Contrasting Behavior of Higher Plant Photosystem I and II Antenna Systems during Acclimation
2007; Elsevier BV; Volume: 282; Issue: 12 Linguagem: Inglês
10.1074/jbc.m606417200
ISSN1083-351X
AutoresMatteo Ballottari, Luca Dall’Osto, Tomas Morosinotto, Roberto Bassi,
Tópico(s)Photoreceptor and optogenetics research
ResumoIn this work we analyzed the photosynthetic apparatus in Arabidopsis thaliana plants acclimated to different light intensity and temperature conditions. Plants showed the ability to acclimate into different environments and avoid photoinhibition. When grown in high light, plants had a faster activation rate for energy dissipation (qE). This ability was correlated to higher accumulation levels of a specific photosystem II subunit, PsbS. The photosystem II antenna size was also regulated according to light exposure; smaller antenna size was observed in high light-acclimated plants with respect to low light plants. Different antenna polypeptides did not behave similarly, and Lhcb1, Lchb2, and Lhcb6 (CP24) are shown to undergo major levels of regulation, whereas Lhcb4 and Lhcb5 (CP29 and CP26) maintained their stoichiometry with respect to the reaction center in all growth conditions. The effect of acclimation on photosystem I antenna was different; in fact, the stoichiometry of any Lhca antenna proteins with respect to photosystem I core complex was not affected by growth conditions. Despite this stability in antenna stoichiometry, photosystem I light harvesting function was shown to be regulated through different mechanisms like the control of photosystem I to photosystem II ratio and the association or dissociation of Lhcb polypeptides to photosystem I. In this work we analyzed the photosynthetic apparatus in Arabidopsis thaliana plants acclimated to different light intensity and temperature conditions. Plants showed the ability to acclimate into different environments and avoid photoinhibition. When grown in high light, plants had a faster activation rate for energy dissipation (qE). This ability was correlated to higher accumulation levels of a specific photosystem II subunit, PsbS. The photosystem II antenna size was also regulated according to light exposure; smaller antenna size was observed in high light-acclimated plants with respect to low light plants. Different antenna polypeptides did not behave similarly, and Lhcb1, Lchb2, and Lhcb6 (CP24) are shown to undergo major levels of regulation, whereas Lhcb4 and Lhcb5 (CP29 and CP26) maintained their stoichiometry with respect to the reaction center in all growth conditions. The effect of acclimation on photosystem I antenna was different; in fact, the stoichiometry of any Lhca antenna proteins with respect to photosystem I core complex was not affected by growth conditions. Despite this stability in antenna stoichiometry, photosystem I light harvesting function was shown to be regulated through different mechanisms like the control of photosystem I to photosystem II ratio and the association or dissociation of Lhcb polypeptides to photosystem I. Plants are exposed to an environment where light and temperature conditions are largely variable. Because they have no possibility of moving to a more favorable environment, plants have evolved several mechanisms of acclimation. Among all the parameters, light intensity has a major influence on plants life; it is a limiting growth factor at dusk or under dense canopy, but it can easily be in excess at midday or under full sun, thus leading to oxidative stress and photoinhibition (1Demmig-Adams B. Adams W.W. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1992; 43: 599-626Crossref Scopus (2081) Google Scholar, 2Barber J. Andersson B. Trends Biochem. Sci. 1992; 17: 61-66Abstract Full Text PDF PubMed Scopus (847) Google Scholar). Low temperature is known to play a synergistic role with excess illumination by limiting electron transport and carbon fixation rates. In these conditions, even a weak light can exceed energy utilization rate and become photoinhibitory (3Huner N.P.A. Oquist G. Sarhan F. Trends Plant Sci. 1998; 3: 224-230Abstract Full Text Full Text PDF Scopus (810) Google Scholar). Mechanisms of photo-protection can be classified according to the time scale of activation upon establishment of the stress. Illumination rapidly activates the dissipation of the excitation energy as heat, a process called nonphotochemical quenching (NPQ) 2The abbreviations used are: NPQ, nonphotochemical quenching; α(β)-DM, n-dodecyl-α(β)-d-maltoside; Chl, chlorophyll; CP, chlorophyll protein; Lhca (b), light harvesting complex of photosystem I (II); LHCI (LHCII), antenna complex of photosystem I (II); PSI (II), photosystem I (II); RC, reaction center; ROS, reactive oxygen species;μE, microeinstein; HPLC, high pressure liquid chromatography; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Rubisco, ribulose-bisphosphate carboxylase/oxygenase; qP, photochemical quenching; VDE, violaxanthin de-epoxidase; Ctrl, control; LL, low light; HL, high light; cLL, cold low light; cHL, cold high light; ZE, zeaxanthin epoxidase. (4Van Kooten O. Snel J.F.H. Photosynth. Res. 1990; 25: 147-150Crossref PubMed Scopus (2072) Google Scholar, 5Niyogi K.K. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 333-359Crossref PubMed Scopus (1619) Google Scholar). Its fastest component is called qE, and it is known to depend on the presence of the PSII subunit PsbS (6Li X.P. Bjorkman O. Shih C. Grossman A.R. Rosenquist M. Jansson S. Niyogi K.K. Nature. 2000; 403: 391-395Crossref PubMed Scopus (1177) Google Scholar). NPQ also includes components activated on a slower time scale such as the synthesis of zeaxanthin from violaxanthin (7Niyogi K.K. Grossman A.R. Björkman O. Plant Cell. 1998; 10: 1121-1134Crossref PubMed Scopus (765) Google Scholar, 8Demmig-Adams B. Biochim. Biophys. Acta. 1990; 1020: 1-24Crossref Scopus (1400) Google Scholar, 9Dall'Osto L. Caffarri S. Bassi R. Plant Cell. 2005; 17: 1217-1232Crossref PubMed Scopus (204) Google Scholar) with zeaxanthin also active in scavenging ROS (10Havaux M. Niyogi K.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8762-8767Crossref PubMed Scopus (568) Google Scholar). These mechanisms are effective in reducing light damage during fast changes in illumination. However, when excess light is experienced during long periods, plants activate other mechanisms of response, such as the modulation of the light harvesting apparatus and metabolic energy sinks. In fact, in high light, the stoichiometry of electron carriers and enzymes of the Calvin cycle increases (11Seeman R.J. Sharkey T.D. Wang J. Osmond B. Plant Physiol. 1987; 84: 796-802Crossref PubMed Google Scholar, 12Gray G.R. Savitch L.V. Ivanov A.G. Huner N. Plant Physiol. 1996; 110: 61-71Crossref PubMed Scopus (159) Google Scholar, 13Walters R.G. Horton P. Planta. 1994; 195: 248-256Crossref Scopus (141) Google Scholar), whereas the relative abundance of antenna proteins with respect to the reaction center complexes decreases (14Anderson J.M. Andersson B. Trends Biochem. Sci. 1988; 13: 351-355Abstract Full Text PDF PubMed Scopus (172) Google Scholar, 15Melis A. Biochim. Biophys. Acta. 1991; 1058: 87-106Crossref Scopus (442) Google Scholar). Here we have extended previous works on the acclimation changes of the model plant Arabidopsis thaliana upon growth in different light and temperature conditions. Functional analysis confirmed that plants were indeed acclimated as shown by the absence of PSII photoinhibition, the modulation of oxygen evolution, and the photochemical capacity in high light. Long term exposure to high light was also shown to induce the accumulation of PsbS and a correlated increase in the qE component of NPQ. Of particular interest is the regulation of the antenna system; the adaptation to different light and temperature conditions leads to extensive changes within the PSII antenna system. Each of the different Lhcb components is tuned in their relative amount with the exception of Lhcb4 and Lhcb5 whose stoichiometry with respect to RCII remained substantially constant. Lhcb6 is peculiar because it undergoes the highest level of modulation, being virtually absent in HL conditions. On the contrary, in PSI no changes were detected in the stoichiometry of any of the Lhca proteins with respect to the reaction center. This suggests that PSI-LHCI behaves as the reference complex with respect to which PSII-LHCII undergoes regulation to maintain chloroplast redox state and ensure photoprotection. Plant Growth and Light Temperature Treatments—A. thaliana plants (Columbia ecotype) were grown for 4 weeks at 100 μE, 19 °C, 90% humidity, and 8 h of daylight. Afterward, they were moved under different light and temperature conditions for an additional 3 weeks. The conditions used are the following: control, 21 °C, 100 μE; low light (LL), 21 °C, 25 μE; high light (HL), 21 °C, 1600 μE; cold low light (cLL), 10 °C, 25 μE; and cold high light (cHL), 10 °C, 600 μE. In all measurements only fully expanded mature leaves were used, and they belonged to the 4th to the 7th leaf pair depending on the time and condition. Chlorophyll Fluorescence and Photosynthetic Parameter Measurements—Chlorophyll fluorescence was measured at room temperature on intact leaves of acclimated plants with a PAM-101 fluorimeter with a saturating light at 4500 μE and actinic light at six different intensities as follows: 100, 360, 640, 1200, 1600, and 2000 μE. Before measurements, plants were dark-adapted for at least 30 min at room temperature. The same measurements were also performed with actinic light of 2000 μE but with an overnight dark adaptation. The parameters Fv/Fm, NPQ, and photochemical quenching (qP) were calculated as (Fm - Fo)/Fo,(Fm - Fm′)/Fm′, and (Fm′-F)/(Fm′-Fo) (16Demmig-Adams B. Adams W.W. Barker D.H. Logan B.A. Bowling D.R. Verhoeven A.S. Physiol. Plant. 1996; 98: 253-264Crossref Scopus (752) Google Scholar). Chloroplast Isolation and Purifications of PSI-LHCI and LHCI—Chloroplasts were isolated from adapted plants by homogenizing leaves in a solution with 0.1 m Tricine/KOH, pH 7.8, 0.4 m sorbitol, 0.5% powder milk. The chloroplasts were then isolated by precipitation at 1500 × g. PSI-LHCI complexes were purified from all plants with the method described previously (17Croce R. Zucchelli G. Garlaschi F.M. Bassi R. Jennings R.C. Biochemistry. 1996; 35: 8572-8579Crossref PubMed Scopus (149) Google Scholar, 18Croce R. Morosinotto T. Castelletti S. Breton J. Bassi R. Biochim. Biophys. Acta. 2002; 1556: 29-40Crossref PubMed Scopus (151) Google Scholar). Spectroscopy and Pigment Analysis—The absorption spectra were recorded using an SLM-Aminco DK2000 spectrophotometer in 5 mm Tricine, pH 7.8, 0.5 m sucrose, and 0.03% β-DM. HPLC analysis was performed according to Ref. 19Gilmore A.M. Yamamoto H.Y. Plant Physiol. 1991; 96: 635-643Crossref PubMed Scopus (218) Google Scholar. Chlorophyll to carotenoid ratio and Chl a/b ratio were independently measured by fitting the spectrum of acetone extracts with the spectra of individual purified pigments (18Croce R. Morosinotto T. Castelletti S. Breton J. Bassi R. Biochim. Biophys. Acta. 2002; 1556: 29-40Crossref PubMed Scopus (151) Google Scholar). CD spectra were measured at 10 °C on a Jasco 600 spectropolarimeter. Chloroplast fluorescence spectra at 77 K were measured with Varian Cary Eclipse and corrected for instrumental response. Samples were in glycerol 60% (v/v) and 10 mm HEPES, pH 7.5. Emission spectra were measured with a 475 nm excitation, whereas excitation spectra were measured with a 735 nm emission. Short Term High Light Stress—Leaves from acclimated plants were transferred in glass tubes in N2 atmosphere and illuminated with light at 1800 μE for 7, 15, and 30 min (20Dainese P. Marquardt J. Pineau B. Bassi R. Murata N. Research in Photosynthesis. Vol. I. Kluwer Academic Publishers Group, Dordrecht, Netherlands1992: 287-290Crossref Google Scholar). Pigment extraction with 80% acetone was then performed on leaf disks frozen in liquid nitrogen. SDS-PAGE Analysis and Coomassie Stain Quantification— PSI-LHCI complexes were analyzed with SDS-PAGE as described in Ref. 21Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar but using a acrylamide/bisacrylamide ratio of 75:1 and a total concentration of acrylamide + bisacrylamide of 4.5 and 15.5%, respectively, for the stacking and running gel, where 6 m urea was also incorporated (22Ballottari M. Govoni C. Caffarri S. Morosinotto T. Eur. J. Biochem. 2004; 271: 4659-4665Crossref PubMed Scopus (56) Google Scholar). The staining for the densitometry was obtained with 0.05% Coomassie Blue R-250 in 25% isopropyl alcohol, 10% acetic acid to improve linearity of the stain with respect to protein amount (23Ball E.H. Anal. Biochem. 1986; 155: 23-27Crossref PubMed Scopus (103) Google Scholar). The protein amount was evaluated after SDS-PAGE by quantifying the stain bound to each band by colorimetry. We acquired the gel image using Bio-Rad GS710 scanner, and the picture was then analyzed with Gel-Pro Analyzer© software, which quantifies the staining of the bands as integrated optical density on the area of the band. At least five repetitions of each sample were analyzed to achieve sufficient accuracy. For the evaluation of Rubisco content, leaf disks were cut, and Chl content was determined with high accuracy. Then the amount of Rubisco, on a Chl basis, was determined by SDS-PAGE and Coomassie staining after identification of the band of Rubisco large subunit by Western blotting. Immunoblot Assays and Western Blotting Quantifications— For the quantification of PsbS, VDE, ZE, Lhcb3–6, and CP47 in different acclimated plants, leaves were homogenized in liquid nitrogen with a solution with 62.5 mm Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 5% β-mercaptoethanol. The chlorophyll content was then quantified by absorption spectra. For each sample 0.2, 0.4, 0.8, 1, and 1.2 μg of chlorophylls have been loaded on SDS-PAGE. Immunoblot assays with antibodies against different polypeptides were performed as described previously (24Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar). To avoid any deviation between different immunoblots, samples were compared only when loaded in the same gel. Deriphat PAGE Analysis—Nondenaturing Deriphat-PAGE was performed following the method described previously (25Peter G.F. Thornber J.P. J. Biol. Chem. 1991; 266: 16745-16754Abstract Full Text PDF PubMed Google Scholar), but using 3.5% (w/v) acrylamide (38:1 acrylamide/bisacrylamide) in the stacking gel and in the resolving gel an acrylamide concentration gradient from 4.5 to 11.5% (w/v) stabilized by a glycerol gradient from 8 to 16%. Thylakoids concentrated at 1 mg/ml chlorophylls were solubilized with a final 0.8% β-DM, and 30 μg of chlorophylls were loaded. The gel images were then analyzed with Gel-Pro Analyzer©. The integrated optical density measured in each band was checked to linearly correlate to the chlorophyll amounts present in each complex. Tocopherol Quantification—Tocopherols were quantified as described previously (26Garcia-Plazaola J.I. Becerril J.M. Phytochem. Anal. 1999; 10: 307-313Crossref Scopus (150) Google Scholar). After extraction with 100% methanol and separation by HPLC, tocopherol was detected with a fluorescence detector using an excitation wavelength at 295 nm and an emission wavelength was 340 nm. Tocopherol standards were obtained from Sigma. Arabidopsis Plants Acclimate to Different Light Conditions and Avoid Photoinhibition—In this work we analyzed the photosynthetic apparatus of Arabidopsis thaliana plants grown in different light and temperature conditions. Plants grown for 4 weeks at 21 °C and 100 μE have been treated for a further 3 weeks at different conditions. Two different light intensities were combined with two temperatures, yielding a total of five different treatments, including the control condition: low light, high light, cold low light, and cold high light (growth conditions are detailed under “Experimental Procedures”). PSII quantum efficiency (Fv/Fm) gives an indication of the functionality of PSII reaction centers; Fv/Fm is normally around 0.80, but when PSII is photo-inhibited this value decreases to 0.4 or less. We followed the PSII quantum efficiency, always measured in fully expanded mature leaves, during the light treatment as reported in Table 1. After the 1st week from the modification of light conditions, HL and cHL plants showed PSII photoinhibition, as evidenced by the reduction of Fv/Fm. In the following weeks, however, despite the prolongation of light treatment, plants recovered from photoinhibition (Table 1). At the moment of the harvesting, all plants showed the same PSII quantum efficiency, irrespective from growth conditions. Thus, after 3 weeks PSII reaction centers are not photo-inhibited in any treated plant, despite both HL and cHL conditions caused at first a photoinhibitory stress (Table 1). This behavior is consistent with the literature of the field that describes that an environmental perturbation first causes a stress response and later leads to a stable long term response defined acclimation (3Huner N.P.A. Oquist G. Sarhan F. Trends Plant Sci. 1998; 3: 224-230Abstract Full Text Full Text PDF Scopus (810) Google Scholar).TABLE 1Photosynthetic parameters of Arabidopsis plants grown in different light conditions PSII quantum efficiency value (Fv/Fm) measured during the acclimation time, showing the initial photoinhibition and the successive recovery. Other fluorescence parameters reported are as follows: qE, the quenching relaxed after 100 s in the dark; and qI, the fraction of NPQ that is not relaxed after 19 min of darkness. Measurements were performed on plants at the end of the treatment with an actinic light of 2000 μE as in Fig. 1A.CtrlLLHLcLLcHLFv/Fm (after 1 week)0.86 ± 0.010.85 ± 0.020.76 ± 0.040.87 ± 0.010.76 ± 0.04Fv/Fm (after 2 weeks)0.85 ± 0.020.85 ± 0.010.82 ± 0.040.87 ± 0.020.82 ± 0.02Fv/Fm (after 3 weeks)0.86 ± 0.010.85 ± 0.010.84 ± 0.010.87 ± 0.010.84 ± 0.01qE (after 3 weeks)2.03 ± 0.092.00 ± 0.222.69 ± 0.122.11 ± 0.122.59 ± 0.25qI (after 3 weeks)0.83 ± 0.080.92 ± 0.030.20 ± 0.170.75 ± 0.120.12 ± 0.01 Open table in a new tab Following treatment, plants exposed to low light conditions did not show an obvious phenotype apart from a slightly reduced growth at both temperatures. Plants grown in high light were also smaller and also showed accumulation of anthocyanins, especially at low temperature, probably the result of the stress perceived by these plants in the 1st week. In conclusion, because PSII is known to be a major photoinhibition target, the Fv/Fm values measured suggest that plants analyzed in this work are able to respond to the different light conditions when they are exposed to strong illumination for a long enough time. Plants Acclimated to High Light Increase Photosynthetic Electron Transport Rate—To confirm that our plants were indeed acclimated, we measured an additional photosynthetic parameter, qP. qP indicates the fraction of 1Chl a excited states, detected from their fluorescence emission, which is quenched by the activity of photochemical reactions (27Genty B. Briantais J.-M. Baker N.R. Biochim. Biophys. Acta. 1989; 990: 87-92Crossref Scopus (7061) Google Scholar). The comparison of this parameter between the different plants thus gives an indication of their efficiency in using light for photochemistry (1Demmig-Adams B. Adams W.W. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1992; 43: 599-626Crossref Scopus (2081) Google Scholar, 4Van Kooten O. Snel J.F.H. Photosynth. Res. 1990; 25: 147-150Crossref PubMed Scopus (2072) Google Scholar). Photochemical quenching values were measured with different light intensities (100, 360, 640, 1200, 1600, and 2000 μE). After 21 min of illumination, qP is clearly higher in plants adapted to HL with respect to both LL and Ctrl. This difference increases with the intensity of actinic light employed in the following measurement: at 1200 μE, qP values are 0.7, 0.35, and 0.14, respectively for HL, Ctrl, and LL (all values are reported in the supplemental figures). Thus, the growth in stronger light stimulates the ability to use energy for photosynthesis, in agreement with previous reports (12Gray G.R. Savitch L.V. Ivanov A.G. Huner N. Plant Physiol. 1996; 110: 61-71Crossref PubMed Scopus (159) Google Scholar, 28Bailey S. Walters R.G. Jansson S. Horton P. Planta. 2001; 213: 794-801Crossref PubMed Scopus (319) Google Scholar) confirming that plants analyzed activate an acclimative response. To verify that qP values measured were indeed because of an increase of Calvin cycle enzymes content, we quantified one of them, the Rubisco, in the leaves acclimated to different conditions. With respect to Ctrl and cLL plants, LL plants contained less Rubisco (60%) whereas in HL plants its content is increased by 250% (200% in cHL). Thus, the observed increase in photochemical efficiency in high light plants was correlated with the presence of a higher content of Calvin cycle enzymes that utilize NADPH and ATP produced from light reactions, as observed previously in acclimation studies (12Gray G.R. Savitch L.V. Ivanov A.G. Huner N. Plant Physiol. 1996; 110: 61-71Crossref PubMed Scopus (159) Google Scholar, 28Bailey S. Walters R.G. Jansson S. Horton P. Planta. 2001; 213: 794-801Crossref PubMed Scopus (319) Google Scholar, 29Hurry V.M. Malmberg G. Gardestrom P. Oquist G. Plant Physiol. 1994; 106: 983-990Crossref PubMed Scopus (119) Google Scholar). A further confirmation that plants analyzed are indeed acclimated comes from the analysis of oxygen evolution rates. Plants grown in high light showed a larger ability to evolve oxygen. In HL leaves it was around 2.5 times with respect to the control, but this activity reached four times the control in cHL plants, again consistent with previous data on Arabidopsis (28Bailey S. Walters R.G. Jansson S. Horton P. Planta. 2001; 213: 794-801Crossref PubMed Scopus (319) Google Scholar, 30Oquist G. Hurry V.M. Huner N. Plant Physiol. 1993; 101: 245-250Crossref PubMed Scopus (109) Google Scholar). We thus showed that the plants analyzed responded to different light conditions by modulating the size of their metabolic sinks, photosynthetic rates, and their ability to use light for photochemistry. All parameters analyzed are consistent with the present literature on acclimation. This deduction, together with the observed absence of PSII photoinhibition after 3 weeks of treatment, is a fundamental starting point for all the following analyses, because it demonstrates that we are observing the effects of acclimation to different light intensities rather than of photo-damage. High Light Acclimated Plants Respond More Promptly to Illumination—A third fluorescence parameter, the NPQ, was also measured in all plants. Its value gives an indication on the fraction of fluorescence quenched nonphotochemically as heat (4Van Kooten O. Snel J.F.H. Photosynth. Res. 1990; 25: 147-150Crossref PubMed Scopus (2072) Google Scholar). In Fig. 1A, NPQ kinetics measured with a 2000 μE illumination in differently acclimated plants are reported, showing a clearly distinct behavior; plants grown in high light are faster in their response, and NPQ reaches its maximum level within 6–7 min. In control and low light-grown plants, instead, NPQ amplitude continues to rise until the light was switched off after 21 min. These results are in agreement with previous reports of larger NPQ in HL acclimated plants with respect to control plants (31Demmig-Adams B. Plant Cell Physiol. 1998; 39: 474-482Crossref Scopus (208) Google Scholar, 32Bailey S. Horton P. Walters R.G. Planta. 2004; 218: 793-802Crossref PubMed Scopus (101) Google Scholar). This faster response can be estimated by calculating qE, the fastest component of NPQ; values reported in Table 1 indicate how this component is clearly larger in HL and cHL plants. These plants are not only faster in responding to illumination but also in relaxing quenching after the light is switched off. The quenching not relaxed after 19 min of dark (qI (4Van Kooten O. Snel J.F.H. Photosynth. Res. 1990; 25: 147-150Crossref PubMed Scopus (2072) Google Scholar)) is far lower in HL plants with respect to Ctrl and LL, as reported in Table 1. In this short dark period in fact, NPQ in HL plants almost relaxed to zero. When NPQ measurements were performed by using different intensities of actinic light, interesting differences were observed in their kinetics. In Fig. 1B, results obtained using the lowest light intensity (360 μE) are reported; the NPQ level continuously rises in plants adapted to LL, whereas it is saturated in a few minutes in cLL and control plants. In HL-adapted plants instead, NPQ is rapidly activated when plants are switched from dark to light but starts to decline after a few minutes, still under actinic light. This suggests that photochemical reactions, once activated, efficiently use absorbed light, whereas nonphotochemical quenching mechanisms are concomitantly inhibited. NPQ kinetics thus suggest that HL plants are able to respond quicker to illumination by activating mechanisms for the dissipation of energy as heat. The fastest component of NPQ, defined as qE, or feedback de-excitation, was shown to be dependent on the PSII subunit PsbS (6Li X.P. Bjorkman O. Shih C. Grossman A.R. Rosenquist M. Jansson S. Niyogi K.K. Nature. 2000; 403: 391-395Crossref PubMed Scopus (1177) Google Scholar). To verify if the increased response rate of HL plants was because of a different accumulation of this subunit, we measured the relative PsbS content by immunoblot titration. Five different dilutions of thylakoid membranes, corresponding to 0.2, 0.4, 0.8, 1, and 1.2 μgof chlorophyll, were loaded in the same SDS-polyacrylamide gel. Fig. 2A shows the results of detection with the antibody against PsbS, whose signal was quantified by densitometry. To avoid saturation, signal linearity through different dilutions was checked in all samples. In Fig. 2B two examples of the possible layout of linearity check is shown; in the case of an LL sample, all points can be fitted with a straight line with good accuracy. In the case of an HL sample, where PsbS content is higher, the last point clearly reaches saturation and stands out of the linear fit. Data points falling out of linearity, as in this case, were discarded. The results of the quantification, shown in Fig. 2C, demonstrate that PsbS indeed accumulates in HL plants with respect to control. Instead, in LL plants, PsbS content is slightly reduced. It is interesting to mention that the cLL sample, where PsbS content is close to control, also showed very similar NPQ kinetics. The content in PsbS thus strongly correlates with observed overall NPQ kinetics and amplitude. In the same samples we also quantified with the same method a PSII core subunit, CP47. If PsbS content is normalized to RCII content, differences between samples are not drastically changed, and the only significant difference with the data shown is that the difference between LL and control is smaller. Carotenoid Biosynthesis Is Regulated during Acclimation— Fluorescence kinetic measurements reported above are a good tool for the evaluation of photosynthetic performances during short light exposure. Long term modifications of photosynthetic apparatus can also be analyzed from changes in the thylakoid pigment compositions. As reported in Table 2, plants grown in distinct conditions showed a difference in their Chl a/b ratio as follows: increased in HL plants (both at 21 and 4 °C) and decreased in LL plants, irrespective to temperature conditions. Because Chl b is specifically bound to antenna proteins (Lhc), this modification in Chl a/b ratio suggests different accumulation levels of these polypeptides: higher in LL plants and lower HL, as shown previously (13Walters R.G. Horton P. Planta. 1994; 195: 248-256Crossref Scopus (141) Google Scholar, 28Bailey S. Walters R.G. Jansson S. Horton P. Planta. 2001; 213: 794-801Crossref PubMed Scopus (319) Google Scholar, 33Lichtenthaler H.K. Kuhn G. Prenzel U. Meier D. Physiol. Plant. 1982; 56: 183-188Crossref Scopus (79) Google Scholar, 34Leong T.-Y. Anderson J.M. Photosynth. Res. 1984; 5: 105-115Crossref PubMed Scopus (168) Google Scholar, 35Maenpaa P. Andersson B. Z. Naturforsch. 1989; 44: 403-406Crossref Scopus (28) Google Scholar). In HL acclimated plants the carotenoid content on a Chl basis is also 30% higher. It is worth noting that the increase in carotenoids occurs in conditions with a lower antenna size, suggesting extra carotenoids can be found free in the membrane. Not all carotenoid species, however, were increased to the same level; on a Chl basis, in fact neoxanthin and β-carotene levels are essentially unaffected. Violaxanthin and lutein content instead undergoes a significant increase in HL conditions together with the accumulation of significant amounts of antheraxanthin and zeaxanthin. Despite the lutein increase, however, components of the β-β branch of carotenoid biosynthetic pathway (β-carotene, zeaxanthin, violaxanthin, and neoxanthin) account for the highest part of the increase in carotenoid content with respect to the β-ϵ branch.TABLE 2Pigment composition of plants treated with different conditions Leaf pigment compositions of plants treated with different light/temperature conditions are reported. Data are normalized to 100 total Chl (a + b) molecules. Standard deviation is also reported.SampleChl a/bNeoxanthinViolaxanthinAntheraxanthinLuteinZeaxanthinβ-CaroteneTotal carotenoidsControl3.0 ± 0.13.4 ± 0.12.9 ± 0.29.9 ± 0.36.7 ± 1.022.9 ± 1.5LL2.6 ± 0.12.9 ± 0.32.7 ± 0.49.7 ± 0.36.2 ± 0.821.6 ± 1.0HL3.5 ± 0.13.1 ± 0.33.5 ± 0.51.5 ± 0.310.7 ± 1.02.7 ± 0.38.3 ± 0.429.9 ± 1.9cLL2.7 ± 0.23.4 ± 0.33.5 ± 0.110.2 ± 1.36.1 ± 0.723.3 ± 2.3Chl3.6 ± 0.13.4 ± 0.54.9 ± 0.62.0 ± 0.412.3 ± 0.82.3 ± 0.47.6 ± 0.632.5 ± 3.4 Open table in a new tab Zeaxanthin Production Rate Decreases in HL Acclim
Referência(s)