Water use efficiency in cereal crops is governed by molecular processes operating across biological scales that are connected imperfectly and non-linearly, from guard cell ion channel gating to canopy evapotranspiration. Despite the global urgency of improving yield under water limitation, no existing synthesis has integrated the molecular machinery of stomatal regulation, mesophyll CO2 conductance, root hydraulic conductance, isohydric and anisohydric stomatal strategies, carbon isotope discrimination as a proxy tool, and specific genetic targets for improvement within a single cereal-focused mechanistic framework. This review advances that synthesis, examining WUE primarily through the lens of non-stomatal carbon fixation rather than stomatal regulation alone. The ABA-dependent stomatal closure cascade and the opposing blue-light opening pathway are conserved in core architecture across cereals, but their calcium-dependent and H+-ATPase branches remain characterised mainly by sequence homology rather than direct cereal evidence. Mesophyll conductance and Rubisco carboxylation efficiency represent underexploited, genetically undefined targets for improving carbon gain. Root hydraulic conductance connects soil water to canopy transpiration on timescales of minutes to days, while whole-plant isohydric to anisohydric strategy is best understood as an emergent, environment-dependent outcome of these faster processes rather than a fixed trait. Carbon isotope discrimination remains a practical relative-ranking proxy rather than a direct measurement. The EPFL-ERECTA stomatal density pathway and the H⁺-ATPase aperture system show the strongest cereal-specific experimental support among engineering targets, though compensatory anatomical and physiological responses mean anatomical change alone is insufficient evidence of durable benefit. Nitrogen-water signalling through CEP-CEPR1 peptide signalling likely shapes WUE as a downstream emergent property, though direct experimental validation is lacking. Five specific research priorities are identified to advance WUE improvement from mechanistic understanding toward field-level translation.
Chlorophylls (Chls) and bacteriochlorophylls (BChls) in photosynthetic organisms are embedded in pigment–protein complexes, mostly through the coordination of peptidyl residues to their central metal atoms. An asymmetry occurs at the axially coordinated metal centers of (B)Chls. The stereochemistry of the ligand coordination toward 42 molecules of Chls-c, the fully π-conjugated porphyrin pigments lacking C17- and C18-chiralities and a long ester chain in the C17-substituent, was surveyed across 21 pigment–protein complexes registered in the Protein Data Bank. It was found that 24 of 42 Chl-c molecules in these complexes were coordinated from the β-side of the macrocyclic π-plane, revealing a tendency opposite to the preferential α-ligation observed for the structurally different (B)Chls with a (bacterio)chlorin π-system in other proteins (129/163 surveyed sites). The most common binding sites for Chl-c molecules in the proteins correspond to the a603 and a612 sites in Chl-a/b-binding light-harvesting complex II (LHCII), both of which accommodate β-epimers. In some cases, a carotenoid molecule contacts Chl-c to occupy the space where the C172-ester group of the Chl-a/b molecule is situated at the corresponding sites in LHCII, and its position of the carotenoid molecule is related to the macrocycle side of the axial ligand coordination of Chl-c. The absence or presence of chirality and bulky substituents at the C17-position of (B)Chls may be concerned with the stereochemistry of the axial coordination and the formation of these pigment–protein complexes.
A new type of kinetic chlorophyll fluorometer (Klughammer et al. 2024, Photosynth Res 161:151–176) which enables measuring the changes of rel. fluorescence yield during application of saturating single-turnover 15µs flashes (ST-kinetics, STK) simultaneously with the PAM relaxation kinetics induced by the same flash, was applied on purified PSII core complexes (PSII CC) isolated from Thermosynechococcus vestitus (Lambertz et al. 2023, Biochim Biophys Acta 1864:148953). While the main text concentrates on purified PSII dimers, under Supplementary Materials also comparative measurements with purified monomers and heterodimers are presented. Freshly solubilized, non-preilluminated samples display extremely high ratios of maximal/minimal fluorescence yields up to 26, corresponding to Fv/Fm = 0.96. Both PAM and STK techniques reveal period-4 oscillations in presence of 2 µM dichlorobenzoquinone (DCBQ). Period-2 oscillations are observed in the PAM relaxation kinetics after relaxation of donor-side dependent quenching (DQ). Maximal fluorescence yields in STK responses are lowered with respect to the corresponding PAM responses by High Intensity Quenching (HIQ), consisting of DQ and carotenoid triplet quenching (TQ). The DQ observed in presence of 20µM DCMU lowers flash-induced Fv by about 30
State transition (ST) is a key regulatory mechanism that balances excitation energy between photosystem II (PSII) and photosystem I (PSI). Although its general principles have been known for decades, methodological constraints have limited precise quantification of ST kinetics and of the associated changes in photosystem absorption cross sections. We present a PCA‑assisted fluorescence spectral analysis that enables in vivo separation of PSI and PSII chlorophyll fluorescence at physiological temperatures. Applied to intact Nicotiana tabacum leaves undergoing ST, this method provides more accurate estimates of both the rates and the extent of the process than earlier approaches. The half‑time of the State 1 to State 2 (St1→St2) transition was approximately 1 min, nearly twice as fast as values inferred from traditional Ffar‑red/Fred fluorescence ratios measured at room temperature or 77 K. The analysis also clarifies how PSI and PSII fluorescence changes relate to variations in their absorption cross sections. Accounting for PSII‑to‑PSI spillover, we found that the mobile LHCII fraction associating with PSI during the St1→St2 transition increased the PSI absorption cross section by 31
Due to fluctuations in sunlight intensity, balancing light-harvesting efficiency and photoprotection is critical for plant fitness. To achieve this balance, plants have evolved a highly regulated mechanism, non-photochemical quenching (NPQ), which is activated under excess light to dissipate absorbed energy and deactivated under low light to preserve photosynthetic efficiency. A key component that allows NPQ to respond rapidly to changing light conditions is photosystem II subunit S (PsbS). Several hypotheses have been proposed to explain the working mechanism of PsbS, including the idea that it modulates thylakoid membrane macrostructure and protein mobility by altering membrane fluidity. To test this hypothesis, we employed a proteoliposome system containing only thylakoid lipids, PsbS, and the fluorescent probe laurdan, enabling direct detection of PsbS–lipid interactions under different pH conditions. Our results show that the presence of PsbS increases membrane fluidity, but the magnitude of this change in the overall bilayer is small compared with temperature-induced effects. These findings indicate that direct interactions between PsbS and thylakoid lipids are insufficient to account for NPQ regulation and suggest that interactions with other components in the thylakoid membranes are likely essential. This study provides insight into the functional mechanism of PsbS and establishes a framework for further investigation of PsbS-mediated regulation of NPQ and its role in optimizing photosynthetic performance under fluctuating light.
Some carotenoids have a function to quench excitation energy of chlorophyll in photosynthetic apparatuses. This is a part of the photoprotective mechanism, non-photochemical quenching (NPQ), which prevents the inhibitory effects of high-light irradiation. In the green sulfur bacterium Chlorobaculum tepidum, which is a strictly anaerobic photosynthetic bacteria, a glycosylated carotenoid (Car-G) has been found to dissipate excitation energy of bacteriochlorophyll (BChl) a in the photochemical reaction center (RC) complex. In this study, we used ultrafast transient absorption spectroscopy to investigate excitation energy transfer (EET) mediated by Car-G in the reaction center complex of C. tepidum. Two spectral changes in carotenoids were identified when a core-antenna BChl a was selectively excited. One was sensitive to detergent replacement, while the other was not. The latter was the formation of a triplet-excited state of carotenoid (3Car) and observed when the primary electron donor accumulated in the oxidized state. The corresponding signal was absent in the RC complex from the ΔcruC mutant strain lacking Car-G. Kinetic analyses and structural insights revealed that 3Car is formed on a tightly-bound Car-G through triplet-to-triplet (TT)-EET from one of the excited states of the core-antenna BChl a. This TT-EET to 3Car is responsible for the anaerobic energy dissipation in the RC complex of C. tepidum.
High light stress seriously compromises crop yields and food security. Protein phosphorylation plays a key regulatory role in high light responses; however, their complexity hinders a full understanding of these processes. To address this, we performed a system-wide quantitative phosphoproteomic analysis in maize and rice over a four-hour high light exposure. By comparing site-specific phosphorylation dynamics between the two species, we identified both conserved and species-specific phosphorylation events associated with light harvesting, electron transport, metabolism, ROS scavenging, and signal transduction. Enhanced phosphorylation of LHCB4 at a conserved phosphosite was observed for both plants, indicating a shared mechanism for light-harvesting complex remodeling. Specifically, maize showed unique phosphorylation regulation of D1 and the PSI subunit PsaE, implying a mechanism for restoring photodamaged reaction centers. In contrast, rice rapidly attenuated electron supply via reduced plastocyanin phosphorylation, concurrent with the early induced phosphorylation of phototropin and a receptor-like kinase. Furthermore, we found that phosphorylation dynamics of metabolic enzymes affect their activities, maize preferentially enhanced photosynthetic efficiency, whereas rice exhibited more tunable regulation across primary metabolism. Overall, this study provides a comprehensive phospho-signaling atlas, revealing convergent and divergent phosphorylation process that underlie differential high light acclimation in maize and rice.
Bryopsidales green macroalgae can induce non-photochemical quenching (NPQ) only slowly, presumably due to the lack of the proton gradient-induced component (qE) of NPQ and xanthophyll cycle. Here, two morphologically rather similar siphonous macroalgae, a Bryopsidales alga Bryopsis sp. and a Dasycladales alga Acetabularia acetabulum, latter of which is capable of qE, were given high light treatments with both constant and fluctuating intensity. No differences in the rate of photoinhibition of Photosystem II (PSII), estimated with the chlorophyll a fluorescence parameter FV/FM, in the absence or presence of lincomycin, were observed between constant and fluctuating light, nor between the two algae. Bryopsis sp. showed slower PSII recovery than A. acetabulum, possibly reflecting a regulatory response rather than increased oxidative stress, as the recovery rates increased with increasing amounts of PSII photoinhibition in both algae. In Bryopsis sp., however, high light treatments led to decreased electron transfer rates, estimated by both chlorophyll a fluorescence and net oxygen production, whereas a stimulation was observed in A. acetabulum. Nigericin, which prevents the formation of qE, increased photoinhibition in A. acetabulum but not in Bryopsis sp. Microoxic conditions as well as inhibitors of plastid terminal oxidase and mitochondrial respiration, on the other hand, enhanced photoinhibition only in Bryopsis sp., suggesting that, in the absence of qE, oxygen-dependent pathways (including flavodiiron proteins) are important for photoprotection. Near-infra-red absorption measurements suggest decreased Photosystem I (PSI) donor side limitation in Bryopsis sp., compared to A. acetabulum, and a lower capacity to keep P700 oxidised.
The D2 protein of Photosystem II has five transmembrane helices (A-E). An extended loop, connecting helices D and E, contributes to the binding environments of the primary quinone electron acceptor Q(A), and that of the bicarbonate bound to the non-heme iron between Q(A) and the secondary quinone electron acceptor Q(B). The residues from Ala240 to Thr243 are within a conserved sequence ((240)AEETYSMVTAN(250)) that contributes to the stabilization of both Q(A) and bicarbonate. We have created the A240D, E241A, E242A, E242D and T243A mutants to study the role of these residues. The mutations in the A240D and E241A strains had little impact on PS II performance, except addition of formate altered chlorophyll a fluorescence decay in the E241A mutant following a single-turnover actinic flash. Measurements of variable chlorophyll a fluorescence and thermoluminescence showed the E242A and E242D mutants had impaired acceptor side electron transport consistent with a reduced redox gap between Q(A) and Q(B). In addition, the T243A mutant exhibited a heightened susceptibility to photodamage. These data show that mutations introduced between (241)Glu-Thr(243) of D2 impair PS II activity but are less detrimental than mutations between the corresponding (243)Glu-Thr(245) residues of D1 in the vicinity of the Q(B)-binding site.
Understanding the rapid adjustments of plants to high-light exposure remains challenging, as multiple excitation and de-excitation pathways are simultaneously activated. In this study, we examined carotenoid pigment conversions at the second-scale in three tree species in parallel with high temporal resolution (<1 s) in vivo fluorescence and absorption spectroscopy. Our results reveal that both β-branch (violaxanthin, antheraxanthin, zeaxanthin) and α-branch (lutein, lutein epoxide) xanthophylls exhibit remarkably fast and oscillating pool dynamics within the first 20 seconds of illumination, reaching even maximal values in that timeframe. Prompt (0–20 s) conversion of the lutein is observed at the expense of both lutein epoxide and α-carotene in certain species, while accumulation of antheraxanthin and zeaxanthin is seen both prompt (0–20 s) and slower (>30 s). Interestingly, mirror trends between whole α- and β-branch carotenoids seem to indicate balancing trends, involving dynamic precursor shifts between α- and β-carotenes. Further, we observe that quick xanthophyll changes match the kinetic trends of fitted Gaussian-modeled absorbance peaks (approx. at 520, 535, 560 nm) within the early seconds. These quick changes in photon absorption are followed by slower-triggered non-photochemical de-excitation through a particular xanthophyll, seen from the dominant 535-nm peak, and likely attributed to antheraxanthin or zeaxanthin. The quick xanthophylls conversion redistributing the excessive excitation energy while quenching fluorescence (EET phase) is shown as one of the first responses to excessive light, before regulated energy dissipation as heat is initiated. These observations invite to interpret the non-steady state conditions and their parametrization more carefully, considering different photoprotective strategies across species.
In photosystem II (PSII) chlorophyll (Chl) fluorescence yield (F) rises during low-to-high light induction. Based on a critical review of the literature, the following evidence is summarized. (1) As the primary acceptor quinone QA gets reduced, fluorescence immediately rises to Fc =1.8 Fo. (2) During microseconds of illumination following QA reduction, Fc rises to Ff where excitation is terminated via carotenoid triplet states (3Car). (3) After many milliseconds of illumination, as the secondary acceptor quinone QB is reduced, fluorescence rises to a maximum Fm, wherein excitation is still terminated via 3Car. This dual phase fluorescence rise is driven by protein conformation changes. The two phases suggest that the C2S2M2 structure of the PSII dimer (Caffarri et al. 2009) may be rewritten as 2SCM such that for a monomer the arrangement is S - CP43 - D1D2 - CP47 - M. The D1- and D2- antenna branches are excitonically separated. Fluorescence of the D1 pigment-protein branch rises during microseconds after QA reduction, that of the D2 branch rises on a time scale of milliseconds during QB reduction. A hypothesis is proposed here based on electrostatic profiling of photosynthetic pigments (Sirohiwal et al. 2021; Saito et al. 2023). When acceptor quinones are oxidized excitation flows through the stromal Chl layer of Chl-proteins CP43 and CP47 to the photochemical Pheophytin/Chl based reaction center. As QA gets reduced during microseconds in CP43, and QB gets reduced during milliseconds in CP47, an associated dynamic signal induces protein conformation changes shifting excitations from the stromal to the lumenal Chl layer. With this, excitation becomes diverted from the photosynthetic reaction center and connected to the characteristically lumenal ChlZD1 and ChlZD2. Together with their nearby CarD1 and CarD2 these ChlsZ form quenching centers, terminating excitation via 3Car with a lifetime of about 1 ns, sufficient to induce the maximum Fm fluorescence.
Photosynthetic microorganisms may show extracellular electron transfer (EET), in which some of the electrons generated by photosynthesis or respiration are lost from the cell. Most studies have focussed on cyanobacteria, with very few on eukaryotic algae. Here we demonstrate EET from the eukaryotic dinoflagellate alga Symbiodinium microadriaticum, a species that can form symbiosis with corals and other Cnidaria. We show that the EET involves diffusible electroactive species, which may represent a previously unsuspected route for communication between symbionts and hosts. We show that EET can be used to study photosynthetic and respiratory functions in the dinoflagellate. We also show that it can provide information on the effects of environmental stresses including changes in temperature (linked to coral bleaching), pH and light intensity. The electrochemical platform outlined in this study offers a novel tool for studying dinoflagellate physiology, the coral-dinoflagellate symbiosis, and the molecular mechanisms of bleaching.
We develop the exciton model of the major light-harvesting complex LHCII from higher plants based on complex time-dependent Redfield (ctR) theory. A simultaneous quantitative fit of the absorption, linear dichroism, and fluorescence spectra allowed us to obtain the site energies of 14 chlorophylls (Chl) within monomeric subunit of the trimeric LHCII antenna. These site energies are noticeably different from those defined in the former exciton model of the complex based on modified Redfield (mR) theory. At the same time there are similarities in distribution of the pigment participations to the exciton states in the ctR and mR models. In both models the blue-most is the Chl b606 site, the Chl a604 pigment is a long-lived intermediate peaking between the Chls b and Chls a bands, and the red-most trap is located at Chl a610. We suggest that the site energies and other parameters emerging from the present ctR model should be used in order to get more realistic (comparing to mR) treatment of the spectra and excitation dynamics in LHCII antenna.
In photosynthetic proteins, pigments at a higher energy level funnel excitation energy to pigments at a lower energy level. Specifically, in Photosystem II (PSII), energy is transferred downhill to the reaction center (RC), where water splitting occurs. However, the lowest-energy state in PSII is not the RC, but the F695 state, which can be observed using low-temperature spectroscopy. This lowest-energy state is typically assigned to a monomeric pigment, Chl B16 (ligated by His114), but this assignment has been called into question based on theoretical fits to low-temperature spectra. In this study, we set out to test concretely whether the F695 state is localized on Chl B16 using site-directed mutagenesis and 77 K fluorescence spectroscopy. To reduce spectral congestion for whole-cell PSII studies, we developed a background strain (PSI-kd/ Δ PBS) that combines a Photosystem I (PSI) knockdown with a Phycobilisome (PBS) knockout. In this background strain, we made site-directed mutations at site Thr5 in the PsbH subunit, which forms a hydrogen bond with the 13^1 -keto group of Chl B16. All mutants were capable of heterotrophic growth (without noticeable differences from wild-type), indicating the PSII function remains intact. As expected for Chl B16-localized fluorescence, the Thr5 → Arg mutation red-shifted the F695 state due to the strengthening of the hydrogen bond, while the Thr5 → Ala mutation exhibits a blue shift as the hydrogen bond is eliminated. Taken together, these findings provide strong confirmation that Chl B16 is responsible for the lowest-energy state.
While natural light in plant canopies often fluctuates and shows a large range of possible intensities, the rate of photosynthetic induction is often measured at only two light intensities. This prompts the question as to how the relationship between different light intensities and the rate of photosynthetic induction can be characterized. Using hundreds of measurements of photosynthetic induction at various light intensities in leaves of tomato (Solanum lycopersicum), we found linear relationships between the time to reach 50
Anthocyanins are non-photosynthetic pigments that absorb photons and thus decrease photosynthesis. Several studies have characterized the reduction in photosynthesis from anthocyanins by comparing high and low anthocyanin genotypes under single light intensity. We hypothesized that high anthocyanin levels would result in a greater reduction in photosynthesis in low than at high PPFD because anthocyanin-induced screening would have a stronger impact under limited conditions. We quantified the reduction of light-saturated gross photosynthesis (Pg) and quantum yield of CO2 (QYCO2) across eight lettuce cultivars ranging from 3.4 to 34 mg m− 2 anthocyanins, and report the photosynthetic effects over a range of wavelengths from 400 to 700 nm. We used spectral reflectance to quantify normalized difference anthocyanin index (NDAI), which ranged from − 0.24 to + 0.12. As expected, photosynthesis decreased as anthocyanin concentration increased, but surprisingly, the photosynthetic reduction was similar over a range of PPFDs from 50 to 1500 µmol m− 2 s− 1. The positive correlation between the absorptance at green wavelengths and NDAI suggest that anthocyanins filtered primarily green photons. Due to the screening effect of the green photons by anthocyanins, Pg and QYCO2 decreased from 45 to 82
The role of chloride on the donor side of Photosystem II (PSII) is considered to be associated with proton removal. These protons are generated at the water oxidizing complex (WOC) during oxidation of water and transferred through specific “water channels” to the lumen. While structural studies have confirmed the presence of chloride at consistent sites near the WOC, the complete mechanism by which chloride ions influence PSII function and stability is not fully resolved. This work utilizes the hypercarbonate-requiring filamentous cyanobacterium Limnospira maxima to investigate these roles through bromide substitution, probing the interdependencies between halide ions and proton transfer in an unusually bicarbonate-rich environment. Bicarbonate has been posited to be a participant in the proton removal process, but also plays roles on the acceptor side of PSII. Low temperature (77 K) spectrofluorometry suggests that halide substitution results in reduced exciton transfer from the phycobilisome to the PSII reaction center. Chlorophyll fast repetition rate (FRR) fluorometry further reveals diminished PSII quantum efficiency under bromide conditions compared to the native chloride containing system. While the rates of electron transfer from QA− to QB were found to be faster in bromide substituted cells, P700 and plastocyanin (PC) redox kinetics reveal a shift in the baseline redox balance of the photosynthetic electron transport chain. Overall, these findings demonstrate that bromide substitution influences the efficiency of proton removal from the WOC despite the elevated bicarbonate concentration in Limnospira maxima, which in turn modulates the distribution of electrons across the photosynthetic apparatus.
We verify various perturbative methods frequently used for modeling of the excitation dynamics within photosynthetic LH2 (B800-B850) antenna by comparing them with an exact (nonperturbative) hierarchical equation (HEOM) approach. We have found that secular theories (like coherent modified Redfield, and combined Redfield-Förster approaches) fail to describe the dynamics, giving too fast transfers within the B800 and B850 bands and too fast B800→B850 conversion due to the neglect of the population-to-coherence transfers. On the other hand, the standard Redfield theory with the full relaxation tensor is capable to give a quantitatively correct picture consistent with the HEOM solution (with an accuracy of some deviations from the exact kinetics due to absence of dynamic localization associated with the B800→B850 pathways). Being computationally inexpensive this theory can be used for modeling of linear and nonlinear spectral responses of various LH2 complexes with averaging over energetic disorder in the cases when the use of nonperturbative methods is limited due to their numerical complexity.
Analysis of in vivo fluorescence yield measured in the red and far-red regions is widely used to estimate photosynthetic parameters related to photosystem II (PSII). Besides PSII, photosystem I (PSI) and phycobilisomes (PBS), the latter present in cyanobacteria, red algae, and glaucophytes, also emit fluorescence. Real-time fluorescence responses of PSI and PBS have not been fully characterized, as practical methods to resolve fluorescence contributions from PSII, PSI, and PBS are limited. To resolve the contributions of PSII, PSI, and PBS to the in vivo fluorescence yield, an analytical workflow is proposed based on fluorescence induction curves and variable fluorescence kinetics measured across four detection wavebands. The fluorescence induction curves and variable fluorescence kinetics were measured using blue and amber lights, which preferentially excited the photosystems (PSII and PSI) and PBS, respectively, in the unicellular red alga Cyanidioschyzon merolae. Both PSI and PBS contributed small but measurable portions to the fluorescence induction curves, enabling estimation of the relative fluorescence amplitudes across detection wavebands through constrained non-negative matrix factorization (NMF). The relative fluorescence amplitudes were used to estimate the relative fluorescence yields of PSII, PSI, and PBS, as well as the PSII-related parameter ΦPSII through least-squares fitting of variable fluorescence data. The resulting kinetics obtained from the optimal NMF models revealed multiple processes associated with nonphotochemical quenching of PSII and provided insights into the regulation of excitonic energy transfer among PSII, PSI, and PBS. The analytical workflow serves as an effective approach to uncover PSI and PBS fluorescence yield kinetics across diverse photosynthetic organisms.
Facultative CAM plants possess a unique ability to reversibly initiate and suppress β-carboxylation, a metabolic pathway characterised by nocturnal stomatal opening and high water-use efficiency. In the present study, we focused on determining how continuous light modifies the interaction of the Mesembryanthemum crystallinum, a model CAM facultative species, with salinity stress – an ever-growing global-scale plant diversity and productivity threat. Expression of stress-dependent PEPC form and RbcL, chlorophyll a fluorescence, pigment composition and antioxidant enzyme activity were analysed. Expression of both mentioned genes was predictably influenced by salinity stress, with stress-dependent PEPC amount increase accompanied by a concurrent RbcL decline. However, exposing plants already affected by salinity stress to continuous light resulted in a limited RbcL decline – a side-effect exclusively induced by salinity stress. Moreover, a limited expression of salinity-dependent PEPC was concomitantly observed; in M. crystallinum leaves, the content of cryptoxanthin, β-carotene, α-carotene, zeaxanthin, violaxanthin, antheraxanthin and neoxanthin has increased, especially in the case of salinity with continuous light treatments. The total SODs activity was increased in plants exposed to salinity combined with continuous light. This study unravels novel evidence that continuous light treatment may alleviate the detrimental effects of high salinity in facultative CAM plants; these conclusions, however, may potentially be extrapolated to other plant groups, including glycophytes.
