll a fl uorescence measurement (Mini-PAM, Waltz) was used to quantify several parameters of photosystem II (PSII) effi ciency: the maximum quantum yield of photosys- tem II (Fv/Fm), the effective quantum yield of photosystem II (ΔF/F’m) and non-photochemical quenching (NPQ) at 800 μmol m–2 s–1 (Schreiber et al. 1995). A Student t-test as well as factorial analysis of variance followed by least sig- nifi cance difference (LSD) test was performed to analyze statistical signifi cance (Statistica 7.1.). Results and discussion Chloroplast biogenesis, usually seen as greening (Fig. 1), is characterized by coordinated biosynthesis of photo- synthetic pigments and other molecules essential for the as- sembly of a fully functional photosynthetic apparatus. Since functional chloroplasts contain up to 4 thousand dif- ferent proteins, most of them encoded by nuclear genes, it is likely that different environmental signals (e.g. light or temperature) would have a great infl uence on signaling dur- ing chloroplast biogenesis (Fey et al. 2005). In order to quantify green color appearance after a certain time of greening, changes in Chl a and b concentrations were mea- sured. Our results (Tab. 1) revealed a permanent increase of Chl a during 24 h of greening at temperatures of 20 and 30 °C, as well as a constant increase of Chl b at 30 °C. Also, delay in Chl a accumulation was observed at 10 °C, as well as in Chl b accumulation at 10 and 20 °C during the fi rst 6 h of greening. Further, Chl b accumulation at 10 and 20 °C was slowed down after 12 h of greening. Generally, inferior chlorophyll biosynthesis was revealed in greening cotyle- dons at lower temperatures, especially at 10 °C. This was also evident from the value of Chl a/b ratio after 24 h of greening, which was lower in cotyledons that were green- ing at 10 °C in comparison to these that were greening at 20 and 30 °C (Tab. 1). Tewari and Tripathy (1998) revealed 90% inhibition of Chl biosynthesis during greening of etio- lated cucumber seedlings at 7 °C caused by the inhibition of enzymes involved in protoporphyrin IX biosynthesis, which directly decreased the biosynthesis of protochloro- phyllide. So, the lower chlorophyll concentrations in such cases would be the result of diminished biosynthesis. Fur- ther, low temperatures diminish CO2 fi xation and thus in- crease the chance for over-reduction of the electron-trans- port chain and consequently for photo-damage of PSII due to inhibition of the repair cycle of D1 protein, the essential protein for optimal PSII functioning (Giardi et al. 1997). Fig. 1. Greening of etiolated sunfl ower cotyledons (0 h) exposed to different temperatures (10 °C, 20 °C, 30 °C) and the light inten- sity of 100 μmol m–2 s–1 for 24 h. Color changes were recorded af- ter 3, 6, 12 and 24 h. Tab. 1. Changes in concentrations of chlorophyll a (Chl a), chlo- rophyll b (Chl b) and chlorophyll a/b ratio (Chl a/b ) in sunfl ower cotyledons, measured after 3, 6, 12 and 24 h of the greening period, at 10 °C, 20 °C and 30 °C. Data represent arithmetic means±stan- dard deviations of ten replications. Factorial analysis of variance followed by least signifi cance difference test was done separately for each temperature. Different lowercase letters indicate a signifi - cant difference at P<0.05. FW – fresh weight. G re en in g te m pe ra tu re L ig ht ex po su re Chl a (mg g–1 FW) Chl b (mg g–1 FW) Chl a/b 10 °C 3 h 0.034±0.012a 0.047±0.022a 0.828±0.372a 6 h 0.027±0.007a 0.039±0.015a 0.747±0.214a 12 h 0.053±0.013b 0.080±0.024b 0.691±0.172a 24 h 0.078±0.013c 0.084±0.025b 0.958±0.172a 20 °C 3 h 0.065±0.010a 0.057±0.019a 1.311±0.586a 6 h 0.122±0.033b 0.064±0.022a 2.018±0.526b 12 h 0.253±0.048c 0.097±0.034b 2.747±0.654c 24 h 0.336±0.079d 0.113±0.035b 3.048±0.374c 30 °C 3 h 0.082±0.011a 0.066±0.024a 1.352±0.383a 6 h 0.172±0.037b 0.097±0.042b 1.916±0.493b 12 h 0.372±0.084c 0.125±0.024c 2.967±0.264c 24 h 0.440±0.046d 0.166±0.025d 2.707±0.473c TEMPERATURE DEPENDENT CHLOROPLAST BIOGENESIS ACTA BOT. CROAT. 76 (1), 2017 109 Changes in maximum quantum yield of PSII (Fv/Fm) are shown in Fig. 2. Continuous increase of Fv/Fm was observed during fi rst 12 h of greening at 10 °C (Fig. 2A). However, the upcoming arrest of PSII assembly that was observed af- ter 24 h of greening at 10 °C (Fig. 2) was most likely due to the inability of thylakoid membranes to accumulate de novo synthesized chloroplast encoded proteins (such as D1) rela- tively to nuclear encoded ones (Allen and Ort 2001). This would cause improper chlorophyll-protein complex assem- bly as well as the formation of fully competent grana (Ru- dowska et al. 2012) required for Fv/Fm values usually ob- served in mature leaves. Such a scenario consequently led to the irreversible photoinhibition that was revealed (Fig. 2A). Values of the Fv/Fm in fully competent and healthy photosynthetic leaves are usually reported to be about 0.83 (Schreiber et al. 1995). Bolhar-Nordenkampf et al. (1989) reported the Fv/Fm of 0.75 as the lowest boundary value of PSII functionality. Judging from our results (Fig. 2B, C), cotyledons that were greening at 20 and 30 °C developed fully functional PSII. Muraja Ljubičić et al. (1998) reported parallel increase of light-harvesting proteins of PSII (LH- CII) with chlorophyll accumulation during greening of po- tato. At the temperature of 30 °C PSII functionality was achieved after as little as 6 h of greening (Fv/Fm=0.77), while at the temperature of 20 °C it needed a longer period (24 h for Fv/Fm to be 0.76). Increased accumulation of Chl a and b, as well as increased Chl a/b ratio in these cotyledons compared to those grown at 10 °C was required for the as- sembly of functional PSII. Since the measurement of Fv/Fm revealed functional PSII in cotyledons subjected to greening at 20 and 30 °C, additional characterization of PSII effi ciency was per- formed. After 24 h of greening cotyledons were exposed to short-term high irradiation (800 μmol m–2 s–1) and the effec- tive quantum yield of PSII (ΔF/F’m) as well as non-photo- chemical quenching (NPQ) were measured (Fig. 3). Al- though there were no differences in ΔF/F’m upon exposure to high irradiation between cotyledons that were greening at 20 and 30 °C, cotyledons that were greening at 30 °C re- vealed a better capacity for NPQ than cotyledons that were greening at 20 °C (Fig. 3). NPQ represents the measure for dissipation of the excess excitation energy as heat relative to dark adapted state (Maxwell and Johnson 2000). Precise mechanisms of NPQ are under constant debate and new evidence is being continuously provided (Jurić et al. 2013). Basically, suffi cient amounts of xanthophyll cycle carot- enoids and structural rearrangement of PSII antennae are essential for effi cient NPQ. Bilger and Björkman (1991) showed up-regulation of the rate of violaxanthin de-epoxi- dation by temperature increase. It seems that higher tem- perature during greening process (30 °C) of etiolated sun- fl ower cotyledons favored zeaxanthin formation, thereby enabling more effi cient heat dissipation by NPQ. It can be concluded that low temperature (10 °C) de- creased biosynthesis of Chl a and Chl b in comparison to two higher investigated temperatures, which was directly linked with impaired assembly of PSII after a prolonged pe- riod of greening (24 h) at 10 °C. Lebkuecher et al. (1999) reported that assembly of PSII during the transition from etioplast to chloroplast in sunfl ower cotyledons at 25 °C was accompanied by small initial LHCII complexes and re- action centers (RCs) that are defi cient in QA to QB electron transport. Further greening in their experiment (up to 12 h) enabled conversion of QB-nonreducing to QB-reducing RCs and effi cient electron transport. Hence, it is most likely that the arrest of PSII assembly after 24 h of greening at 10 °C that we have observed was due to over-reduction damage in the PSII reaction center. Also, fully developed PSII at tem- Fig. 2. Changes in maximum quantum yield of PSII (Fv/Fm) in sunfl ower cotyledons, measured after 3, 6, 12 and 24 h of the greening period, at 10 °C (A), 20 °C (B) and 30 °C (C). Data rep- resent arithmetic means±standard deviations of fi ve replications. Differences between presented values were evaluated using least signifi cance difference test. Different lowercase letters indicate a signifi cant difference at P<0.05. Fig. 3. The effect of short-term high irradiation (800 μmol m–2 s–1) on effective quantum yield of PSII (ΔF/F’m) and non-photochemi- cal quenching (NPQ) of sunfl ower cotyledons, after 24 h of green- ing at different temperatures (20 °C and 30 °C). Data represent arithmetic means±standard deviations of fi ve replications. Differ- ences between presented values were evaluated using Student’s t- test. Asterisk (*) indicate a signifi cant difference at P<0.05. LEPEDUŠ H., JAKOPEC M., ANTUNOVIĆ DUNIĆ J., KRIZMANIĆ G., OSMANOVIĆ S., CESAR V. 110 ACTA BOT. 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