Light harvesting complexes in chlorophyll c-containing algae

Besides the so-called ‘green lineage’ of eukaryotic photosynthetic organisms that include vascular plants, a huge variety of different algal groups exist that also harvest light by means of membrane intrinsic light harvesting proteins (Lhc). The main taxa of these algae are the Cryptophytes, Haptophytes, Dinophytes, Chromeridae and the Heterokonts, the latter including diatoms, brown algae, Xanthophyceae and Eustigmatophyceae amongst others. Despite the similarity in Lhc proteins between vascular plants and these algae, pigmentation is significantly different since no Chl b is bound, but often replaced by Chl c, and a large diversity in carotenoids functioning in light harvesting and/or photoprotection is present. Due to the presence of Chl c in most of the taxa the name ‘Chl c-containing organisms’ has become common, however, Chl b-less is more precise since some harbour Lhc proteins that only bind one type of Chl, Chl a. In recent years huge progress has been made about the occurrence and function of Lhc in diatoms, so-called fucoxanthin chlorophyll proteins (FCP), where also the first molecular structure became available recently. In addition, especially energy transfer amongst the unusual pigments bound was intensively studied in many of these groups. This review summarises the present knowledge about the molecular structure, the arrangement of the different Lhc in complexes, the excitation energy transfer abilities and the involvement in photoprotection of the different Lhc systems in the so-called Chl c-containing organisms.This article is part of a Special Issue entitled Light harvesting, edited by Dr. Roberta Croce.     

Calcium-induced formation of chlorophyll b and light-harvesting chlorophyll complex in cucumber cotyledons in the dark

Dark-grown cucumber seedlings were exposed to intermittent light (2 min light and 98 min dark) and then cotyledons were incubated with 50 mM CaCl₂ in the dark. Chlorophyll (Chl) a was selectively accumulated under intermittent light and Chl b was accumulated during the subsequent dark incubation with CaCl₂. The change in chlorophyll-protein complexes during Chl b accumulation induced by CaCl₂ in the dark was investigated by SDS-polyacrylamide gel electrophoresis. Chlorophyll-protein complex I and free chlorophyll were major chlorophyll-containing bands of the cotyledons intermittently illuminated 10 times. When these cotyledons were incubated with CaCl₂ in the dark, the light-harvesting Chl complex was formed. When the number of intermittent illumination periods was extended to 55, small amounts of Chl b and light-harvesting Chl complex were recognized at the end of intermittent light treatment, and these two pigments were further increased during the subsequent incubation of the cotyledons with CaCl₂ in the dark compared to water controls.     

Action spectra for chlorophyll formation during greening of wheat leaves in continuous light

The contents of Chl a and Chl b in dark-grown leaves of wheat ( Triticum aestivum L. cv. Starke II, Weibull) irradiated for 20 h with light of different wavelengths and intensities have been measured. The amount of Chl b formed revealed a higher maximum in the Chl b /Chl a ratio for light most strongly absorbed by the phytochrome system and giving a high [P_fr]/[P_tot] ratio. This indicates a stronger enhancing effect of P_fr on Chl b formation than on Chl a formation. Similarities between action spectra for Chl a and b formation indicate that Chl b originates from Chl a . A comparatively high effectiveness of light within the wavelength range 500–550 nm as compared to the action spectrum for PChl→Chl phototransformation shows that PChl₆₃₆, in addition to PChl₆₅₀, takes part in the greening process.     

Endolithic chlorophyll d-containing phototrophs

Cyanobacteria in the genus Acaryochloris are the only known oxyphototrophs that have exchanged chlorophyll a (Chl a) with Chl d as their primary photopigment, facilitating oxygenic photosynthesis with near infrared (NIR) light. Yet their ecology and natural habitats are largely unknown. We used hyperspectral and variable chlorophyll fluorescence imaging, scanning electron microscopy, photopigment analysis and DNA sequencing to show that Acaryochloris-like cyanobacteria thrive underneath crustose coralline algae in a widespread endolithic habitat on coral reefs. This finding suggests an important role of Chl d-containing cyanobacteria in a range of hitherto unexplored endolithic habitats, where NIR light-driven oxygenic photosynthesis may be significant.     

Calcium-induced formation of chlorophyll b and light-harvesting chlorophyll a/b-protein complex in cucumber cotyledons in the dark

Dark-grown cucumber seedlings were exposed to intermittent light (2 min light and 98 min dark) and then cotyledons were incubated with 50 mM CaCl₂ in the dark. Chlorophyll (Chl) a was selectively accumulated under intermittent light and Chl b was accumulated during the subsequent dark incubation with CaCl₂. The change in chlorophyll-protein complexes during Chl b accumulation induced by CaCl₂ in the dark was investigated by SDS-polyacrylamide gel electrophoresis. Chlorophyll-protein complex I and free chlorophyll were major chlorophyll-containing bands of the cotyledons intermittently illuminated 10 times. When these cotyledons were incubated with CaCl₂ in the dark, the light-harvesting Chl a/b-protein complex was formed. When the number of intermittent illumination periods was extended to 55, small amounts of Chl b and light-harvesting Chl a/b-protein complex were recognized at the end of intermittent light treatment, and these two pigments were further increased during the subsequent incubation of the cotyledons with CaCl₂ in the dark compared to water controls.     

Widespread occurrence and unexpected diversity of red-shifted chlorophyll producing cyanobacteria in humid subtropical forest ecosystems

Discovery of red-shifted chlorophyll d and f in cyanobacteria has opened up new avenues to estimate global carbon fixation driven by far-red light. Shaded habitats in humid subtropical forest ecosystems contain an increased proportion of far-red light components relative to residual white light. After an extensive survey of shaded ecosystems within subtropical forests, wide occurrence of red-shifted chlorophyll-producing cyanobacteria was demonstrated by isolated Chl f-producing and Chl d-containing cyanobacteria. Chl f-producing cyanobacteria were classified into the genera of Aphanocapsa and Chroococcidiopsis and two undescribed genera within Leptolyngbyaceae. Newly isolated Chl d-containing Acaryochloris sp. CCNUM4 showed the closest phylogenetic relationship with Acaryochloris species isolated from marine environments. Acaryochloris sp. CCNUM4 produced Chl d as major photopigment, and Chl f-producing cyanobacteria use Chl a under white light conditions but Chl a + f under far-red light conditions. Their habitats are widely distributed in subtropical forest ecosystems and varied from mosses on limestone to macrophyte and freshwater in the streams and ponds. This study presents a significant advance in the knowledge of distribution and diversity of red-shifted chlorophyll-producing cyanobacteria in terrestrial ecosystems. The results suggest that Chl f-producing and Chl d-containing cyanobacteria might be important primary producers in far-red light dominant niches worldwide.     

Origin of the chlorophyll b formyl oxygen in Chlorella vulgaris.

Chlorophyll (Chl) b is an accessory light-harvesting pigment of plants and chlorophyte algae. Chl b differs from Chl a in that the 3-methyl group on ring B of chl a is replaced by a 3-formyl group on Chl b. The present study determined the biosynthetic origin of the Chl b formyl oxygen in in vivo labeling experiments. A mutant strain of the unicellular chlorophyte Chlorella vulgaris, which can not synthesize Chls when cultured in the dark but rapidly greens when transferred to the light, was grown in the dark for several generations to deplete Chls, and then the cells were transferred to the light and allowed to form Chls in a controlled atmosphere containing 18O2. Chl a and Chl b were purified from the cells and analyzed by high-resolution mass spectroscopy. Analysis of the mass spectra indicated that over 76% of the Chl a molecules had incorporated an atom of 18O. For Chl b, 58% of the molecules had incorporated an atom of 18O at one position and 34% of the molecules had incorporated an atom of 18O at a second position. These results demonstrate that the isocyclic ring keto oxygen of both Chl a and Chl b, as well as the formyl oxygen of Chl b, is derived from O2.     

Use of the atLEAF+ chlorophyll meter for a nondestructive estimate of chlorophyll content

At present, chlorophyll meters are widely used for a quick and nondestructive estimate of chlorophyll (Chl) contents in plant leaves. Chl meters allow to estimate the Chl content in relative units - the Chl index (CI). However, using such meters, one can face a problem of converting CI into absolute values of the pigment content and comparing data acquired with different devices and for different plant species. Many Chl meters (SPAD-502, CL-01, CCM-200) demonstrated a high degree of correlation between the CI and the absolute pigment content. A number of formulas have been deduced for different plant species to convert the CI into the absolute value of the photosynthetic pigment content. However, such data have not been yet acquired for the atLEAF+ Chl meter. The purpose of the present study was to assess the applicability of the atLEAF+ Chl meter for estimating the Chl content. A significant species-specific exponential relationships between the atLEAF value (corresponding to CI) and extractable Chl a, Chl b, Chl (a+b) for Calamus dioicus and Cleistanthus sp. were shown. The correlations between the atLEAF values and the content of Chl a, Chl b, and Chl (a+b) per unit of leaf area was stronger than that per unit of dry leaf mass. The atLEAF value- Chl b correlation was weaker than that of atLEAF value-Chl a and atLEAF value-Chl (a+b) correlations. The influence of light conditions (Chl a/b ratio) on the atLEAF value has been also shown. The obtained results indicated that the atLEAF+ Chl meter is a cheap and convenient tool for a quick nondestructive estimate of the Chl content, if properly calibrated, and can be used for this purpose along with other Chl meters.     

Leaf and canopy photosynthesis of a chlorophyll deficient soybean mutant

The photosynthetic, optical, and morphological characteristics of a chlorophyll‐deficient (Chl‐deficient) “yellow” soybean mutant (MinnGold) were examined in comparison with 2 green varieties (MN0095 and Eiko). Despite the large difference in Chl content, similar leaf photosynthesis rates were maintained in the Chl‐deficient mutant by offsetting the reduced absorption of red photons by a small increase in photochemical efficiency and lower non‐photochemical quenching. When grown in the field, at full canopy cover, the mutants reflected a significantly larger proportion of incoming shortwave radiation, but the total canopy light absorption was only slightly reduced, most likely due to a deeper penetration of light into the canopy space. As a consequence, canopy‐scale gross primary production and ecosystem respiration were comparable between the Chl‐deficient mutant and the green variety. However, total biomass production was lower in the mutant, which indicates that processes other than steady state photosynthesis caused a reduction in biomass accumulation over time. Analysis of non‐photochemical quenching relaxation and gas exchange in Chl‐deficient and green leaves after transitions from high to low light conditions suggested that dynamic photosynthesis might be responsible for the reduced biomass production in the Chl‐deficient mutant under field conditions.     

The involvement of phytochrome in controlling chlorophyll and 5-aminolevulinate formation in a gymnosperm seedling (Pinus sylvestris)

Chlorophyll a (Chl a) accumulation in the cotyledons of Scots pine seedlings (Pinus sylvestris L.) is much higher in the light than in darkness where it ceases 6 days after germination. When these darkgrown seedlings are treated with continuous white light (3,500 lx) a 3 h lag phase appears before Chl a accumulation is resumed. The lag phase can be eliminated by pretreating the seedlings with 7 h of weak red light (0.14 Wm^-2) or with 14 red light pulses separated by relatively short dark periods (<100 min). The effect of 15s red light pulses can be fully reversed by 1 min far-red light pulses. This reversibility is lost within 2 min. In addition, the amount of Chl a formed within 27 h of continuous red light is considerably reduced by the simultaneous application of far-red (RG 9) light. It is concluded that phytochrome (P_fr) is required not only for the elimination of the lagphase but also to maintain a high rate of Chl a accumulation in continuous light. Since accumulation of 5-aminolevulinate (ALA) responds in the same manner as Chl a accumulation to a red light pretreatment it is further concluded that ALA formation is the point where phytochrome regulates Chl biosynthesis in continuous light. No correlation has been found between ALA and Chl a formation in darkness. This indicates that in a darkgrown pine seedling ALA formation is not rate limiting for Chl a accumulation.Abbreviations Chl chlorophyll(ide) - PChl protochlorophyll(ide) - ALA 5-aminolevulinate - P_r the red absorbing form of phytochrome - P_fr the far-red absorbing form of phytochrome - P_tot total phytochrome ([P_r]+[P_fr])     

Formyl group modification of chlorophyll a: a major evolutionary mechanism in oxygenic photosynthesis

We discuss recent advances in chlorophyll research in the context of chlorophyll evolution and conclude that some derivations of the formyl side chain arrangement of the porphyrin ring from that of the Chl a macrocycle can extend the photosynthetic active radiation (PAR) of these molecules, for example, Chl d and Chl f absorb light in the near‐infrared region, up to ～750 nm. Derivations such as this confer a selective advantage in particular niches and may, therefore, be beneficial for photosynthetic organisms thriving in light environments with particular light signatures, such as red‐ and near‐far‐red light‐enriched niches. Modelling of formyl side chain substitutions of Chl a revealed yet unidentified but theoretically possible Chls with a distinct shift of light absorption properties when compared to Chl a.     

Interaction of the photosystem 1 reaction center with antenna chlorophyll in a pigment-protein complex isolated from pea chloroplasts

Using a specially developed phosporoscopic attachment to spectropolarimeter, light induced spectra of circular dichroism (CD) in region 600-750 nm were measured for a pigment protein complex of photosystem 1 (PC-1) isolated from pea chloroplast (chlorophyll : P700 = 40). Minor components at 672 and 678 nm are observed in light induced spectra besides the components of dimer splitting of P700 Qy transition at 691 and 698 nm. Haussian deconvolution of light induced CD spectra of P700 and low temperature CD spectrum of PC-1 indicates that minor components are due to forms of antenna chlorophylls Chl672 and Chl678, rotational strength of that is changed by 2-4% as a result of P700 oxidation. Long term incubation of PC-1 with Triton X-100 inhibits P700 and destroys longwave optically active chlorophyll forms. A strong relation between dichroic density of 693 nm band in CD spectrum of PC-1 and the value of light induced absorption change at 698 nm could be used to determine P700 concentration on the basis of CD spectrum of PC-1. Such a relation shows that Chl693 is an important component of photo-system 1 reaction center. It is suggested that P700 is not an isolated dimer but it is included in the local complex from 8-10 chlorophyll molecules (Chl672, Chl678, Chl686, Chl693).     

Regulation by light of chlorophyll synthesis in the cotyledons of Scots pine (Pinus sylvestris) seedlings

Seedlings of gymnosperms, unlike angiosperms, synthesize chlorophyll(ide) (Chl) in darkness (D). In Scots pine cotyledons ( Pinus sylvestris L.) Chl accumulation ceases in D at a low level but Chl accumulation is strongly increased by light, red light (R) being more effective than blue light (B), whereas in Pinus maritima Chi synthesis is almost light-independent. In Scots pine the capacity to form Chl can be increased by R pulses, fully reversible by far-red light, demonstrating the involvement of phytochrome. However, when B- or R–grown seedlings were transferred to D, Chl accumulation stopped immediately irrespective of the level of P_fr (far-red light absorbing form of phytochrome), indicating that the conversion of protochlorophyllide (PChl) is light-dependent. Dose response curves in R and B and simultaneous irradiation with R and B show that R and B are perceived by separate photoreceptors. The immunodetected NADPH-dependent protochlorophyllide oxidoreductase (POR, EC 1.6.99.1), assumed to regulate light-dependent Chl synthesis in angiosperms, is not correlated with the capacity of gymnosperm Chi accumulation in darkness. While two FOR bands could be separated in extracts from dark grown material (38 and 36 kDa) of Pinus sylvestris and P. maritima , only the 38 kDa band disappeared consistently in the light. However. the significance of the more light resistant 36 kDa band for chlorophyll synthesis remains unclear as well.     

The presence of chlorophyll b in Synechocystis sp. PCC 6803 disturbs tetrapyrrole biosynthesis and enhances chlorophyll degradation

Both chlorophyll (Chl) a and b accumulate in the light in a Synechocystis sp. PCC 6803 strain that expresses higher plant genes coding for a light-harvesting complex II protein and Chl a oxygenase. This cyanobacterial strain also lacks photosystem (PS) I and cannot synthesize Chl in darkness because of the lack of chlL. When this PS I-less/chlL(-)/lhcb(+)/cao(+) strain was grown in darkness, small amounts of two unusual tetrapyrroles, protochlorophyllide (PChlide) b and pheophorbide (pheide) b, were identified. Accumulation of PChlide b trailed that of PChlide a by several days, suggesting that PChlide a is an inefficient substrate of Chl a oxygenase. The presence of pheide b in this organism suggests a breakdown of Chl b via a pathway that does not involve conversion to a-type pigments. When the PS I-less/chlL(-) control strain was grown in darkness, Chl degradation was much slower than in the PS I-less/chlL(-)/lhcb(+)/cao(+) strain, suggesting that the presence of Chl b leads to more rapid turnover of Chl-binding proteins and/or a more active Chl degradation pathway. Levels and biosynthesis kinetics of Chl and of its biosynthetic intermediates are very different in the PS I-less/chlL(-)/lhcb(+)/cao(+) strain versus in the control. Moreover, when grown in darkness for 14 days, upon the addition of delta-aminolevulinic acid, the level of magnesium-protoporphyrin IX increased 60-fold in the PS I-less/chlL(-)/lhcb(+)/cao(+) strain (only approximately 2-fold in the PS I-less/chlL(-) control strain), whereas the PChlide and protoheme levels remained fairly constant. We propose that a b-type PChlide, Chl, or pheide in the PS I-less/chlL(-)/lhcb(+)/cao(+) strain may bind to tetrapyrrole biosynthesis regulatory protein(s) (for example, the small Cab-like proteins) and thus affect the regulation of this pathway.     

A novel role of water-soluble chlorophyll proteins in the transitory storage of chorophyllide

All chlorophyll (Chl)-binding proteins involved in photosynthesis of higher plants are hydrophobic membrane proteins integrated into the thylakoids. However, a different category of Chl-binding proteins, the so-called water-soluble Chl proteins (WSCPs), was found in members of the Brassicaceae, Polygonaceae, Chenopodiaceae, and Amaranthaceae families. WSCPs from different plant species bind Chl a and Chl b in different ratios. Some members of the WSCP family are induced after drought and heat stress as well as leaf detachment. It has been proposed that this group of proteins might have a physiological function in the Chl degradation pathway. We demonstrate here that a protein that shared sequence homology to WSCPs accumulated in etiolated barley (Hordeum vulgare) seedlings exposed to light for 2 h. The novel 22-kD protein was attached to the outer envelope of barley etiochloroplasts, and import of the 27-kD precursor was light dependent and induced after feeding the isolated plastids the tetrapyrrole precursor 5-aminolevulinic acid. HPLC analyses and spectroscopic pigment measurements of acetone-extracted pigments showed that the 22-kD protein is complexed with chlorophyllide. We propose a novel role of WSCPs as pigment carriers operating during light-induced chloroplast development.     

A cyanobacterium that contains chlorophyll f - a red-absorbing photopigment

A Chl f-containing filamentous cyanobacterium was purified from stromatolites and named as Halomicronema hongdechloris gen., sp. nov. after its phylogenetic classification and the morphological characteristics. Hongdechloris contains four main carotenoids and two chlorophylls, a and f. The ratio of Chl f to Chl a is reversibly changed from 1:8 under red light to an undetectable level of Chl f under white-light culture conditions. Phycobiliproteins were induced under white light growth conditions. A fluorescence emission peak of 748 nm was identified as due to Chl f. The results suggest that Chl f is a red-light inducible chlorophyll.     

NPQ activation reduces chlorophyll triplet state formation in the moss Physcomitrella patens

Plants live in variable environments in which light intensity can rapidly change, from limiting to excess conditions. Non-photochemical quenching (NPQ) is a regulatory mechanism which protects plants from oxidative stress by dissipating excess Chl singlet excitation. In this work, the physiological role of NPQ was assessed by monitoring its influence on the population of the direct source of light excess damage, i.e., Chl triplets ((3)Chl*). (3)Chl* formation was evaluated in vivo, with the moss Physcomitrella patens, by exploiting the high sensitivity of fluorescence-detected magnetic resonance (FDMR). A dark adapted sample was compared with a pre-illuminated sample in which NPQ was activated, the latter showing a strong reduction in (3)Chl* yield. In line with this result, mutants unable to activate NPQ showed only a minor effect in (3)Chl* yield upon pre-illumination.The decrease in (3)Chl* yield is equally experienced by all the Chl pools associated with PSII, suggesting that NPQ is effective in protecting both the core and the peripheral antenna complexes. Moreover, the FDMR results show that the structural reorganization in the photosynthetic apparatus, required by NPQ, does not lead to the formation of new (3)Chl* traps in the LHCs. This work demonstrates that NPQ activation leads to effective photoprotection, promoting a photosystem II state characterized by a reduced probability of (3)Chl* formation, due to a decreased singlet excited state population, while maintaining an efficient quenching of the (3)Chl* eventually formed by carotenoids.     

Identification and characterization of Arabidopsis mutants with reduced quenching of chlorophyll fluorescence

Regulation of nonradiative dissipation of absorbed light energy in PSII is an indispensable process to avoid photoinhibition in plants. To dissect molecular mechanisms of the regulation, we identified Arabidopsis mutants with reduced quenching of Chl fluorescence using a fluorescence imaging system. By analyses of Chl fluorescence induction pattern in the light and quantum yield of both photosystems, 37 mutants were classified into three groups. The first group was characterized by an extremely high level of minimum Chl fluorescence at the open PSII center possibly due to a defect in PSII. Mutants with significant reduction in the nonphotochemical quenching formation but not in quantum yield of both photosystems were classified into the second group. Mutants in the third group showed reduction in quantum yield of both photosystems possibly due to a defect in the electron transport activity. Mutants in the second and third groups were further characterized by light intensity dependence of Chl fluorescence parameters and steady state redox level of P700.     

Effects of light and circadian clock on growth and chlorophyll accumulation of Nannochloropsis gaditana

Circadian clocks synchronize various physiological, metabolic and developmental processes of organisms with specific phases of recurring changes in their environment (e.g. day and night or seasons). Here, we investigated whether the circadian clock plays a role in regulation of growth and chlorophyll (Chl) accumulation in Nannochloropsis gaditana, an oleaginous marine microalga which is considered as a potential feedstock for biofuels and for which a draft genome sequence has been published. Optical density (OD) of N. gaditana culture was monitored at 680 and 735 nm under 12:12 h or 18:6 h light‐dark (LD) cycles and after switching to continuous illumination in photobioreactors. In parallel, Chl fluorescence was measured to assess the quantum yield of photosystem II. Furthermore, to test if red‐ or blue‐light photoreceptors are involved in clock entrainment in N. gaditana, some of the experiments were conducted by using only red or blue light. Growth and Chl accumulation were confined to light periods in the LD cycles, increasing more strongly in the first half than in the second half of the light periods. After switching to continuous light, rhythmic oscillations continued (especially for OD₆₈₀) at least in the first 24 h, with a 50% decrease in the capacity to grow and accumulate Chl during the first subjective night. Pronounced free‐running oscillations were induced by blue light, but not by red light. In contrast, the photosystem II quantum yield was determined by light conditions. The results indicate interactions between circadian and light regulation of growth and Chl accumulation in N. gaditana.