James Barber, known to colleagues and friends as Jim, passed away in January 2020 after a long battle against cancer. During his long and distinguished career in photosynthesis research, Jim made many outstanding contributions with the pinnacle achieving his dream of determining the first detailed structure of the Mn cluster involved in photosynthetic water oxidation. Here, colleagues and friends remember Jim and reflect upon his scientific career and the impact he had on their lives and the scientific community.

Jim Barber FRS, President of the International Society of Photosynthesis Research 2007–2010, died on 5 January 2020 peacefully after a long illness. Jim kept going as long as he could in his incredible energetic way, organizing meetings and writing papers after his cancer diagnosis in 2012. He was desperately trying to write a paper for the Biochemical Society (as he had been awarded the Heatley Medal and Prize for 2020) when I visited him last December. He knew his time had come, but he wanted to keep going.Jim left school at 16 and later studied for his A-levels at night school. After gaining his BSc in Chemistry at Swansea University, he went to the University of East Anglia for his MSc and PhD, also completing a 1 year post-doc at Leiden University. Jim was appointed lecturer in the Department of Botany and Plant Technology at Imperial College London in 1968 and a full professor in 1979. He then moved to the Biochemistry Department at Imperial, which he headed from 1989 to 1999.He spent his whole career specializing in research on the light reactions of photosynthesis, culminating in 2004 in the first refined crystallographic structure of Photosystem II, the water-splitting enzyme, in collaboration with Professor So Iwata. He was awarded many honours for his achievements, including Member of Academia Europaea (1989), Foreign Member of the Royal Swedish Academy of Sciences (2003) and Fellow of the Royal Society (2005). In addition to his research, he edited countless books and organized many meetings on photosynthesis. In later years, he focused on artificial photosynthesis in order to develop a technology for solar fuel production, to which end he established the Biosolar lab at the Politecnico di Torino and the Solar Fuels lab in NTU Singapore.Jim truly was someone who wanted to get on in life and finally fought a battle against cancer. I will never forget that during my last visit to see him before Christmas he was trying to calculate how much ATP he needed to keep going!Jim is survived by his wife, Lyn, his two children, Neil and Julie, and six grandchildren.

Article Figures and data Abstract eLife digest Introduction Results and discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Carotenoids are essential in oxygenic photosynthesis: they stabilize the pigment–protein complexes, are active in harvesting sunlight and in photoprotection. In plants, they are present as carotenes and their oxygenated derivatives, xanthophylls. While mutant plants lacking xanthophylls are capable of photoautotrophic growth, no plants without carotenes in their photosystems have been reported so far, which has led to the common opinion that carotenes are essential for photosynthesis. Here, we report the first plant that grows photoautotrophically in the absence of carotenes: a tobacco plant containing only the xanthophyll astaxanthin. Surprisingly, both photosystems are fully functional despite their carotenoid-binding sites being occupied by astaxanthin instead of β-carotene or remaining empty (i.e. are not occupied by carotenoids). These plants display non-photochemical quenching, despite the absence of both zeaxanthin and lutein and show that tobacco can regulate the ratio between the two photosystems in a very large dynamic range to optimize electron transport. eLife digest Most life on Earth depends on photosynthesis, the process used by plants and many other organisms to store energy from sunlight and produce oxygen. The first steps of photosynthesis, the capture and conversion of sunlight into chemical energy, happen in large assemblies of proteins containing many pigment molecules called photosystems. In plants, the pigments involved in photosynthesis are green chlorophylls and carotenoids. In addition to harvesting light, carotenoids have an important role in preventing damage caused by overexposure to sunlight There are over one thousand different carotenoids in living beings, but only one, β-carotene, is present in every organism that performs the type of photosynthesis in which oxygen is released, and is thought to be essential for the process. However, this could never be proved because it is impossible to remove β-carotene from cells using typical genetic approaches without affecting all other carotenoids. Xu et al. used genetic engineering to create tobacco plants that produced a pigment called astaxanthin in place of β-carotene. Astaxanthin is a carotenoid from salmon and shrimp, not normally found in plants. These plants are the first living things known to perform photosynthesis without β-carotene and demonstrate that this pigment is not essential for photosynthesis as long as other carotenoids are present. Xu et al. also show that the photosystems can adapt to using different carotenoids, and can even operate with a reduced number of them. Xu et al’s findings show the high flexibility of photosynthesis in plants, which are able to incorporate non-native elements to the process. These results are also important in the context of increasing the photosynthetic efficiency, and thus the productivity of crops, since they show that a radical redesign of the photosynthetic machinery is feasible. Introduction Carotenoids form a large class of natural pigments responsible for the yellow, orange, and red colors of fruits and leaves (Stange, 2016). In the photosynthetic membranes, they are mainly associated with proteins, forming pigment–protein complexes. Their large absorption cross-section in the blue region of the solar spectrum makes them ideal light-harvesting pigments, especially for aquatic organisms (Croce and van Amerongen, 2014). However, the primary role of carotenoids in photosynthesis is photoprotection. Their capacity to quench chlorophyll (Chl) triplets (thus avoiding their reaction with molecular oxygen and the production of singlet oxygen), and to scavenge singlet oxygen make them essential for the survival of the organism (Frank and Cogdell, 1996; Borth, 1975; Havaux, 1998). In addition, carotenoids are involved in the quenching of singlet excited state Chls in a process known as non-photochemical quenching (NPQ), which controls the level of excited states in the membrane, thus protecting the photosynthetic apparatus from high light damage (Ruban et al., 2012). Two species of carotenoids are present in the photosynthetic membranes: carotenes and their oxygenated derivatives, xanthophylls. The main carotene, β-carotene (β-car), is associated with the core of photosystems I and II (Umena et al., 2011; Qin et al., 2015), and is present in all organisms performing oxygenic photosynthesis. The xanthophylls (in plants mainly lutein (Lut), neoxanthin (Neo), violaxanthin (Vio) and zeaxanthin (Zea)), instead, are bound to the light-harvesting complexes (LHCs) that act as peripheral antennae increasing the absorption cross-section of both photosystems (Qin et al., 2015; Su et al., 2017). LHCs are able to accommodate different xanthophylls, but they cannot fold in the presence of β-carotene only (Croce et al., 1999; Phillip et al., 2002). Also, PSII assembly has been suggested to require the presence of carotenes (Masamoto et al., 2004), while PSI is stable also in the absence of carotenoids (Masamoto et al., 2004; Santabarbara et al., 2013). While mutants lacking individual or all xanthophylls but still containing carotenes have been identified for several organisms (e.g. Dall'Osto et al., 2013; Ware et al., 2016; Niyogi et al., 1997; Pogson et al., 1998; Domonkos et al., 2013; Schäfer et al., 2005), mutants lacking carotenes have only been isolated in cyanobacteria and the green alga Chlamydomonas reinhardtii when these organisms were grown heterotrophically (Santabarbara et al., 2013; Sozer et al., 2010; Tóth et al., 2015). In these mutants, no PSII was assembled. This finding, together with the fact that no PSII complexes without carotenes have ever been observed, have suggested that carotenes have a vital role not only in photosynthesis but also for the survival of the plant cell (Dall’Osto et al., 2014). However, this assumption could never be verified, because available mutants without carotenes completely lack carotenoids. In this work, we have analyzed tobacco (Nicotiana tabacum) plants in which the carotenoid biosynthetic pathway was engineered (by stable transformation of the chloroplast genome) to only produce the ketocarotenoid astaxanthin (Lu et al., 2017; Figure 1). The physiological characteristics and the autotrophic growth of these plants demonstrate that photosynthesis without carotenes is possible, at least when plants are grown in laboratory conditions. Figure 1 with 1 supplement see all Download asset Open asset Phenotype of wild-type (WT) and astaxanthin-synthesizing tobacco plants (Asta) and pigment analysis. WT (A) and Asta (B) plants, 7 and 21 weeks old, respectively. Note that the older leaves in the mutant are less orange than the young leaves. See also Figure 1—figure supplement 1. (C) Chromatographic profiles of the pigments extracted from leaves normalized to the Chl a peak. Neo, neoxanthin; Vio, violaxanthin; Lut, lutein; β-car, β-carotene; Chl, chlorophyll; 4-keto, 4-ketoanteraxanthin; Ast, astaxanthin; Ado, adonixanthin; Can, Canthaxanthin. Results and discussion The leaves of the tobacco mutant are orange at an early stage and become greener with age (see Figure 1, Table 1). This is likely due to the high expression of the chloroplast genome in young leaves (Edwards et al., 2010), which in the mutant results in the massive production of astaxanthin. Most of the astaxanthin is present in the form of crystals or aggregates in the chloroplast (Lu et al., 2017). The high-level synthesis of astaxanthin uses a substantial part of the plant’s energy budget and fixed carbon and may contribute to the slow growth of the plants. This negative effect on growth is likely exacerbated by the fact that astaxanthin absorbs most of the incident light, decreasing the number of photons available for photosynthesis. Indeed, the greening of the leaves corresponds to an increase in the growth rate of the plants. Both young and mature leaves of the mutant plants contain only 20% of the Chls per fresh weight as compared to the wild-type (WT), but have a similar (mature leaves) or far higher (young leaves) carotenoid content (Table 1). However, the mutant, at all stages of growth, only contains astaxanthin and traces of by-products of astaxanthin synthesis (Hasunuma et al., 2008) and does not accumulate (<0.005 times the WT) the carotenoids that are usually present in the WT (Figure 1). This result is different from the analysis of previously generated astaxanthin-producing plants that still contained WT carotenoids, although in reduced amounts (Hasunuma et al., 2008; Fujii et al., 2016; Röding et al., 2015). Thus, our engineered tobacco (hereafter referred to as Asta) represents the first organism showing autotrophic growth in the virtual absence of carotenes. In the following, we report the experiments performed on mature leaves, which have a Chl/car similar to the WT. Table 1 Pigment composition of leaves. SamplesChl a/bChl/carChl/fresh wt (mg/g)Chl/leaf area # (mg)WT3.79 ± 0.094.21 ± 0.202.63 ± 0.390.0312 ± 0.0026Asta mature leaves3.01 ± 0.103.02 ± 0.220.58 ± 0.070.0062 ± 0.0008Asta young leaves3.16 ± 0.190.76 ± 0.130.55 ± 0.090.0049 ± 0.0011 (#50 mm2; Average values ± SD are shown. n = 10 biological replicas). Since violaxanthin and lutein are considered to be necessary for the folding of the antenna complexes (Dall'Osto et al., 2006), and β-carotene was thought to be required for PSII assembly and photosynthetic activity (Santabarbara et al., 2013; Sozer et al., 2010; Tóth et al., 2015), we analyzed the effect of their absence on the composition and organization of the photosynthetic apparatus. 2D gel electrophoresis (Figure 2) and immunoblot analyses (Figure 2—figure supplement 1) of thylakoid membranes show that all of the main photosynthetic proteins are present in Asta plants, but the PSII/PSI ratio is far higher than in the WT (Figure 2—figure supplement 1). The LHC/PSII ratio is, however, similar, except for the antenna protein Lhcb5 that is strongly reduced, and for PsbS, the main protein involved in NPQ (Li et al., 2000), which is increased in the mutant (Figure 2—figure supplement 1). PSI-LHCI, ATP synthase and cytochrome b6f have the same mobility in native gels as the WT complexes, indicating that they are stable and have the same supramolecular organization. By contrast, the stability of PSII seems to be affected as the bands corresponding to PSII supercomplexes and LHCII trimers, which are well defined in the WT, are substituted by a smear in the mutant, suggesting that the PSII complexes are more heterogeneous, incompletely assembled or less stable than in the WT (Figure 2). Figure 2 with 1 supplement see all Download asset Open asset Protein composition and supramolecular organization of the photosynthetic complexes in WT and Asta plants. (A) Thylakoids were solubilized with 1% α-DDM and loaded on a blue-native gel. Second dimension SDS-PAGE of the WT (B) and the Asta mutant (C). The immunoblotting analysis is shown in Figure 2—figure supplement 1. Next, we investigated the effects of the change in carotenoid composition on the properties of the individual complexes that were isolated from thylakoid membranes and separated by sucrose density gradient ultracentrifugation (Figure 3—figure supplement 1). Pigment analysis (Table 2 and Figure 3—figure supplement 2) confirmed that astaxanthin is the only carotenoid associated with all pigment-binding complexes in Asta plants, while β-carotene is present in PSI in a highly substoichiometric amount (0.15 β-carotene molecules per complex) and it is virtually absent in PSII (0.03 molecules per complex). This means that most of the PSI and PSII complexes in the mutant plants do not contain β-carotene at all. Normalized to Chl, Asta-LHCs contain the same number of carotenoids as the WT monomers, but instead of binding lutein, neoxanthin and violaxanthin, they only bind astaxanthin, indicating that all carotenoid-binding sites are promiscuous and can accommodate different xanthophylls. The pigment analysis also showed that β-carotene can be substituted by astaxanthin in both PSII and PSI cores. However, the higher Chl/car ratio in the isolated Asta complexes compared to the WT complexes indicates that not all sites that are occupied by β-carotene in the WT are occupied by astaxanthin in the mutant complexes, but some are left ‘empty’ in that they are not occupied by carotenoids. Although we cannot exclude the possibility that some of the astaxanthin molecules are more loosely bound and thus are lost during purification, the fact that both PSI and PSII complexes can be purified with a large number of ‘empty’ sites indicates that their occupancy by carotenoids is not crucial for the stability of the complexes. Table 2 Pigment composition of isolated thylakoidal complexes. SamplesChl a/bChl/carLute+neo+violaβ-caroAsta and its bypass productsTotal chls*WT Lhcb Monomers2.41 ± 0.014.1 ± 0.032.86 ± 0.010.06 ± 0.01n.d.12WT LHCII trimer1.40 ± 0.003.60 ± 0.013.90 ± 0.01n.d.n.d.14Asta-Lhcb mon1.48 ± 0.014.50 ± 0.03n.d.n.d.3.1 ± 0.0214WT PSII8.88 ± 0.125.00 ± 0.023.47 ± 0.083.93 ± 0.08n.d.37Asta PSII7.74 ± 0.278.90 ± 0.17n.d.0.03 ± 0.014.12 ± 0.0137WT PSI-LHCI9.29 ± 0.124.60 ± 0.0214.37 ± 0.1619.56 ± 0.16n.d.156Asta-PSI-LHCI5.44 ± 0.198.10 ± 0.18n.d.0.15 ± 0.0319.11 ± 0.03156 *Total Chls are based on values reported in the literature for the WT complexes (Qin et al., 2015; Su et al., 2017). The chromatograms are shown in Figure 3—figure supplement 2. (Average values ± SD are shown. n ≥ 3 biological replicas, n.d. = not detected). Interestingly, absorption (Figure 3—figure supplement 3A) and circular dichroism (Figure 3—figure supplement 3B) spectra of LHCs and PSII core complexes from the WT and the mutant are very similar (see Figure 3—figure supplement 3 for a more detailed explanation) in the Chl absorption regions. This indicates that there are no significant changes in the pigment organization of the complexes and thus in their three-dimensional structure. The only exception is Asta-PSI-LHCI, the fluorescence emission of which showed a 6 nm shift to shorter wavelengths as compared to the WT complex (Figure 3—figure supplement 3C and Figure 3—figure supplement 4). Since the PSI emission at 77 K originates mostly from two specific Chls (called far-red Chls) of Lhca3 and Lhca4 (Morosinotto et al., 2003), we can conclude that the interaction between these Chls is slightly changed in the mutant. Carotenoids are known to be required for the stability of the pigment-binding holoproteins (Paulsen et al., 1993). Our data measured on the isolated complexes show that the difference in composition between WT and mutant complexes influences the denaturation temperature by only 5–10°C (Figure 3A). This is surprising considering that several of the carotenoid-binding sites in the isolated PSI and PSII are not occupied by carotenoids and indicates that only some of them play a crucial role in protein stability. Figure 3 with 5 supplements see all Download asset Open asset Thermal and photo-stability of photosynthetic complexes. (A) Thermal denaturation was monitored by following the CD signals in the Qy (Lhcb: 610–700 nm, PSII core: 640–700 nm, PSI-LHCI: 675–735 nm) region at increasing temperature. (B) Photobleaching is measured as the decrease in absorption of the area in the Qy region (600–750 nm) as a function of the length of the high light treatment (Average values ± SD are shown for n = 3 technical replicas.). The purification of the complexes is shown in Figure 3—figure supplement 1. The pigment analysis of the purified complexes is shown in Figure 3—figure supplement 2. The absorption, CD, and fluorescence emission spectra of the complexes are shown in Figure 3—figure supplement 3 for comparison the fluorescence spectra of the thylakoids are shown in Figure 3—figure supplement 4. The raw data used for this figure are provided in Figure 3—figure supplement 5. Photoprotection via Chl triplet quenching and singlet oxygen scavenging is the primary role of carotenoids in photosynthesis (Siefermann-Harms, 1987). Photobleaching experiments (Figure 3B) show that, while the photostability of LHCs and PSII core is only partially affected by the change in carotenoid composition, Asta-PSI-LHCI is far more sensitive to light than the WT complex. It is likely that this effect on PSI is due to the reduced number of carotenoid molecules associated with the complex, which results in less efficient Chl triplet quenching. However, it is worth noting that, even with a large part of the carotenoid-binding sites not occupied by carotenoids, PSI is more photostable than PSII-WT, in agreement with the fact that, in PSII, carotenoids cannot provide protection by quenching singlet oxygen formed via P680 triplet because of the very high oxidizing potential of PSII (Telfer, 2014). Next, we investigated the effect of the substitution of carotenes with astaxanthin on the light-harvesting and trapping properties of the photosynthetic complexes in vivo by performing time-resolved fluorescence measurements at 20°C on intact leaves (Figure 4 and Figure 4—figure supplement 1). The PSI kinetics is very similar in the WT (70 ps) and in the mutant (65 ps), and the small difference can be ascribed to the reduced far-red Chl content of Asta-PSI-LHCI, which is known to influence the PSI trapping time (Croce and van Amerongen, 2013). The PSII kinetics in the mutant leaves changes in the presence versus absence of photochemistry (measurements performed with the reaction center (RC) open and closed, respectively) as it does in the WT, indicating that excitation energy transfer occurs in the mutant and the harvested energy is used for photochemistry. However, all the kinetics are faster and the difference between closed and open RC is smaller in mutant than in WT leaves, suggesting that the antenna complexes of the mutant plants are statically quenched in vivo. Measurements on isolated Asta-Lhcb show that this is indeed the case: these complexes are strongly quenched (lifetime of 0.87 ns vs. 3.5 ns in the WT; Figure 4—figure supplement 2) due to the presence of astaxanthin (Liguori et al., 2017). It has also been shown that part of the astaxanthin population can transfer excitation energy to the chlorophylls, thus also acting as light-harvesting pigment (Liguori et al., 2017). Figure 4 with 2 supplements see all Download asset Open asset Normalized fluorescence decay traces measured at room temperature on WT and Asta leaves in the presence (F0) or absence (FM) of photochemistry. Fluorescence was detected at (A) λ = 685 nm and (B) λ = 720 nm. respectively. Note that, although the decay at 680 nm is dominated by PSII and at 720 nm by PSI, both complexes contribute to the decay at both wavelengths (see Figure 4—figure supplement 1 for the analysis of the complete data set with spectral resolution). The fluorescence decay and analysis of the purified LHCII are shown in Figure 4—figure supplement 2. The presence of excitation energy transfer from the antenna to the RC in mutant leaves indicates that, although the interactions between the building blocks of the PSII supercomplex are not strong enough to survive purification (see Figure 2), the supercomplexes are functional in vivo meaning that, in the membrane, the subunits are close enough to each other to ensure the delivery of the harvested energy to the reaction center. Indeed, the short excited state lifetime of the antenna (indicative of a constitutively quenched antenna) in the mutant can fully account for the lower maximum quantum efficiency of PSII (FV/FM; Table 3) in mutant plants, which is mainly the result of low fluorescence emission in the absence of photochemistry (FM). Table 3 In vivo photosystem II parameters. FV/FM*PSII:PSI ratio (ECS)#PSII:PSI antenna size (ECS)#Relative PSII antenna size (fluorescence)§§WT0.82 ± 0.011.09 ± 0.121 ± 0.591 ± 0.04Asta0.43 ± 0.032.6 ± 0.330.33 ± 0.050.33 ± 0.02 (Average values ± SD are shown. *n = 15, #n = 4, §§n = 3 leaves/plants). Finally, we analyzed the photosynthetic performance of the Asta plants. Electrochromic shift (ECS) of the carotenoid absorption is commonly used to study the function of all major photosynthetic complexes (Bailleul et al., 2015). We verified that the mutant plants exhibit an ECS signal and we determined its light-induced difference spectrum, which agreed with the prediction that astaxanthin is solely responsible for this in vivo Stark effect (Figure 5—figure supplement 1). Using ECS, we observed that the functional PSII/PSI RC ratio was far larger in the mutant than in the WT, in qualitative agreement with our biochemical data (Table 3; Figure 2—figure supplement 1). The difference in the values obtained with the two methods is partially due to the limited quantitative power of immunoblots, but also suggests that some of the PSII cores are not functional. The high PSII/PSI ratio in the mutant seems to be a compensation mechanism for the decrease in the relative functional PSII/PSI antenna size (measured with two independent methods; Table 3 and Figure 5—figure supplement 2) observed in the Asta plants, which is due to the presence of static quenching. Indeed, comparison of the steady-state photochemical yields of PSII and PSI revealed that, at all light intensities, in both WT and mutant plants, the balance between PSII and PSI photochemistry is maintained (Figure 5A and Figure 5—figure supplements 3 and 4), meaning that the plants are able to compensate for the strong decrease in the PSII functional antenna size by decreasing the PSI/PSII ratio. This means that these plants have the capacity to modulate the PSI/PSII ratio in a large dynamic range. Finally, transient QA reduction and reoxidation kinetics suggest that no significant differences in the PSII electron transfer occur in the mutant plants (Figure 5—figure supplements 2 and 5). Figure 5 with 7 supplements see all Download asset Open asset Photosynthetic performance and photoprotection. (A) Relation between qL and ΦPSI measured at various light intensities (70–1030 μmol photons m−2 s−1). (B) NPQ kinetics upon transition from the dark-to-light (upward arrow,) and subsequent relaxation in the dark (downward arrow). Average values ± SD are shown for n = 3 leaves/plants. The inset shows amplitude-normalized kinetics. See also Figure 5—figure supplements 1–7. The full operational capacity of the electron transport chain permitted us to verify whether the photoprotective regulation is maintained in the mutant plants. As expected, the NPQ amplitude was largely reduced in the mutant (Figure 5B and Figure 5—figure supplement 6), because the ΔpH-induced, PsbS-dependent quenching has to compete with the strong, constitutive astaxanthin quenching in these plants. Note that the difference in NPQ level (1.8 in the WT vs. 0.3 in the mutant) can be fully ascribed to the presence of the static quencher in the mutant, which strongly reduces the maximal fluorescence in both dark (FM) and light (FM’) states. This is supported by the NPQ(t) calculation, which permits to correct the apparent NPQ for the presence of a pre-existing quenching, assuming that a decrease of the FV/FM value is solely due to this static quenching. The data show that NPQ(t) is even larger in the mutant than in the WT (Figure 5—figure supplement 7). Importantly, despite the difference in apparent NPQ amplitude, the kinetics of onset and recovery are identical to those of the WT (inset in Figure 5B) and consistent with qE characteristics. This outcome is particularly striking if one considers that Asta plants lack both lutein and zeaxanthin, which are believed to be essential for NPQ (Niyogi et al., 1998). It is likely that the high amount of PsbS in the mutant (Figure 2—figure supplement 1) can compensate for the lack of the xanthophyll cycle, or that astaxanthin can also be responsible for the dynamic quenching. Whatever the reason for the presence of NPQ in the mutant, our Asta plants clearly show that lutein and zeaxanthin are not absolutely necessary for it. In summary, we have shown that the carotenoid-binding sites of the core complexes of PSI and PSII are promiscuous. Although they bind carotenes in all known photosynthetic organisms, our data demonstrate that they can also accommodate xanthophylls. This is at variance with the LHCs that can bind various xanthophylls but cannot fold with carotenes. More importantly, we show that both PSI and PSII are stable while most of their carotenoid-binding sites are not occupied by carotenoids and the rest is occupied by an alien xanthophyll. These results indicate that the core complexes are even more robust than the outer antennae and can endure radical changes even in one of their main components. In this respect, it is important to realize that the difference in growth rate between WT and mutant plants is not due to the absence of carotenes, but rather to the presence of astaxanthin that stabilizes the LHCs in a quenched conformation. In conclusion, the substitution of carotenes with the xanthophyll astaxanthin does not impair the functional assembly of the photosynthetic apparatus, nor does it impede efficient electron transfer and NPQ, demonstrating that carotenes are not essential neither for the biosynthesis of the photosynthetic apparatus nor for its function. This finding has important implications not only for our understanding of the structure and function of the photosynthetic apparatus but also for future efforts to design synthetic photosystems with novel and improved properties. Materials and methods Tobacco growth and thylakoid isolation Request a detailed protocol Seeds from mutant and WT plants were sown on moist filter paper and synchronized at 4°C for 2–3 days before being moved to room temperature until germination. The seedlings were transferred to soil and grown at 22°C under 150–200 μmol photons m−2 s−1 for the WT and 80–120 μmol photons m−2 s−1 for the mutant with 14 hr of light per day. Plants were fed with commercial fertilizer each week. Leaves from WT (5–6 weeks old) and Asta plants (around 20 weeks for younger leaves, 24–30 weeks for older leaves) were used for physiological measurements and thylakoid isolation. WT thylakoid isolation was performed as described in Xu et al., 2015. The centrifuge speed was increased to 4000 g for the first step of the isolation from mutant tobacco. Pigment analysis Request a detailed protocol Pigments from isolated protein–pigment complexes or leaves were extracted with 80% acetone. HPLC was performed as in Xu et al., 2015 with the modification that buffer B was linearly increased from 0 to 100% in 9.2 min. Chlorophyll a/b ratios and chlorophyll/carotenoid ratios were calculated by fitting their individual absorption spectra to measured spectra (Xu et al., 2015). Examples of the fitting of total thylakoids and isolated Lhcbs are shown in Figure 3—figure supplement 2 panels C and D. Blue-native gel electrophoresis, SDS-PAGE, immunoblotting, and sucrose density gradient centrifugation Request a detailed protocol Blue-native gels were performed as described in Järvi et al., 2011 with the modifications described in Bielczynski et al., 2016. The second dimension and the SDS-PAGE were performed as described in Schägger, 2006. For immunoblot analysis, total protein extracts were separated by SDS-PAGE and transferred to a Protran 0.45 mm nitrocellulose membrane. Specific primary antibodies (Agrisera) were used to detect the target proteins. Chemiluminescence was detected using an ImageQuant LAS 4000 imaging system. For sucrose density gradient fractionation, thylakoids equivalent to 0.2 mg total chlorophyll were washed with 5 mM EDTA and resuspended in 200 μL 10 mM Hepes (pH 7.5). An equal volume of 1.2% α-DDM was added, mixed gently, and the solubilized thylakoids were centrifuged at 14,000 rpm for 10 min at 4°C. The supernatant was loaded on a 0–1 M sucrose gradient (10 mM Hepes, pH 7.5, 0.03% α-DDM) and centrifuged at 288,000 g for 17 hr. The separated bands were collected with a syringe. State-steady spectroscopy measurements Absorption and CD Request a detailed protocol Absorption spectra were measured at room temperature with a Varian Cary 4000 UV-Vis-spectrophotometer. CD spectra were recorded using a Chirascan-Plus spectropolarimeter (Applied Photophysics) at 20°C. The OD of the samples was 0.8–1/cm at the maximum of the Qy region. 77 K fluorescence emission Request a detailed protocol Low-temperature fluorescence emission spectra were recorded using a Fluorolog 3.22 spectrofluorometer (Jobin Yvon-Spex). For 77 K measurements, a home-built liquid nitrogen-cooled device was used. The samples were excited at 440 nm and the fluorescence emission was detected in the 600–800 nm range. Excitation and emission slit widths were set to 3 nm. All measurements were performed in the same buffers used for the sucrose gradients. Photobleaching and protein stability Photobleaching Request a detailed protocol The samples were diluted to an absorbance of around 0.8 at the maximum in the Qy region. The protein–pigment complexes were illuminated with white light (7100 μmol photons m−2 s−1) from a halogen lamp with optic fiber arm. After each interval, the cuvette was removed from the light beam, and the absorption spectra were recorded with a Varian Cary 4000 UV-Vis-spectrophotometer in the range between 600 and 750 nm (Croce et al., 1999). Protein stability Request a detailed protocol The stabil

Research on mutant tobacco plants shows that a pigment called β-carotene is not necessary for photosynthesis.

Research on mutant tobacco plants shows that a pigment called beta-carotene is not necessary for photosynthesis.

Photosystems I and II convert solar energy into the chemical energy that powers life. Chlorophyll a photochemistry, using red light (680 to 700 nm), is near universal and is considered to define the energy "red limit" of oxygenic photosynthesis. We present biophysical studies on the photosystems from a cyanobacterium grown in far-red light (750 nm). The few long-wavelength chlorophylls present are well resolved from each other and from the majority pigment, chlorophyll a. Charge separation in photosystem I and II uses chlorophyll f at 745 nm and chlorophyll f (or d) at 727 nm, respectively. Each photosystem has a few even longer-wavelength chlorophylls f that collect light and pass excitation energy uphill to the photochemically active pigments. These photosystems function beyond the red limit using far-red pigments in only a few key positions.

Transient absorption studies conducted on in vitro lycopene aggregates, as well as on lycopene crystalloids inside tomato chromoplasts, reveal the appearance of a long-lived excited state, which we unambiguously identified as lycopene triplet. These triplet states must be generated by singlet exciton fission, which occurs from the lycopene (2)A(g) state. This is the first time the singlet fission process has ever been shown to occur in a biological material. We propose that the formation of carotenoid assemblies in chromoplasts may constitute a photoprotective process during chromoplast maturation, in addition to their function in signaling processes.

Photosystems I and II convert solar energy into the chemical energy that powers life. Both photosystems use chlorophyll-a photochemistry, absorbing almost the same color of light and thus accessing comparable amounts of energy. This energy is considered the “red-limit” for oxygenic photosynthesis. Here we report that a trait that is common in cyanobacteria extends the photochemical red-limit, with both photosystems using ~110 meV less energy. This ~45 nm upshift of the photochemically active chlorophylls may represent an extended red-limit relevant to stable environments. Studies of these unprecedented low-energy photosystems, with their farred photochemistry but largely red antenna, could solve key long-standing mechanistic and energetic questions in photosynthesis, some of which are relevant to the hunt for life on planets in other solar systems. Understanding the bioenergetics and resilience of these systems could provide design principles and feasibility assessments for projects aimed at improving photosynthetic efficiency by engineering long-wavelength photosystems. One Sentence Summary: Long-wavelength chlorophylls in key positions in Photosystem I and II reflect an extension of the photochemical red-limit for photosynthesis.

A standing building survey in 2001 recorded a section of the former southern wall of the West India (middle) Export Dock, in East London, built between 1803 and 1806. This revealed the extent of skilful engineering involved in construction of the wall; the main brick build incorporated a curving or banana-shaped section, both for structural stability and to accommodate the profile of ships' hulls. This was laid onto a wooden frame, previously unrecorded, for which coniferous timbers were used, supported by a series of wooden piles, driven into the natural gravel. The wall is also the earliest known example of reinforced brickwork. This new study provides considerable additional information on the construction of the wall, and of Georgian engineering in this form.

In this review, I outline the indirect evidence for the formation of singlet oxygen (1O2) obtained from experiments with the isolated PSII reaction center complex. I also review the methods we used to measure singlet oxygen directly, including luminescence at 1,270 nm, both steady state and time resolved. Other methods we used were histidine-catalyzed molecular oxygen uptake (enabling 1O2 yield measurements), and dye bleaching and difference absorption spectroscopy to identify where quenchers of 1O2 can access this toxic species. We also demonstrated the protective behavior of carotenoids bound within Chl–protein complexes which bring about a substantial amount of 1O2 quenching within the reaction center complex. Finally, I describe how these techniques have been used and expanded in research on photoinhibition and on the role of 1O2 as a signaling molecule in instigating cellular responses to various stress factors. I also discuss the current views on the role of 1O2 as a signaling molecule and the distance it might be able to travel within cells.

The electronic properties of carotenoid molecules underlie their multiple functions throughout biology, and tuning of these properties by their in vivo locus is of vital importance in a number of cases. This is exemplified by photosynthetic carotenoids, which perform both light-harvesting and photoprotective roles essential to the photosynthetic process. However, despite a large number of scientific studies performed in this field, the mechanism(s) used to modulate the electronic properties of carotenoids remain elusive. We have chosen two specific cases, the two β-carotene molecules in photosystem II reaction centers and the two luteins in the major photosystem II light-harvesting complex, to investigate how such a tuning of their electronic structure may occur. Indeed, in each case, identical molecular species in the same protein are seen to exhibit different electronic properties (most notably, shifted absorption peaks). We assess which molecular parameters are responsible for this in vivo tuning process and attempt to assign it to specific molecular events imposed by their binding pockets.

Light-induced electron transfer reactions in the chlorophyll a/d-binding Photosystem I reaction centre of Acaryochloris marina were investigated in whole cells by pump-probe optical spectroscopy with a temporal resolution of ~5ns at room temperature. It is shown that phyllosemiquinone, the secondary electron transfer acceptor anion, is oxidised with bi-phasic kinetics characterised by lifetimes of 88±6ns and 345±10ns. These lifetimes, particularly the former, are significantly slower than those reported for chlorophyll a-binding Photosystem I, which typically range in the 5-30ns and 200-300ns intervals. The possible mechanism of electron transfer reactions in the chlorophyll a/d-binding Photosystem I and the slower oxidation kinetics of the secondary acceptors are discussed.

Fluorescence line-narrowing and resonance Raman properties of various chlorophylls have been measured in organic solvents. Resonance Raman spectroscopy is already a well-established method for the study of photochemical reactions in the various pigment-protein complexes involved in photosynthesis, while fluorescence line-narrowing is still an emerging technique for such systems. Interpretation of these vibrational spectra requires accurate comparative data on the pure isolated pigments. By comparing three different chlorophylls, a, b, and d, which have different substituents on the porphyrin ring, the various spectral lines associated with vinyl and formyl groups on the X and Y electronic axes could be distinguished. The difference between five- and six-coordination of the central Mg atom in FT-Raman spectra was determined by varying the organic solvent used. These chlorophylls are important in photosynthesis: all three in light-harvesting and energy transfer and, in the case of a and d, also in electron transfer. The assignment of spectral bands which we provide here, along with the description of their behavior with respect to the conformation and state of interaction of the pigment molecule, constitutes an essential step if these vibrational techniques are to be exploited to their full potential.

The oxidation potential of the primary electron donor P740 of photosystem I (PS I) of the chlorophyll d dominated cyanobacterium, Acaryochloris marina, has been reinvestigated. We find a midpoint potential of about 450 mV in a photosystem I-enriched membrane fraction as well as in thylakoids, which is significantly higher than that originally reported by Hu et al. (1998). This value, which is, indeed, very similar to that found for P700 in chlorophyll a dominated organisms, is in agreement with results of Bailleul et al. (this Congress). In addition, the extinction difference coefficient for the oxidation of the primary donor has been determined and a value of 45 (±4) mM−1 cm−1 at 740 nm was obtained.

Absorbance difference spectroscopy and redox titrations have been applied to investigate the properties of photosystem I from the chlorophyll d containing cyanobacterium Acaryochloris marina. At room temperature, the (P740(+)-P740) and (F(A/B)(-)-F(A/B)) absorbance difference spectra were recorded in the range between 300 and 1000 nm while at cryogenic temperatures, (P740(+)A(1)(-)-P740A(1)) and ((3)P740-P740) absorbance difference spectra have been measured. Spectroscopic and kinetic evidence is presented that the cofactors involved in the electron transfer from the reduced secondary electron acceptor, phylloquinone (A(1)(-)), to the terminal electron acceptor and their structural arrangement are virtually identical to those of chlorophyll a containing photosystem I. The oxidation potential of the primary electron donor P740 of photosystem I has been reinvestigated. We find a midpoint potential of 450+/-10 mV in photosystem I-enriched membrane fractions as well as in thylakoids which is very similar to that found for P700 in chlorophyll a dominated organisms. In addition, the extinction difference coefficient for the oxidation of the primary donor has been determined and a value of 45,000+/-4000 M(-1) cm(-1) at 740 nm was obtained. Based on this value the ratio of P740 to chlorophyll is calculated to be 1 : to approximately 200 chlorophyll d in thylakoid membranes. The consequences of our findings for the energetics in photosystem I of A. marina are discussed as well as the pigment stoichiometry and spectral characteristics of P740.

We have investigated the photosynthetic properties of Acaryochloris marina, a cyanobacterium distinguished by having a high level of chlorophyll d, which has its absorption bands shifted to the red when compared with chlorophyll a. Despite this unusual pigment content, the overall rate and thermodynamics of the photosynthetic electron flow are similar to those of chlorophyll a-containing species. The midpoint potential of both cytochrome f and the primary electron donor of photosystem I (P740) were found to be unchanged with respect to those prevailing in organisms having chlorophyll a, being 345 and 425 mV, respectively. Thus, contrary to previous reports (Hu, Q., Miyashita, H., Iwasaki, I. I., Kurano, N., Miyachi, S., Iwaki, M., and Itoh, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13319–13323), the midpoint potential of the electron donor P740 has not been tuned to compensate for the decrease in excitonic energy in A. marina and to maintain the reducing power of photosystem I. We argue that this is a weaker constraint on the engineering of the oxygenic photosynthetic electron transfer chain than preserving the driving force for plastoquinol oxidation by P740, via the cytochrome b6f complex. We further show that there is no restriction in the diffusion of the soluble electron carrier between cytochrome b6f and photosystem I in A. marina, at variance with plants. This difference probably reflects the simplified ultrastructure of the thylakoids of this organism, where no segregation into grana and stroma lamellae is observed. Nevertheless, chlorophyll fluorescence measurements suggest that there is energy transfer between adjacent photosystem II complexes but not from photosystem II to photosystem I, indicating spatial separation between the two photosystems.