The acclimative response of the main light-harvesting chlorophyll a/b-protein complex of photosystem II (LHCII) to elevated irradiances at the level of trimeric subunits

The changes in structural organization of the major light-harvesting chlorophyll a/b–protein complex of photosystem II (LHC II) at the level of trimeric subcomplexes were studied in spinach plants grown under low light conditions (50 μmol quanta m⁻² s⁻¹) and then acclimated to elevated irradiances. By monitoring photochemical quenching of fluorescence yield (q_P), photosystem II (PS II) functional status was assessed in leaves of plants acclimated to a range of elevated irradiances. Three separate acclimative irradiances were selected for the experiments, reflecting: limiting light conditions (150 μmol quanta m⁻² s⁻¹), near to the inflexion point on the irradiance curve conditions (300 μmol quanta m⁻² s⁻¹) and an excessive light, causing a moderate stress in the form of down regulation of PS II (450 μmol quanta m⁻² s⁻¹). An immunoblot analysis showed that there was a clear decline in an abundance on chlorophyll basis of Lhcb1-3 apoproteins as an acclimative irradiance increased from 50 to 450 μmol quanta m⁻² s⁻¹, with Lhcb1 decreasing to a lesser extent than Lhcb2 and Lhcb3 (only at excessive irradiance). When analyzed by non-denaturing isoelectric focusing BBY membrane fragments (PSII-enriched, stacked thylakoid membranes) isolated from low light-grown plants were resolved into nine fractions, seven of which (labelled 3–9) were established by us previously [Jackowski and Pielucha, J. Photochem. Photobiol. B: Biol. 64 (2001) 45] to be LHC II subcomplexes representing mixed populations of closely similar trimers, comprising permutations of Lhcb1 and Lhcb2 (subcomplexes 3–7) or Lhcb1-3 (subcomplexes 8 and 9). A heterogeneity with regard to accumulation behaviour of LHC II subcomplexes in response to elevated irradiances was revealed. The subcomplexes 5 and 6 were accumulating at similar level, regardless of the light irradiance experienced. Another group consisting of the subcomplexes 3 and 4 (the most basic ones) showed a progressive increase in relative abundance with increasing an irradiance intensity whereas the subcomplexes 7–9 (the most acidic ones) exhibited a progressive decline in their relative abundance during an acclimation of spinach plants to elevated irradiances thus they may collectively represent an elevated irradiance-responsive subunit of LHCII.     

CHLOROPLAST PIGMENTS and THEIR BIOSYNTHESIS IN RELATION TO LIGHT INTENSITY*

Abstract— Depending on the light intensity that they received during growth, radish seedlings altered not only the pigment and quinone composition of the thylakoid membrane but also the chloroplast ultrastructure. In strong light, sun chloroplasts of radish were very similar to those from sun leaves of beech trees, while those developed under under dim light possessed a typical shade chloroplast. Radish shade chloroplasts contained a higher chlorophyll content and a higher concentration of xanthophylls resulting in a lower xanthophyll to carotene ratio as compared to sun chloroplasts. Chloroplasts from radish grown in strong light showed a much higher activity in their terpenoid metabolism than plastids from shade plants. Chlorophylls and carotenoids which are involved in the absorption of light and the transfer of energy during photosynthesis were labeled by [³H]-mevalonate to a much higher degree in plastids from sun leaves as compared to plastids from shade leaves. This shows that in strong light where pigments are continuously broken down and resynthesized in order to maintain photosynthesis, chlorophylls and carotenoids exhibit a much higher turnover rate than the pigments of shade plants.     

THE EFFECT OF LEVULINIC ACID ON THE LIGHT INDUCED DEVELOPMENT OF PHOTOSYSTEM I AND II ACTIVITIES IN GREENING MAIZE LEAVES*

Photosystem I and Photosystem II activities were measured in chloroplasts isolated after 0–20 h illumination from etiolated maize leaves in which chlorophyll synthesis was specifically inhibited by levulinic acid. In control leaves not treated with levulinic acid, Photosystem I activity/chlorophyll developed rapidly during the first 2h in light, then fell off, and reached a constant level after 6h of illumination. In levulinic acid treated leaves, in which chlorophyll accumulation was inhibited up to 60%, a similar initial rise in Photosystem I activity was observed. However, the decrease in activity was much slower and continued for at least 20 h. The development of Photosystem I activity calculated on a leaf fresh weight basis was similar for control leaves or leaves treated with levulinic acid. This indicates that development of Photosystem I activity may not be related to chlorophyll accumulation during greening. Photosystem II activity/chlorophyll in leaves treated with or without levulinic acid increased similarly during the first 6h and then remained constant. Activity of Photosystem II per leaf fresh weight increased linearly, after the first h, for 20 h in the control leaves; in levulinic acid treated leaves this development was reduced by about 60%. Thus, development of Photosystem II activity can be related to chlorophyll accumulation. SDS gel electrophoresis of plastid membranes from control leaves illuminated for 12 h showed the presence of chlorophyll-protein complex I as well as Chl-protein 11; in the case of levulinic acid treated leaves only Chl-protein complex I was detectable, while Chl-protein complex II was markedly reduced.     

Color of illumination during growth affects LHCII chiral macroaggregates in pea plant leaves

To determine whether the color of illumination under which plants are grown, affects the structure of photosynthetic antennae, pea plants were grown under either blue-enriched, red-enriched, or white light. Carotenoid content of isolated chloroplasts was found to be insensitive to the color of illumination during growth, while chlorophyll a/b ratio in chloroplasts isolated from young illuminated leaves showed susceptibility to color. Color of illumination affects the LHCII chiral macroaggregates in intact leaves and isolated chloroplasts, providing light-induced alteration of the handedness of the LHCII chiral macroaggregate, as measured with circular dichroism and circularly polarized luminescence. The susceptibility of handedness to current illumination (red light excitation of chlorophyll fluorescence) is dependent on the color under which the plants were grown, and was maximal for the red-enriched illumination. We propose the existence of a long-term (growth period) color memory, which influences the susceptibility of the handedness of LHCII chiral macroaggregates to current light.     

Heterogeneity of the main light-harvesting chlorophyll a/b-protein complex of photosystem II (LHCII) at the level of trimeric subunits.

To study organization of the main light-harvesting chlorophyll a/b-protein complex of photosystem II (LHCII) from spinach thylakoid membranes at the level of trimeric subcomplexes, we have applied non-denaturing isoelectric focusing (ndIEF) in vertical, slab polyacrylamide gels. When analyzed by two consecutive ndIEF/electroelution runs, spinach BBY membrane preparations (PSII(alpha)-enriched, stacked thylakoid membranes) were resolved into nine fractions of 100% purity, labelled 1-9 in order of decreasing pI values. Seven of these fractions (3-9) were shown by absorption spectroscopy to stand for LHCII subcomplexes. The subcomplexes were established - by monitoring their circular dichroism spectra and comparing them to the spectra of native LHCII trimers and monomers - to be structurally intact trimers. The analysis of polypeptide composition of the subcomplexes in terms of apparent molecular masses and Lhcb genes' products led us to the conclusion that each of the subcomplexes might be a mixed population of closely similar individual trimers, comprising of permutations of Lhcb1 and Lhcb2 (subcomplexes 3-7) or Lhcb1, Lhcb2 and Lhcb3 (subcomplexes 8 and 9).     

Non-invasive Monitoring of Biochemical Response of Wheat Seedlings Toward Titanium Dioxide Nanoparticles Treatment Using Attenuated Total Reflectance Fourier Transform Infrared and Laser Induced Fluorescence Spectroscopy

Widespread commercial application of titanium dioxide nanoparticles leads to their dispersion in the environment and inevitable interaction with living organisms. Their presence necessitates the monitoring of nanoparticle interactions with plants using advanced techniques that are capable of noninvasively and sensitively estimating the changes involved in the biochemical profile. The current study aims to investigate the effects of titanium dioxide nanoparticles on biochemicals of wheat leaves using label free, nondestructive, rapid, sensitive, and advanced spectroscopic probes: laser induced fluorescence and attenuated total reflectance Fourier transform infrared spectroscopy coupled with multivariate analysis. The fluorescence and infrared spectra of control and titanium dioxide nanoparticle treated wheat leaves were acquired in the region from 400 to 800?nm and 4000 to 485?cm^?1. The treatment of titanium dioxide nanoparticles decreases the chlorophyll content and the concentrations of cellulose, hemicellulose, xyloglucans, pectin, and lignin indicating interferences in the biosynthesis and structure of cell walls of the wheat leaves. The level of amide I, carbonyl, and methylene groups also increases following the treatment of titanium dioxide nanoparticles indicating lipid and protein peroxidation and the accumulation of carbonyl compounds. The changes in the integrated area ratios of the amide II/amide I, carbonyl/methyl, and methylene/amide II bands demonstrate disorder in the membrane integrity. This study establishes the efficiency of noninvasive, label-free, and rapid protocols based on attenuated total reflectance Fourier transform infrared and laser induced fluorescence to monitor the interactions of nanoparticles with plants at early stage of plant growth before visual signs of toxicity appear.     

Changes in the room-temperature emission spectrum of chlorophyll during fast and slow phases of the Kautsky effect in intact leaves

Changes in the room-temperature emission spectrum of chlorophyll (Chl) were analyzed using fast diode-array recordings during the Kautsky effect in mature and in greening barley leaves. In mature leaves, the comparison of F(O) (basal level of fluorescence yield at transient O) and F(M) (maximum level of fluorescence yield at transient M) spectra showed that the relative amplitude of total variable fluorescence was maximal for the 684 nm Photosystem II (PSII) band and minimal for the 725 nm Photosystem I band. During the increase from F(O) to F(M), a progressive redshift of the spectrum of variable fluorescence occurred. This shift reflected the different fluorescence rise kinetics of different layers of chloroplasts inside the leaf. This was verified by simulating the effect of screening on the emission spectrum of isolated chloroplasts and by experiments on greening leaves with low Chl content. In addition, experiments performed at different greening stages showed that the presence of uncoupled Chl at early-greening stages and light-harvesting complex II (LHCII) at later stages have detectable but minor effects on the shape of room-temperature emission spectra. When strong actinic light was applied to mature green leaves, the slow fluorescence yield, which declined from F(M) to F(T) (steady-state level of fluorescence yield at transient T), was accompanied by a slight redshift of the 684 nm PSII band because of nonphotochemical quenching of short-wavelength-emitting Chl ascribed to LHCII.     

REGULATION OF CHLOROPLAST DEVELOPMENT BY RED AND BLUE LIGHT

There are specific differences between red and blue light greening of etiolated seedlings of Hordevm vulgare L. Blue light results in a different prenyl lipid composition of chloroplast as compared to red light of equal quanta density. This is documented by a much higher prenylquinone content, higher chlorophyll a/b ratios, and lower values for the ratio xanthophylls to carotenes (x/c). The photosynthetic activity of “blue light” chloroplasts (Hill reaction) is higher than that of “red light” chloroplasts. These differences in prenylquinone composition and Hill-activity are associated with a different ultrastructure of chloroplasts. “Red light” chloroplasts exhibit a much higher grana content than “blue light” chloroplasts. The difference in thylakoid composition, photosynthetic activity and chloroplast structure found between blue and red light greening are similar to those found between sun and shade leaves and those between plants grown under high and low light intensities.     

TEMPERATURE DEPENDENCE OF DELAYED LIGHT EMISSION IN THE 6 to 340 MICROSECOND RANGE AFTER A SINGLE FLASH IN CHLOROPLASTS

Abstract. The delayed light emission decay rate (up to 120 μs) and the rise in chlorophyll a fluorescence yield (from 3 to 35 μs) in isolated chloroplasts from several species, following a saturating 10 ns flash, are temperature independent in the 0–35°C range. However, delayed light in the 120–340 μs range is temperature dependent. Arrhenius plots of the exponential decay constants are: (a) linear for lettuce and pea chloroplasts but discontinuous for bush bean (12–17°C) and spinach (12–20°C) chloroplasts; (b) unaffected by 3-(3,4 dichlorophenyl)-1,1-dimethylurea (inhibitor of electron flow), gramicidin D (which eliminates light-induced membrane potential) and glutaraldehyde fixation (which stops gross structural changes).
The discontinuities, noted above for bush bean and spinach chloroplasts, are correlated with abrupt changes in (a) the thylakoid membrane lipid fluidity (monitored by EPR spectra of 12 nixtroxide stearate, 12NS) and (b) the fluidity of extracted lipids (monitored by differential calorimetry and EPR spectra of 12 NS). However, no such discontinuity was observed in (a) chlorophyll a fluorescence intensity of thylakoids and (b) fluorescence of tryptophan residues of delipidated chloroplasts.
Microsecond delayed light is linearly dependent on light intensity at flash intensities as low as one quantum per 2 times 10⁴ chlorophyll molecules. We suggest that this delayed light could originate from a one quantum process in agreement with the hypothesis that recombination of primary charges leads to this light emission. A working hypothesis for the energy levels of Photosystem II components is proposed involving a charge stabilization step on the primary acceptor side, which is in a lipid environment.
Finally, the redox potential of P680 (the reaction center for chlorophyll of system II) is calculated to be close to 1.0–1.3 V.     

Protochlorophyllide and chlorophyll forms in dark-grown stems and stem-related organs

Protochlorophyllide contents and the spectral properties together with photoactivities of native protochlorophyllide forms have been studied in dark-forced stems of 26 and epicotyls or hypocotyls of 9 plant species. The 77 K fluorescence emission spectra show that a form emitting at 629-631 nm is general in these organs. Besides this short-wavelength form, other protochlorophyllide forms emitting at 636, 645 and around 650-655 nm are found with various relative amplitudes. The pigment contents show good correlation with the ratio of short- to long-wavelength forms, i.e., the higher this ratio is, the less protochlorophyllide is detected. In addition to protochlorophyllide, several dark-grown plants also contain chlorophylls. In some cases only one chlorophyll form appears with emission maximum at 678-680 nm; other plants have forms characteristic of the fully developed photosynthetic apparatus (with maxima at 685, 695 and 730-740 nm). Flash illumination can transform only the 645 and 650-655 nm protochlorophyllide forms, the shorter-wavelength-emitting forms being inactive. Plant species with dominating 629-636 nm protochlorophyllide forms cannot accumulate chlorophyll on continuous illumination of natural intensity, and they became photodamaged. The structural or molecular background of the appearance of the different protochlorophyllide and chlorophyll forms and the reasons for their photosensitivity are discussed.     

BASF 13.338 INDUCED CHANGES IN STRUCTURE and FUNCTION OF THE PHOTOSYNTHETIC APPARATUS OF WHEAT SEEDLINGS

Abstract— Growing wheat seedlings in the presence of BASF 13.338 [4-chloro-5-dimethylamino-2-phenyl-3(2H)pyridazinone], a PS II inhibitor of the pyridazinone group, brought about notable changes in the structure and functioning of photosynthetic apparatus. In BASF 13.338 treated plants, there was a decrease in the ratio of Chi a/Chl b, an increase in xanthophyll/carotene ratio and an increase in the content of Cyt b 559 (HP + LP). Chl/p700 ratio increased when measured with the isolated chloroplasts but not with the isolated PS I particles of the treated plants. The SDS-PAGE pattern of chloroplast preparations showed an increase in the CPII/CP I ratio. The F685/F740 ratio in the emission spectrum of chloroplasts at -196°C increased. The difference absorption spectrum of chloroplasts between the control and the treated plants showed a relative increase of a chlorophyll component with a peak absorption at 676 nm and a relative decrease of a chlorophyll component with a peak absorption at 692 nm for the treated plants. The excitation spectra of these chloroplast preparations were similar. Chloroplasts from the treated plants exhibited a greater degree of grana stacking as measured by the chlorophyll content in the 10 K pellet. The rate of electron transfer through photosystem II at saturating light intensity in chloroplast thylakoids isolated from the treated plants increased (by 50%) optimally at treatment of 125 μM BASF 13.338 as compared to the control. This increase was accompanied by an increase in (a) I₅₀ value of DCMU inhibition of photosystem II electron transfer; (b) the relative quantum yield of photosystem II electron transfer; (c) the magnitude of C550 absorbance change; and (d) the rate of carotenoid photobleaching. These observations were interpreted in terms of preferential synthesis of photosystem II in the treated plants. The rate of electron transfer through photosystems I and through the whole chain (H₂O → methyl viologen) also increased, due to an additional effect of BASF 13.338, namely, an increase in the rate of electron transfer through the rate limiting step (between plastoquinol and cytochrome f). This was linked to an enhanced level of functional cytochrome f. The increase in the overall rate of electron transfer occurred in spite of a decrease in the content of photosystem I relative to photosystem II. Treatment with higher concentrations (> 125 μM) of BASF 13.338 caused a further increase in the level of cytochrome f, but the rate of electron transfer was no greater than in the control. This was due to an inhibition of electron transfer at several sites in the chain.     

Electrophoresis in plant cell organelles

Electric currents applied through living plant tissue induce intracellular transfer of electrode associated molecular complexes which alter the spatial configurations between the cell nucleus and surrounding organelles. This putative transduction process was quantitatively evaluated by microscopically determining the degree of Nucleus-Organelle Clustering (NOC) between the plant cell nuclei and chloroplasts. Type and molecular complexity of the electrode affiliated donor greatly influenced the level of Cf, an empirically derived clustering factor. A low level, well defined NOC was observed in normal non-transduced plant tissue, the nature of which suggested the presence of a natural electric field surrounding the nucleus. Movement of cell organelles into proximity with the nuclear membrane was considered as taking place by means of electrophoretic mechanisms.     

Quality control of Photosystem II: where and how does the degradation of the D1 protein by FtsH proteases start under light stress?--Facts and hypotheses

Degradation of the reaction center-binding D1 protein of Photosystem II is central in photoinhibition of Photosystem II. In higher plant chloroplasts, Photosystem II complexes are abundant in the grana. It has been suggested that the Photosystem II complexes containing photodamaged D1 protein migrate for their repair from the grana to the non-appressed stroma thylakoids, where the photodamaged D1 protein is degraded by a specific protease(s) such as filamentation temperature sensitive H (FtsH) protease. There are several possible ways to activate the FtsH proteases. As FtsH is a membrane-bound ATP-dependent metalloprotease, it requires ATP and zinc as essential part of its catalytic mechanism. It is also suggested that a membrane protein(s) associated with FtsH is required for modulation of the FtsH activity. Here, we propose several possible mechanisms for activation of the proteases, which depend on oligomerization of the monomer subunits. In relation to the oligomerization of FtsH subunits, we also suggest unique distribution of active FtsH hexamers on the thylakoids: hexamers of the FtsH proteases are localized near the Photosystem II complexes at the grana. Degradation of the D1 protein probably takes place in the grana rather than in the stroma thylakoids to circumvent long-distance migration of both the Photosystem II complexes containing the photodamaged D1 protein and the proteases.     

Detection of Vibrational Spectroscopic Biomarkers of the Effect of Gold Nanoparticles on Wheat Seedlings Using Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy

The aim of this study is to evaluate the biochemical changes in the leaves of wheat seedlings exposed to gold nanoparticles (AuNPs) nondestructively and rapidly using attenuated total reflectance Fourier transform infrared spectroscopy and laser-induced fluorescence. The 18?nm size gold nanoparticles are synthesized by citrate reduction. For analyzing the effect of gold nanoparticles on wheat seedlings, the treatment of gold nanoparticles was applied to the seedlings through roots and following the spectroscopic measurement of biochemical signatures. The laser-induced fluorescence measurement has been performed to access the effect of gold nanoparticles on the chlorophyll concentration of wheat seedlings. The decrease in the fluorescence intensity and the fluorescence intensity ratio on the treatment of gold nanoparticles indicates increase in the concentration of chlorophyll in the leaves of wheat seedlings. The attenuated total reflectance Fourier transform infrarred spectroscopy in combination with principal component analysis has been used to visualize the biochemical changes in the cellulose, hemicellulose, pectin, lignin, amino acids, proteins, and lipid of the leaves of wheat seedlings by recording infrared spectra in the region from 4000 to 400?cm^?1. Principal component analysis applied to the preprocessed infrared data clearly distinguishes the spectral variability between control and gold nanoparticle treated seedlings. The study shows that exposure of gold nanoparticles increases the concentrations of cellulose, hemicelluloses, pectin, and lignin in the leaves of wheat seedlings. The increase in these chemicals indicates the modulation of cell walls of the wheat seedlings by the gold nanoparticle treatment. The exposure to gold nanoparticles also enhances the expression of lipid and proteins in the leaves of wheat seedlings.     

Determination of rare earth elements in chloroplasts of Brassia napus by INAA and biochemical separation techniques

Intact chloroplasts were isolated from mesophyll protoplasts of Brassia napus. Concentrations of 8 rare earth elements (REEs) in the chloroplasts were determined by instrumental neutron activation analysis (INAA). The results showed that there were trace amounts of REEs in the chloroplasts, which corresponded to 1 atom of REEs per 2000 chlorophyll molecules. About 30% of the total REEs in the leaves are localized in the chloroplasts and the light REEs were enriched with respect to the heavy elements of the series.     

Chlorophyll Breakdown in Senescent Banana Leaves: Catabolism Reprogrammed for Biosynthesis of Persistent Blue Fluorescent Tetrapyrroles

Chlorophyll breakdown is a visual phenomenon of leaf senescence and fruit ripening. It leads to the formation of colorless chlorophyll catabolites, a group of (chlorophyll‐derived bilin‐type) linear tetrapyrroles. Here, analysis and structure elucidation of the chlorophyll breakdown products in leaves of banana (Musa acuminata) is reported. In senescent leaves of this monocot all chlorophyll catabolites identified were hypermodified fluorescent chlorophyll catabolites (hmFCCs). Surprisingly, nonfluorescent chlorophyll catabolites (NCCs) were not found, the often abundant and apparently typical final chlorophyll breakdown products in senescent leaves. As a rule, FCCs exist only fleetingly, and they isomerize rapidly to NCCs in the senescent plant cell. Amazingly, in the leaves of banana plants, persistent hmFCCs were identified that accounted for about 80 % of the chlorophyll broken down, and yellow leaves of M. acuminata display a strong blue luminescence. The structures of eight hmFCCs from banana leaves were analyzed by spectroscopic means. The massive accumulation of the hmFCCs in banana leaves, and their functional group characteristics, indicate a chlorophyll breakdown path, the downstream transformations of which are entirely reprogrammed towards the generation of persistent and blue fluorescent FCCs. As expressed earlier in related studies, the present findings call for attention, as to still elusive biological roles of these linear tetrapyrroles.     

Ultraviolet-A induced changes in photosystem II of thylakoids: effects of senescence and high growth temperature

Ultraviolet-A (UV-A) radiation induced changes in photosystem II (PS II) of senescing leaves of wheat seedlings were investigated. UV-A radiation did not show any significant effect on the level of photosynthetic pigments. However, the decline in F(v)/F(m) and oxygen evolution rate indicated the damaging effect of the radiation on primary photochemistry of PS II. Modification at the Q(B)-binding site was inferred from the observed downshift of peak temperature of thermoluminescence (TL) B-bands. The UV-A induced changes in PS II of chloroplasts from senescing leaves were found to be synergistically accelerated by high growth temperature.