Photosynthesis Research最新文献

Timelapse microscopy has recently been employed to study the metabolism and physiology of cyanobacteria at the single-cell level. However, the identification of individual cells in brightfield images remains a significant challenge. Traditional intensity-based segmentation algorithms perform poorly when identifying individual cells in dense colonies due to a lack of contrast between neighboring cells. Here, we describe a newly developed software package called Cypose which uses machine learning (ML) models to solve two specific tasks: segmentation of individual cyanobacterial cells, and classification of cellular phenotypes. The segmentation models are based on the Cellpose framework, while classification is performed using a convolutional neural network named Cyclass. To our knowledge, these are the first developed ML-based models for cyanobacteria segmentation and classification. When compared to other methods, our segmentation models showed improved performance and were able to segment cells with varied morphological phenotypes, as well as differentiate between live and lysed cells. We also found that our models were robust to imaging artifacts, such as dust and cell debris. Additionally, the classification model was able to identify different cellular phenotypes using only images as input. Together, these models improve cell segmentation accuracy and enable high-throughput analysis of dense cyanobacterial colonies and filamentous cyanobacteria.

Myriophyllum spicatum, a semi-aquatic plant, can develop heterophylly by forming both submerged and aerial leaves to adapt to water level variations in its habitat. The aerial leaves exhibit shorter and fewer lobes, but thicker cuticle and developed stomata than submerged leaves. The heterophylly exhibited by M. spicatum could be controlled by hormones including abscisic acid, indole-3-acetic acid, and Jasmonic acid, as their levels were consistently higher in aerial leaves than in submerged leaves. Genes responsible for the formation of cuticle and stomata exhibited elevated expression in the aerial leaves, offering a molecular explanation for their structural adaptations to terrestrial environment. Moreover, aerial leaves exhibited greater resistance to intense light, while submerged leaves demonstrated a pronounced capacity of utilizing HCO₃^- for photosynthesis. Differential gene expression patterns pertaining to photosynthesis, carotenoid production, and HCO₃^- utilization elucidated the molecular mechanisms driving M. spicatum's photosynthetic adaptations to aquatic and terrestrial environment. In conclusion, the ability of M. spicatum to withstand changing water levels can be linked to its adaptable phenotype and the genetic characteristics inherited from its terrestrial ancestors, both of which are governed by hormonal regulation. These features may allow M. spicatum to outcompete other macrophytes that are more sensitive to water level fluctuations in their growing surroundings.

Evergreen conifers thrive in challenging environments by maintaining multiple sets of needles, optimizing photosynthesis even under harsh conditions. This study aimed to investigate the relationships between needle structure, photosynthetic parameters, and age along the light gradient in the crowns of Abies alba, Taxus baccata, and Picea abies. We hypothesized that: (1) Needle structure, photochemical parameters, and photosynthetic pigment content correlate with needle age and light levels in tree crowns. (2) The photosynthetic capacity of ageing needles would decline and adjust to the increasing self-shading of branches. Our results revealed a non-linear increase in the leaf mass-to-area ratio. The maximum quantum yield of photosystem II photochemistry decreased linearly with needle age without reaching levels indicative of photoinhibition. Decreased maximum electron transport rates (ETR_max) were linked to declining values of saturating photosynthetic photon flux density and increasing non-photochemical quenching of fluorescence (NPQ), indicating energy losses as heat. The chlorophyll a to chlorophyll b ratio linearly decreased, suggesting older needles sustain high light capture efficiency. These findings offer new insights into the combined effects of needle ageing and self-shading on photochemistry and pigment content. This functional needle balance highlights the trade-off between the costs of long-term needle retention and the benefits of efficient resource utilization. In environments where air temperature is less of a constraint on photosynthesis due to climate warming, evergreen coniferous trees could sustain or enhance their photosynthetic capacity. They can achieve this by shortening needle lifespan and retaining fewer cohorts of needles with higher ETR_max and lower NPQ compared to older needles.

The initial electron transfer (ET) processes in reaction centers (RCs) of Chloroflexus (Cfl.) aurantiacus were studied at 295 K using femtosecond transient absorption (TA) difference spectroscopy. Particular attention was paid to the decay kinetics of the primary electron donor excited state (P^*) and the formation/decay of the absorption band of the monomeric bacteriochlorophyll a anion (B_A^-) at ~ 1035 nm, which reflects the dynamics of the charge-separated state P⁺B_A^-. It was found that in Q_A-depleted RCs containing native bacteriopheophytin a (BPheo) molecules at the H_A and H_B binding sites, the decay of P^* to form the P⁺H_A^- state contains a fast (4 ps; relative amplitude 70%) and a slow (13 ps; relative amplitude 30%) kinetic components. The B_A^- absorption band at ~ 1035 nm was detected only for the fast component. Based on global analysis of the TA data, the results are discussed in terms of the presence of two P^* populations: in one, P^* decays in 4 ps via a dominant two-step activationless P^* → P⁺B_A^- → P⁺H_A^- ET with a contribution of 70% to the overall primary charge separation process, and in the other, P^* decays in 13 ps via a one-step superexchange P^* → P⁺H_A^- ET (contribution of 30%). Similar femtosecond TA measurements on Q_A-depleted-Pheo_A-modified RCs, in which the charge separation energetics was changed by replacing BPheo H_A with plant pheophytin a, suggest the presence of a P^* population where P⁺H_A^- formation can occur via a thermally activated two-step ET process.

Excitation energy transfer between the photochemically active protein complexes is key for photosynthetic processes. Phototrophic organisms like cyanobacteria experience subtle changes in irradiance under natural conditions. Such changes need adjustments to the excitation energy transfer between the photosystems for sustainable growth. Spectroscopic assessments on purified photosystems usually fail to capture these subtle changes. In this study, we examined whole cells from two model cyanobacteria, Synechocystis sp. PCC 6803 and Synechococcus elongatus UTEX 2973, grown under high and low light conditions to decode the high light tolerance of the latter. This allowed us to study photosynthetic machinery in the native state and in this work we particularly focused on the excitation energy transfer within PSII and PSI manifold. Understanding the high-light tolerance mechanism is imperative as it can help design strategies for increasing the light tolerance of cyanobacteria used for carbon neutral bioproduction. Our observations suggest that Synechococcus 2973 employs an uncommon photoprotection strategy, and the absence of hydroxy-echinenone pigment in this strain opens the possibility of an orange carotenoid protein homolog utilizing zeaxanthin as a scavenger of reactive oxygen species to provide photoprotection. Furthermore, the adjustments to the high-light adaptation mechanism involve downregulating the phycobilisome antenna in Synechococcus 2973, but not in Synechocystis 6803. Additionally, the stoichiometric changes to PSII/PSI are more tightly regulated in Synechococcus 2973.

The femtosecond dynamics of energy transfer from light-excited spirilloxanthin (Spx) to bacteriochlorophyll (BChl) a in the reaction centers (RCs) of purple photosynthetic bacteria Rhodospirillum rubrum was studied. According to crio-electron microscopy data, Spx is located near accessory BChl a in the B-branch of cofactors. Spx was excited by 25 fs laser pulses at 490 nm, and difference absorption spectra were recorded in the range 500-700 nm. To reveal the dynamics of individual states, we applied global analysis using different kinetic schemes. We found that the energy transfer Spx → BChl a occurs during 0.22 ps with a low efficiency of ~ 31%. The monomeric BChl a acts as the primary energy acceptor, presumably in the B-branch of cofactors. Then the energy is transferred to the BChl a dimer within 0.25 ps and subsequently used for charge separation. As a result of internal conversion in Spx, the majority (~ 69%) of the excitation energy transfers in 0.2 ps from the singlet-excited state S₂ to the states S₁ and S*, which, in turn, relax to the ground state in 1.5 and 9 ps, ​​respectively. We showed that the S₁ and S* states in Spx are not involved in energy transfer to BChl a. The found parameters of energy transfer Spx→BChl a turned out to be close to those in the light-harvesting complexes LH1 of Rhodospirillum rubrum. The sequence of events in Spx after its excitation is discussed.

Red algae are photosynthetic eukaryotes whose light-harvesting complexes (LHCs) associate with photosystem I (PSI). In this study, we examined characteristics of PSI-LHCI, PSI, and LHCI isolated from the red alga Galdieria sulphuraria NIES-3638. The PSI-LHCI supercomplexes were purified using anion-exchange chromatography followed by hydrophobic-interaction chromatography, and finally by trehalose density gradient centrifugation. PSI and LHCI were similarly prepared following the dissociation of PSI-LHCI with Anzergent 3-16. Polypeptide analysis of PSI-LHCI revealed the presence of PSI and LHC proteins, along with red-lineage chlorophyll a/b-binding-like protein (RedCAP), which is distinct from LHC proteins within the LHC protein superfamily. RedCAP, rather than LHC proteins, exhibited tight binding to PSI. Carotenoid analysis of LHCI identified zeaxanthin, β-cryptoxanthin, and β-carotene, with zeaxanthin particularly enriched, which is consistent with other red algal LHCIs. A Qy peak of chlorophyll a in the LHCI absorption spectrum was blue-shifted compared with those of PSI-LHCI and PSI, and a fluorescence emission peak was similarly shifted to shorter wavelengths. Based on these results, we discuss the diversity of LHC proteins and RedCAP in red algal PSI-LHCI supercomplexes.

Maize (Zea mays L.) performs highly efficient C₄ photosynthesis by dividing photosynthetic metabolism between mesophyll and bundle sheath cells. In vivo physiological measurements are indispensable for C₄ photosynthesis research as photosynthetic activities are easily interrupted by leaf section or cell isolation. Yet, direct in vivo observation regarding bundle sheath cells in the delicate anatomy of the C₄ leaf is still challenging. In the current work, we used two-photon fluorescence-lifetime imaging microscopy (two-photon-FLIM) to access the photosynthetic properties of bundle sheath cells on intact maize leaves. The results provide spectroscopic evidence for the diminished total PSII activity in bundle sheath cells at its physiological level and show that the single PSIIs could undergo charge separation as usual. We also report an acetic acid-induced chlorophyll fluorescence quenching on intact maize leaves, which might be a physiological state related to the nonphotochemical quenching mechanism.

The Orange Carotenoid Protein (OCP) is a unique water-soluble photoactive protein that plays a critical role in regulating the balance between light harvesting and photoprotective responses in cyanobacteria. The challenge in understanding OCP´s photoactivation mechanism stems from the heterogeneity of the initial configurations of its embedded ketocarotenoid, which in the dark-adapted state can form up to two hydrogen bonds to critical amino acids in the protein's C-terminal domain, and the extremely low quantum yield of primary photoproduct formation. While a series of experiments involving point mutations within these contacts helped us to identify these challenges, they did not resolve them. To overcome this, we shifted from classical mutagenesis to the translational introduction of non-canonical amino acid residues into the OCP structure. In this work, we demonstrate that replacing a single meta-hydrogen in tyrosine-201 with a halogen atom (chlorine, bromine, or iodine) leads to targeted modifications in the keto-carotenoid-protein matrix interaction network, both in the dark-adapted state and upon photoactivation. We found that such atomic substitutions allow us to effectively weaken key hydrogen bonds without disrupting protein folding, thereby increasing the yield of OCP photoactivation products. Such genetically encoded chemical modification of individual atoms and their systematic in situ variation in complex protein structures establishes a foundation for transforming OCP into a practical tool for optogenetics and other applications.