The development of antenna complexes of barley (Hordeum vulgare cv. Akcent) under different light conditions as judged from the analysis of 77 K chlorophyll a fluorescence spectra

Using 77 K chlorophyll a (Chl a) fluorescence spectra in vivo, the development was studied of Photosystems II (PS II) and I (PS I) during greening of barley under intermittent light followed by continuous light at low (LI, 50 μmol m⁻² s⁻¹) and high (HI, 1000 μmol m⁻² s⁻¹) irradiances. The greening at HI intermittent light was accompanied with significantly reduced fluorescence intensity from Chl b excitation for both PS II (F685) and PS I (F743), in comparison with LI plants, indicating that assembly of light-harvesting complexes (LHC) of both photosystems was affected to a similar degree. During greening at continuous HI, a slower increase of emission from Chl b excitation in PS II as compared with PS I was observed, indicating a preferred reduction in the accumulation of LHC II. The following characteristics of 77 K Chl a fluorescence spectra documented the photoprotective function of an elevated content of carotenoids in HI leaves: (1) a pronounced suppression of Soret region of excitation spectra (410–450 nm) in comparison with the red region (670–690 nm) during the early stage of greening indicated a strongly reduced excitation energy transfer from carotenoids to the Chl a fluorescing forms within PS I and PS II; (2) changes in the shape of the excitation band of Chl b and carotenoids (460–490 nm) during greening under continuous light confirmed that the energy transfer from carotenoids to Chl a within PS II remained lower as compared with the LI plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effect of norflurazon on protein composition and chlorophyll organization in pigment–protein complex of Photosystem II

The pyridazinone-type herbicide norflurazon SAN 9789 inhibiting the biosynthesis of long-chain carotenoids results in significant decrease in PS II core complexes and content of light-harvesting complex (LHC) polypeptides in the 29.5–21 kDa region. The Chl a forms at 668, 676, and 690 nm that belong to LHC and antenna part of PS I disappear completely after treatment. The intensity of the Chl b form at 648 nm is sharply decreased in treated seedlings grown under 30 or 100 lx light intensity. The bands of carotenoid absorption at 421, 448 (Chl a), 452, 480, 492, 496 (β-carotene), and 508 nm also disappear. The band shift from 740 to 720 nm and decrease in its intensity relative to the 687 nm emission peak in the low-temperature fluorescence spectrum (77 K) suggests a disturbance of energy transfer from LHC to the Chla form at 710–712 nm.     

Characterization of the Porphyridium cruentum Chl a-binding LHC by in vitro reconstitution: LHCaR1 binds 8 Chl a molecules and proportionately more carotenoids than CAB proteins

The Porphyridium cruentum light harvesting complex (LHC) binds Chl a, zeaxanthin and -carotene and comprises at least 6 polypeptides of a multigene family. We describe the first in vitro reconstitution of a red algal light-harvesting protein (LHCaR1) with Chl a/carotenoid extracts from P. cruentum. The reconstituted pigment complex (rLHCaR1) is spectrally similar to the native LHC I, with an absorption maximum at 670 nm, a 77 K fluorescence emission peak at 677 nm (ex. 440 nm), and similar circular dichroism spectra. Molar ratios of 4.0 zeaxanthin, 0.3 -carotene and 8.2 Chl a per polypeptide for rLHCaR1 are similar to those of the native LHC I complex (3.1 zeaxanthin, 0.5 -carotene, 8.5 Chl a). The binding of 8 Chl a molecules per apoprotein is consistent with 8 putative Chl-binding sites in the predicted transmembrane helices of LHCaR1. Two of the putative Chl a binding sites (helix 2) in LHCaR1 were assigned to Chl b in Chl a/b-binding (CAB) LHC II [Kühlbrandt et al. (1994) Nature 367: 614–21]. This suggests either that discrimination for binding of Chl a or Chl b is not very specific at these sites or that specificity of binding sites evolved separately in CAB proteins. LHCaR1 can be reconstituted with varying ratios of carotenoids, consistent with our previous observation that the carotenoid to Chl ratio is substantially higher in P. cruentum grown under high irradiance. Also notable is that zeaxanthin does not act as an accessory light-harvesting pigment, even though it is highly likely that it occupies the position assigned to lutein in the CAB LHCs.This revised version was published online in October 2005 with corrections to the Cover Date.     

Comparison of chlorophyll a spectra in wild-type and mutant barley chloroplasts grown under day or intermittent light

The absorption (640–710 nm) and fluorescence emission (670–710 nm) spectra (77 K) of wild-type and Chl b-less, mutant, barley chloroplasts grown under either day or intermittent light were analysed by a RESOL curve-fitting program. The usual four major forms of Chl a at 662, 670, 678 and 684 nm were evident in all of the absorption spectra and three major components at 686, 693 and 704 nm in the emission spectra. A broad Chl a component band at 651 nm most likely exists in all chlorophyll spectra in vivo. The results show that the mutant lacks not only Chl b, but also the Chl a molecules which are bound to the light-harvesting, Chl a/b, protein complex of normal plants. It also appears that the absorption spectrum of this antenna complex is not modified appreciably by its isolation from thylakoid membranes.Abbreviations Chl chlorophyll - DL daylight - ImL intermittent light - WT wildtype - LHC light-harvesting Chl a/b protein complex - S.E. standard error of the mean DBP-CIW No. 763.     

Which side of the π-macrocycle plane of (bacterio)chlorophylls is favored for binding of the fifth ligand?

Reported crystallographic data and calculated molecular models indicated that chlorophyll (Chl) a and bacteriochlorophyll (BChl) a tend to bind the fifth ligand on the side of the macrocycle where the C13²-(R)-methoxycarbonyl moiety protrudes (denoting the ‘back’ side). The crystal structures of 34 photosynthetic proteins possessing (B)Chl cofactors revealed that most of Chl a and BChl a (and b) are coordinated by any peptidyl residue (e.g., histydyl-imidazolyl group), peptidyl backbone or water from the ‘back’ side. Almost all the cofactors that bind a water molecule as the fifth ligand in these proteins have a ‘back’ configuration. Theoretical model calculations for methyl chlorophyllide a (MeChlid a) and methyl bacteriochlorophyllide a (MeBChlid a) bound to an imidazole molecule indicated that the ‘back’ side is energetically favored for the ligand binding. These results are consistent with the fact that ethyl chlorophyllide a (EtChlid a) dihydrate crystal consists of the ‘back’ complex. The modeling also showed that both removal and stereochemical inverse of the C13²-methoxycarbonyl group affect the relative stability between the ‘back’ and ‘face’ complexes. The effect of the C13²-moiety on the choice of the macrocycle side for the ligand binding is discussed in relation to the function of P700. This revised version was published online in June 2006 with corrections to the Cover Date.     

Relevance of the Diastereotopic Ligation of Magnesium Atoms of Chlorophylls in the Major Light-harvesting Complex II (LHC II) of Green Plants

The recent high-resolution crystal structure of LHC II [Liu et al. (2004) Nature 428: 287–292] makes possible an unprecedented insight into the stereochemical features of how chlorophylls (Chl)s are bound. The diastereotopic ligation generates four structurally different pigment types, two Chl a and two Chl b, which are distinguished not only by the groups in the 7-position (methyl in Chl a and formyl in Chl b) but also by the face of the tetrapyrrole to which the fifth magnesium ligand is bound. Within a LHC II monomer, out of the eight Chl a six have a ‚normal’ α-coordination and two are β-coordinated while out of the six Chl b only one has the ‚special’ β-coordination. In Photosystem I where a more meaningful statistical analysis could be made, out of 96 Chl a only 14 are β-coordinated, again indicating a preference for the ‚normal’ α-coordination [Balaban et al. (2002) Biochim Biophys Acta Bioenerget 1556: 197–207; Oba and Tamiaki (2002a) Photosynth Res 74: 1–10]. Astonishingly, all the special β-Chls are part of the stromal ring of Chls within the LHC II trimers and occupy key positions for the excitation energy transfer. Sequential energy traps are engineered with one hetero- and three homo-dimers. A careful pairing of carotenoids with the special β-Chls, which could quench their triplet states efficiently, implies a functional relevance of this diastereotopic ligation.     

Structural stability and properties of three isoforms of the major light-harvesting chlorophyll a/b complexes of photosystem II

Three isoforms of the major light-harvesting chlorophyll (Chl) a/b complexs of photosystem II (LHCIIb) in the pea, namely, Lhcb1, Lhcb2, and Lhcb3, were obtained by overexpression of apoprotein in Escherichia coli and by successfully refolding these isoforms with thylakoid pigments in vitro. The sequences of the protein, pigment stoichiometries, spectroscopic characteristics, thermo- and photostabilities of different isoforms were analysed. Comparison of their spectroscopic properties and structural stabilities revealed that Lhcb3 differed strongly from Lhcb1 and Lhcb2 in both respects. It showed the lowest Qy transition energy, with its reddest absorption about 2 nm red-shifted, and the highest photostability under strong illuminations. Among the three isoforms, Lhcb 2 showed lowest thermal stability regarding energy transfer from Chl b to Chl a in the complexes, which implies that the main function of Lhcb 2 under high temperature stress is not the energy transfer.     

Energy transfer pathways in the CP24 and CP26 antenna complexes of higher plant photosystem II: a comparative study

Antenna complexes are key components of plant photosynthesis, the process that converts sunlight, CO₂, and water into oxygen and sugars. We report the first (to our knowledge) femtosecond transient absorption study on the light-harvesting pigment-protein complexes CP26 (Lhcb5) and CP24 (Lhcb6) of Photosystem II. The complexes are excited at three different wavelengths in the chlorophyll (Chl) Qy region. Both complexes show a single subpicosecond Chl b to Chl a transfer process. In addition, a reduction in the population of the intermediate states (in the 660-670 nm range) as compared to light-harvesting complex II is correlated in CP26 to the absence of both Chls a604 and b605. However, Chl forms around 670 nm are still present in the Chl a Qy range, which undergoes relaxation with slow rates (10-15 ps). This reduction in intermediate-state amplitude CP24 shows a distinctive narrow band at 670 nm connected with Chls b and decaying to the low-energy Chl a states in 3-5 ps. This 670 nm band, which is fully populated in 0.6 ps together with the Chl a low-energy states, is proposed to originate from Chl 602 or 603. In this study, we monitored the energy flow within two minor complexes, and our results may help elucidate these structures in the future.     

Effects of different excitation and detection spectral regions on room temperature chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence parameters

The effects of different spectral region of excitation and detection of chlorophyll (Chl) a fluorescence at room temperature on the estimation of excitation energy utilization within photosystem (PS) 2 were studied in wild-type barley (Hordeum vulgare L. cv. Bonus) and its Chl b-less mutant chlorina f2 grown under low and high irradiances [100 and 1 000 μmol(photon) m⁻² s⁻¹]. Three measuring spectral regimes were applied using a PAM 101 fluorometer: (1) excitation in the red region (maximum at the wavelength of 649 nm) and detection in the far-red region beyond 710 nm, (2) excitation in the blue region (maximum at the wavelength of 461 nm) and detection beyond 710 nm, and (3) excitation in the blue region and detection in the red region (660– 710 nm). Non-photochemical quenching of maximal (NPQ) and minimal fluorescence (SV₀), determined by detecting Chl a fluorescence beyond 710 nm, were significantly higher for blue excitation as compared to red excitation. We suggest that this results from higher non-radiative dissipation of absorbed excitation energy within light-harvesting complexes of PS2 (LHC2) due to preferential excitation of LHC2 by blue radiation and from the lower contribution of PS1 emission to the detected fluorescence in the case of blue excitation. Detection of Chl a fluorescence originating preferentially from PS2 (i.e. in the range of 660–710 nm) led to pronounced increase of NPQ, SV₀, and the PS2 photochemical efficiencies (F_V/F_M and F_V′/F_M′), indicating considerable underestimation of these parameters using the standard set-up of PAM 101. Hence PS1 contribution to the minimal fluorescence level in the irradiance-adapted state may reach up to about 80 %.     

Characterization of chlorophyll–protein complexes isolated from a Siphonous green alga, Bryopsis corticulans

Six chlorophyll–protein complexes are isolated from thylakoid membranes of Bryopsis corticulans by dodecyl-β-d-maltoside polyacrylamide gel electrophoresis. Unlike that of higher plants, the 77 K fluorescence emission spectrum of the CP1 band, the PSI core complexes of B. corticulans, presents two peaks, one at 675 nm and the other at 715–717 nm. The emission peak at 715–717 nm is slightly higher than that at 675 nm in the CP1 band when excited at 438 or 540 nm. However, the peak at 715 nm is obviously lower than that at 675 nm when excited at 480 nm. The excitation spectra of CP1 demonstrate that the peak at 675 nm is mainly attributed to energy from Chl b while it is the energy from Chl a that plays an important role in exciting the peak at 715–717 nm. Siphonaxanthin is found to contribute to both the 675 nm and 715–717 nm peaks. We propose from the above results that chlorophyll a and siphonaxanthin are mainly responsible for the transfer of energy to the far-red region of PSI while it is Chl b that contributes most of the transfer of energy to the red region of PSI. The analysis of chlorophyll composition and spectral characteristics of LHCP¹ and LHCP³ also indicate that higher content of Chl b and siphonaxanthin, mainly presented in LHCP¹, the trimeric form of LHCII, are evolved by B. corticulans to absorb an appropriate amount of light energy so as to adapt to their natural habitats.     

Alteration of Components of Chlorophyll-Protein Complexes and Distribution of Excitation Energy Between the Two Photosystems in Two New Rice Chlorophyll b-less mutants

Two new yellow rice chlorophyll (Chl) b-less (lack) mutants VG28-1 and VG30-5 differ from the other known Chl b-less mutants with larger amounts of soluble protein and ribulose-1,5-bisphosphate carboxylase/oxygenase small sub-unit and smaller amounts of Chl a. We investigated the altered features of Chl-protein complexes and excitation energy distribution in these two mutants, as compared with wild type (WT) rice cv. Zhonghua 11 by using native mild green gel electrophoresis and SDS-PAGE, and 77 K Chl fluorescence in the presence of Mg²⁺. WT rice revealed five pigment-protein bands and fourteen polypeptides in thylakoid membranes. Two Chl b-less mutants showed only CPI and CPa pigment bands, and contained no 25 and 26 kDa polypeptides, reduced amounts of the 21 kDa polypeptide, but increased quantities of 32, 33, 56, 66, and 19 kDa polypeptides. The enhanced absorption of CPI and CPa and the higher Chl fluorescence emission ratio of F685/F720 were also observed in these mutants. This suggested that the reduction or loss of the antenna LHC1 and LHC2 was compensated by an increment in core component and the capacity to harvest photon energy of photosystem (PS) 1 and PS2, as well as in the fraction of excitation energy distributed to PS2 in the two mutants. 77 K Chl fluorescence spectra of thylakoid membranes showed that the PS1 fluorescence emission was shifted from 730 nm in WT rice to 720 nm in the mutants. The regulation of Mg²⁺ to excitation energy distribution between the two photosystems was complicated. 10 mM Mg²⁺ did not affect noticeably the F685/F730 emission ratio of WT thylakoid membranes, but increased the ratio of F685/F720 in the two mutants due to a reduced emission at 685 nm as compared to that at 720 nm.     

Effect of detergent on aggregation of the light-harvesting chlorophyll a/b complex of photosystem 2 and its impact for carotenoid function and fluorescence quenching

Spectroscopy was used to investigate the fluorescence quenching mechanism in light-harvesting complex 2 (LHC2). The 77 K fluorescence excitation spectroscopy was performed for detection of aggregation state of LHC2 treated with different concentrations of octylphenol poly(ethyleneglycol ether)₁₀ (TX-100). Resonance Raman (RR) spectra excited with 488, 496, and 514 nm provided molecular configuration of neoxanthin, lutein 1, and lutein 2, respectively. At increased concentration of TX-100, the RR signals of xanthophylls were enhanced in the four frequency regions, which was accompanied with increase of fluorescence of chlorophyll (Chl) a. Thus the absorption of the three xanthophyll molecules was inclined to excitation wavelength, which proved that functional configurations of xanthophyll molecules in LHC2 were vital for fast transfer of excitation energy to Chl a molecules. Changes in the v4 region (C-H out-of-plane bending modes, at ∼960 cm⁻¹ in RR spectra) demonstrated that the twist feature of neoxanthin, lutein 1, and lutein 2 molecules existed in LHC2 trimers, however, it was lost in the LHC2 macro-aggregates. In the second derivative absorption spectra of LHC2, neoxanthin absorption was not detected in LHC2 macro-aggregates, while evident absorption was found in LHC2 trimers and this absorption decreased obviously when TX-100 concentration was higher than 1 mM. Hence the neoxanthin molecule had a structural role in formation of LHC2 trimers. The RR and absorption spectra also implied that carotenoid molecules constructed the functional LHC2 trimers via their intrinsic configuration features, which enabled energy transfer to Chl a efficiently and led to lower fluorescence quenching efficiency. In contrast, these intrinsic twist configurations were lost in LHC2 macro-aggregates and led to lower energy transfer efficiency and higher fluorescence quenching efficiency.     

Study on energy transfer between carotenoid and chlorophyll a in cytochrome b 6 f complex from Bryopsis corticulans

The excitation energy transfer between carotenoid and chlorophyll (Chl) in the cytochrome b ₆ f complex from Bryopsis corticulans (B. corticulans), in which the carotenoid is 9-cis-α-carotene, was investigated by means of fluorescence excitation and sub-microsecond time-resolved absorption spectroscopies. The presence of efficient singlet excitation transfer from α-carotene to Chl a was found with an overall efficiency as high as ∼ ∼24%, meanwhile the Chl a-to-α-carotene triplet excitation transfer was also evidenced. Circular dichroism spectroscopy showed that α-carotene molecule existed in an asymmetric environment and Chl a molecule had a certain orientation in this complex.Bin-Xing Li and Ping Zuo contributed equally to this work.     

Kinetics of absorbance and anisotropy upon excited state relaxation in the reaction center core complex of a green sulfur bacterium

Properties of the excited states in reaction center core (RCC) complexes of the green sulfur bacterium Prosthecochloris aestuarii were studied by means of femtosecond time-resolved isotropic and anisotropic absorption difference spectroscopy at 275 K. Selective excitation of the different transitions of the complex resulted in the rapid establishment of a thermal equilibrium. At about 1 ps after excitation, the energy was located at the lowest energy transition, BChl a 835. Time constants varying between 0.26 and 0.46 ps were observed for the energy transfer steps leading to this equilibrium. These transfer steps were also reflected in changes in polarization. Our measurements indicate that downhill energy transfer towards excited BChl a 835 occurs via the energetically higher spectral forms BChl a 809 and BChl a 820. Low values of the anisotropy of about 0.07 were found in the ‘two-color’ measurements at 820 and 835 nm upon excitation at 800 nm, whereas the ‘one-color’ kinetics showed much higher anisotropies. Charge separation occurred with a time constant varying between 20 and 30 ps. This revised version was published online in June 2006 with corrections to the Cover Date.     

The redox state of the plastoquinone pool controls the level of the light-harvesting chlorophyll a/b binding protein complex II (LHC II) during photoacclimation

A cytochrome b ₆ f deficient mutant of Lemna perpusilla maintains a constant and lower level of the light-harvesting chl a/b-binding protein complex II (LHC II) as compared to the wild type plants at low-light intensities. Inhibition of the plastoquinone pool reduction increases the LHC II content of the mutant at both low- and high-light intensities but only at high-light intensity in the wild type plants. Proteolytic activity against LHC II appears during high-light photoacclimation of wild type plants. However, the acclimative protease is present in the mutant at both light intensities. These and additional results suggest that the plastoquinone redox state serves as the major signal-transducing component in the photoacclimation process affecting both, synthesis and degradation of LHC II and appearance of acclimative LHC II proteolysis. The plastoquinol pool cannot be oxidized by linear electron flow in the mutant plants which are locked in a ‘high light’ acclimation state. The cytochrome b ₆ f complex may be involved indirectly in the regulation of photoacclimation via 1) regulation of the plastoquinone redox state; 2) regulation of the redox-controlled thylakoid protein kinase allowing exposure of the dephosphorylated LHC II to acclimative proteolysis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Gradual Disassembly of Photosystem 2 In Vivo Induced by Excess Irradiance. A Hypothesis Based on Changes in 77 K Fluorescence Spectra of Chlorophyll a in Barley Leaves

Effects of short-term exposure to different irradiances on the function of photosystem 2 (PS2) were studied for barley grown at low (LI; 50 μmol m⁻² s⁻¹) and high (HI; 1 100 μmol m⁻² s⁻¹) irradiances. HI barley revealed higher ability to down-regulate the light-harvesting within PS2 after exposure to high irradiance as compared to LI plants. This ability was estimated from the light-induced decreases of F685/F742 and E476/E436 in emission and excitation spectra of 77 K chlorophyll (Chl) a fluorescence in vivo which was 65 and 10 % for HI plants as compared to 30 and 2 % for LI plants, respectively. For LI plants this protective down-regulation of the light-harvesting of PS2 was saturated at 430 μmol m⁻² s⁻¹, and progressive PS2 photodamage was induced at higher irradiances. After exposure of LI segments to 2 200 μmol m⁻² s⁻¹ a pronounced maximum at 700 nm appeared in emission spectrum of 77 K Chl a fluorescence. Based on complementary analysis of 77 K excitation spectra measured at the emission wavelength 685 nm we suggest that this emission maximum may be attributed to the formation of aggregates of light-harvesting complexes of PS2 (LHC2) with part of PS2 core during progressive PS2 photodamage. Our results can be explained assuming different contributions of LHC2 and PS2 core to the total nonradiative dissipation of absorbed excitation energy for the LI and HI barley. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterization of Chlorophyll–protein Complexes Isolated from Two Marine Green Algae, Bryopsis maxima and Ulva pertusa, Growing in the Intertidal Zone

Three Chl–protein complexes were isolated from thylakoid membranes of Bryopsis maxima and Ulva pertusa, marine green algae that inhabit the intertidal zone of the Pacific Ocean off the eastern coast of Japan by dodecyl-β-d-maltoside polyacrylamide gel electrophoresis. The slowest-moving fractions showed low Chl a/b and Chl/P-700 ratios, indicating that this fraction corresponds to complexes in PS I, which is large in both algae. The intermediate and fastest-moving fractions showed the traits of PS II complexes, with some associated Chl a/b–protein complexes and LHC II, respectively. The spectral properties of the separated Chl–proteins were also determined. The absorption spectra showed a shallow shoulder at 540 nm derived from siphonaxanthin in Bryopsis maxima, but not in Ulva pertusa. The 77 K emission spectra showed a single peak in Bryopsis maxima and two peaks in Ulva pertusa. Besides the excitation spectra indicated that the excitation energy transfer to the PS I complexes differed quite a lot higher plants. This suggested that the mechanisms of energy transfer in both of these algae differ from those of higher plants. Considering the light environment of this coastal area, the large size of the antennae of PS I complexes implies that the antennae are arranged so as to balance light absorption between the two photosystems. In addition, we discuss the relationships among the photosystem stoichiometry, the energy transfer, and the distribution between the two photosystems.     

Effects of heat treatment on the protein secondary structure and pigment microenvironment in photosystem 1 complex

The protein secondary structure and pigments' microenvironment in photosystem 1 (PS1) complexes were studied in the temperature range of 25–80 °C using Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy, respectively. Quantitative analysis of the component bands of the amide I band (1 700–1 600 cm⁻¹) showed no significant change below 50 °C. However, apparent conformational changes occurred at 60 °C and further continued at 70 and 80 °C accompanied with transitions of secondary structure mainly from α-helix to the β-sheet structures. CD analysis demonstrated that the regular arrangement, viz. protein microenvironment of pigments of PS1 complexes, was destroyed by heat treatment which might come from the changes of protein secondary structure of PS1. The CD signals at 645 nm contributed by chlorophyll (Chl) b of light-harvesting complex 1 (LHC1) were easily destroyed at the beginning of heat treatment (25–60 °C). When temperature reached 70 and 80 °C, the CD signals at 478 nm contributed mainly by Chl b of LHC1 and 498 nm contributed by carotenoids decreased most rapidly, indicating that LHC1 was more sensitive to high temperature than core complexes. In addition, the oxygen uptake rate decreased by 90.81 % at 70 °C and was lost completely at 80 °C showing that heat treatment damaged the regular function of PS1 complexes. This may be attributed to heat-induced changes of pigment microenvironment and protein secondary structure, especially transmembrane α-helix located in PsaA/B of PS1.