Bryophyta (mosses) are a basal group of plants that lack stomata in their haploid form. Consequently, these plants are defined as poikilohydric, meaning poor control over water loss, and their cytosol is assumed to reach equilibrium with ambient humidity. This classification does not fully align with the diverse strategies observed in mosses. We studied gas exchange in 16 species under controlled dehydration conditions. Our results revealed that the cell wall water potential was below the cytosolic water potential in most species, as reflected by relative humidity at the cell wall surface decreasing to as low as 60% even under optimum hydric conditions. This indicates that cell membranes and/or cell walls involve barriers with higher resistance to water movement than previously assumed (2.7-23 MPa m2 s mmol-1). Additionally, species with better water control also exhibit traits of an avoidance strategy, including elastic tissues, high capacitance, and less negative osmotic pressure. These findings point to a basal, non-stomatal mechanism of water loss control through cell membranes and/or cell walls. Potentially, this mechanism is homologous to the nonstomatal control recently identified in angiosperms, which induces unsaturated conditions in the substomatal cavities. Bryophyta presents a valuable nonstomatal model for further investigating this mechanism.

### Competing Interest Statement The authors have declared no competing interest.

Mesophyll cells exhibit a previously underappreciated capacity to regulate water loss via low plasma membrane conductance ( L p), offering a non-stomatal mechanism for transpiration control. However, the structural basis and regulation of L p remain poorly understood, limiting its integration into predictive models. In this study, we show that L p responds dynamically to changes in cytosolic water potential ( ψ cy), decreasing as ψ cy approaches the turgor loss point. This identifies ψ cy as the primary physiological signal regulating L p. We introduce a predictive, physiologically grounded model linking L p to ψ cy. Our model establishes a mechanistic connection between internal water status, leaf hydraulics, substomatal cavity unsaturation, and gas exchange. This framework opens new avenues for understanding and modelling plant water use under stress. ### Competing Interest Statement The authors have declared no competing interest.

Our understanding of how photosynthetic capacity varies among C4 species and across growth and measurement conditions remains limited. We collated 1696 CO2 response curves of net CO2 assimilation rate (A/Ci curves) from C4 species grown and measured at various environmental conditions and used these data to estimate the apparent maximum carboxylation activity of phosphoenolpyruvate carboxylase (VpmaxA) and CO2-saturated net photosynthetic rate (Amax), two key parameters describing photosynthetic capacity. We examined how VpmaxA and Amax vary with species-specific traits, growth and measurement conditions. We found little systematic variation of VpmaxA and Amax across the classical C4 biochemical subtypes or growth forms, but showed that growth temperature and measurement conditions are major factors determining C4 photosynthetic capacity. We found no evidence that common C4 model species (e.g. maize, sorghum and Setaria viridis) differ in photosynthetic capacity from other C4 species when grown in controlled environments. However, C4 model species showed up to twice the photosynthetic capacity of other C4 species when grown in the field. Our multivariate model accounts for 47-51% of the variation reported in VpmaxA and Amax, and we argue that environmental conditions have a greater influence on C4 photosynthetic capacity than biochemical subtypes or growth forms.

Photosynthesis underpins life on Earth, serving as the primary energy source while regulating global carbon and water cycles, thereby shaping climate and vegetation. Advancing photosynthesis research is essential for improving crop productivity and refining photosynthesis models across scales, ultimately addressing critical global challenges such as food security and environmental sustainability. This minireview synthesizes a selection of recent advancements presented at the 2nd European Congress of Photosynthesis Research, focusing on improving photosynthesis efficiency and modelling across the scales. We explore strategies to optimize light harvesting and carbon fixation, leading to canopy level improvements. Alongside synthetic biology, we examine recent advances in harnessing natural variability in key photosynthetic traits, considering both methodological innovations and the vast reservoir of opportunities they present. Additionally, we highlight unique insights gained from plants adapted to extreme environments, offering pathways to improve photosynthetic efficiency and resilience simultaneously. We emphasize the importance of a holistic approach, integrating dynamic modeling of metabolic processes to bridge these advancements. Beyond photosynthesis improvements, we discuss the progress of improving photosynthesis simulations, particularly through improved parametrization of mesophyll conductance, crucial for enhancing leaf-to-global scale simulations. Recognizing the need for greater interdisciplinary collaboration to tackle the grand challenges put on photosynthesis research, we highlight two initiatives launched at the congress-an open science platform and a dedicated journal for plant ecophysiology. We conclude this minireview with a forward-looking outline, highlighting key next steps toward achieving meaningful improvements in photosynthesis, yield, resilience and modeling.

C2 photosynthesis is a photosynthetic pathway in which photorespiratory CO2 release and refixation are enhanced in leaf bundle sheath (BS) tissues. The evolution of C2 photosynthesis has been hypothesized to be a major step in the origin of C4 photosynthesis, highlighting the importance of studying C2 evolution. In this study, physiological, anatomical, ultrastructural, and immunohistochemical properties of leaf photosynthetic tissues were investigated in six non-C4 Tribulus species and four C4 Tribulus species. At 42°C, T. cristatus exhibited a photosynthetic CO2 compensation point in the absence of respiration (C*) of 21 µmol mol-1, below the C3 mean C* of 73 µmol mol-1. Tribulus astrocarpus had a C* value at 42°C of 55 µmol mol-1, intermediate between the C3 species and the C2 T. cristatus. Glycine decarboxylase (GDC) allocation to BS tissues was associated with lower C*. Tribulus cristatus and T. astrocarpus allocated 86% and 30% of their GDC to the BS tissues, respectively, well above the C3 mean of 11%. Tribulus astrocarpus thus exhibits a weaker C2 (termed sub-C2) phenotype. Increased allocation of mitochondria to the BS and decreased length-to-width ratios of BS cells, were present in non-C4 species, indicating a potential role in C2 and C4 evolution.

Leaf-level gas exchange enables insights into the physiology and in vivo biochemical processes of plants. Advances in infrared gas analysis have resulted in user-friendly off-the-shelf gas exchange systems that allow researchers to collect physiological measurements with the push of a few buttons. Here, I describe how to set up the gas exchange equipment and what to pay attention to while making measurements, and provide some guidelines on how to analyze and interpret the data obtained.

Gas exchange measurements enable mechanistic insights into the processes that underpin carbon and water fluxes in plant leaves which in turn inform understanding of related processes at a range of scales from individual cells to entire ecosytems. Given the importance of photosynthesis for the global climate discussion it is important to (a) foster a basic understanding of the fundamental principles underpinning the experimental methods used by the broad community, and (b) ensure best practice and correct data interpretation within the research community. In this review, we outline the biochemical and biophysical parameters of photosynthesis that can be investigated with gas exchange measurements and we provide step-by-step guidance on how to reliably measure them. We advise on best practices for using gas exchange equipment and highlight potential pitfalls in experimental design and data interpretation. The Supporting Information contains exemplary data sets, experimental protocols and data-modelling routines. This review is a community effort to equip both the experimental researcher and the data modeller with a solid understanding of the theoretical basis of gas-exchange measurements, the rationale behind different experimental protocols and the approaches to data interpretation.

Understanding the short-term responses of mesophyll conductance (gm) and stomatal conductance (gsc) to environmental changes remains a challenging yet central aspect of plant physiology. This review synthesises our current knowledge of these short-term responses, which underpin CO2 diffusion within leaves. Recent methodological advances in measuring gm using online isotopic discrimination and chlorophyll fluorescence have improved our confidence in detecting short-term gm responses, but results need to be carefully evaluated. Environmental factors like vapour pressure deficit and CO2 concentration indirectly impact gm through gsc changes, highlighting some of the complex interactions between the two parameters. Evidence suggests that short-term responses of gm are not, or at least not fully, mechanistically linked to changes in gsc, cautioning against using gsc as a reliable proxy for gm. The overarching challenge lies in unravelling the mechanistic basis of short-term gm responses, which will contribute to the development of accurate models bridging laboratory insights with broader ecological implications. Addressing these gaps in understanding is crucial for refining predictions of gm behaviour under changing environmental conditions.

Measurements of in vivo photosynthesis are powerful tools that probe the largest fluxes of carbon and energy in an illuminated leaf, but often the specific techniques used are so varied and specialized that it is difficult for researchers outside the field to select and perform the most useful assays for their research questions. The goal of this chapter is to provide a broad overview of the current tools available for the study of photosynthesis, both in vivo and in vitro, so as to provide a foundation for selecting appropriate techniques, many of which are presented in detail in subsequent chapters. This chapter will also organize current methods into a comparative framework and provide examples of how they have been applied to research questions of broad agronomical, ecological, or biological importance. This chapter closes with an argument that the future of in vivo measurements of photosynthesis lies in the ability to use multiple methods simultaneously and discusses the benefits of this approach to currently open physiological questions. This chapter, combined with the relevant methods chapters, could serve as a laboratory course in methods in photosynthesis research or as part of a more comprehensive laboratory course in general plant physiology methods.

Cuticular conductance to water (gcw ) is difficult to quantify for stomatous surfaces due to the complexity of separating cuticular and stomatal transpiration, and additional complications arise for determining adaxial and abaxial gcw . This has led to the neglect of gcw as a separate parameter in most common gas exchange measurements. Here, we describe a simple technique to simultaneously estimate adaxial and abaxial values of gcw , tested in two amphistomatous plant species. What we term the 'Red-Light method' is used to estimate gcw from gas exchange measurements and a known CO2 concentration inside the leaf during photosynthetic induction under red light. We provide an easy-to-use web application to assist with the calculation of gcw . While adaxial and abaxial gcw varies significantly between leaves of the same species we found that the ratio of adaxial/abaxial gcw (γn ) is stable within a plant species. This has implications for use of generic values of gcw when analysing gas exchange data. The Red-Light method can be used to estimate total cuticular conductance (gcw-T ) accurately with the most common setup of gas exchange instruments, i.e. a chamber mixing the adaxial and abaxial gases, allowing for a wide application of this technique.

Over half the proteins in the E. coli cytoplasm form homo or hetero-oligomeric structures. Experimentally determined structures are often considered in determining a protein's oligomeric state, but static structures miss the dynamic equilibrium between different quaternary forms. The problem is exacerbated in homo-oligomers, where the oligomeric states are challenging to characterize. Here, we re-evaluated the oligomeric state of 17 different bacterial proteins across a broad range of protein concentrations and solutions by native mass spectrometry (MS), mass photometry (MP), size exclusion chromatography (SEC), and small-angle X-ray scattering (SAXS), finding that most exhibit several oligomeric states. Surprisingly, some proteins did not show mass-action driven equilibrium between the oligomeric states. For approximately half the proteins, the predicted oligomeric forms described in publicly available databases underestimated the complexity of protein quaternary structures in solution. Conversely, AlphaFold multimer provided an accurate description of the potential multimeric states for most proteins, suggesting that it could help resolve uncertainties on the solution state of many proteins.

Cross-linking combined with mass spectrometry (XL-MS) provides a wealth of information about the three-dimensional (3D) structure of proteins and their interactions. We introduce MaxLynx, a novel computational proteomics workflow for XL-MS integrated into the MaxQuant environment. It is applicable to noncleavable and MS-cleavable cross-linkers. For both, we have generalized the Andromeda peptide database search engine to efficiently identify cross-linked peptides. For noncleavable peptides, we implemented a novel dipeptide Andromeda score, which is the basis for a computationally efficient N-squared search engine. Additionally, partial scores summarize the evidence for the two constituents of the dipeptide individually. A posterior error probability (PEP) based on total and partial scores is used to control false discovery rates (FDRs). For MS-cleavable cross-linkers, a score of signature peaks is combined with the conventional Andromeda score on the cleavage products. The MaxQuant 3D peak detection was improved to ensure more accurate determination of the monoisotopic peak of isotope patterns for heavy molecules, which cross-linked peptides typically are. A wide selection of filtering parameters can replace the manual filtering of identifications, which is often necessary when using other pipelines. On benchmark data sets of synthetic peptides, MaxLynx outperforms all other tested software on data for both types of cross-linkers and on a proteome-wide data set of cross-linked Drosophila melanogaster cell lysate. The workflow also supports ion mobility-enhanced MS data. MaxLynx runs on Windows and Linux, contains an interactive viewer for displaying annotated cross-linked spectra, and is freely available at https://www.maxquant.org/.

Photosynthetic manipulation provides new opportunities for enhancing crop yield. However, understanding and quantifying the importance of individual and multiple manipulations on the seasonal biomass growth and yield performance of target crops across variable production environments is limited. Using a state-of-the-art cross-scale model in the APSIM platform we predicted the impact of altering photosynthesis on the enzyme-limited (Ac ) and electron transport-limited (Aj ) rates, seasonal dynamics in canopy photosynthesis, biomass growth, and yield formation via large multiyear-by-location crop growth simulations. A broad list of promising strategies to improve photosynthesis for C3 wheat and C4 sorghum were simulated. In the top decile of seasonal outcomes, yield gains were predicted to be modest, ranging between 0% and 8%, depending on the manipulation and crop type. We report how photosynthetic enhancement can affect the timing and severity of water and nitrogen stress on the growing crop, resulting in nonintuitive seasonal crop dynamics and yield outcomes. We predicted that strategies enhancing Ac alone generate more consistent but smaller yield gains across all water and nitrogen environments, Aj enhancement alone generates larger gains but is undesirable in more marginal environments. Large increases in both Ac and Aj generate the highest gains across all environments. Yield outcomes of the tested manipulation strategies were predicted and compared for realistic Australian wheat and sorghum production. This study uniquely unpacks complex cross-scale interactions between photosynthesis and seasonal crop dynamics and improves understanding and quantification of the potential impact of photosynthesis traits (or lack of it) for crop improvement research.

Increasing [CO2] may influence commercial crop and timber yield. While selection of genotypes sensitive to elevated [CO2] (e[CO2]) appears possible in agricultural crops, there is limited evidence for genotype-by-CO2 (G × CO2) interactions in commercial tree species. We examined [CO2] responsiveness in 124 open-pollinated Eucalyptus globulus ssp. globulus (E. globulus) families with the aim of assessing whether G × CO2 interactions are detectable in seedlings for early-age screening. Plants were grown in ambient (a[CO2]; ~ 405 μmol mol−1) and e[CO2] (640 μmol mol−1) and harvested 25 days after germination. Total, shoot, and root dry weights were determined for each plant. Carbon isotopic discrimination against 13C (Δ13C) was determined at the family level. We observed highly significant (p < 0.0001) increases in mean total, shoot, and root dry weights. Mixed-model equations were used to estimate the main and interaction effects of the G × CO2 for each mass trait. The main effects from the mixed-model output ([CO2] and individual-tree effects) were significant for all traits. However, [CO2]-by-individual tree interactions were non-significant for all traits, indicating little G × CO2 interaction. A secondary aim was to examine the correlation between greenhouse and mature-age growth from breeding trials that use common families conducted under ambient [CO2]. These correlations were non-significant, suggesting early growth is not necessarily indicative of later-age responses. Our results suggest that while early growth of E. globulus is enhanced under e[CO2], genotypes respond relatively uniformly to e[CO2] and little opportunity exists for seedling-based selection at the population level based upon the response of plants during the first weeks of growth.

Spatial separation of ions in the gas phase, providing in-formation about their size as collisional cross-sections, can readily be achieved through ion mobility. The tim-sTOF Pro (Bruker Daltonics) series combines a trapped ion mobility device with a quadrupole, collision cell, and a time-of-flight analyzer to enable the analysis of ions at great speed. Here, we show that the timsTOF Pro is capable of physically separating N-glycopeptides from nonmodified peptides and producing high-quality frag-mentation spectra, both beneficial for glycoproteomics analyses of complex samples. The glycan moieties enlarge the size of glycopeptides compared with non -modified peptides, yielding a clear cluster in the mobilo-gram that, next to increased dynamic range from the physical separation of glycopeptides and nonmodified peptides, can be used to make an effective selection filter for directing the mass spectrometer to analytes of inter-est. We designed an approach where we (1) focused on a region of interest in the ion mobilogram and (2) applied stepped collision energies to obtain informative glyco-peptide tandem mass spectra on the timsTOF Pro:glyco-polygon-stepped collision energy-parallel accumulation serial fragmentation. This method was applied to selected glycoproteins, human plasma- and neutrophil-derived glycopeptides. We show that the achieved physical sep-aration in the region of interest allows for improved extraction of information from the samples, even at shorter liquid chromatography gradients of 15 min. We validated our approach on human neutrophil and plasma samples of known makeup, in which we captured the anticipated glycan heterogeneity (paucimannose, phos-phomannose, high mannose, hybrid and complex glycans) from plasma and neutrophil samples at the expected abundances. As the method is compatible with off-the -shelve data acquisition routines and data analysis software, it can readily be applied by any laboratory with a timsTOF Pro and is reproducible as demonstrated by a comparison between two laboratories.

EDITORIAL article Front. Plant Sci., 22 October 2021Sec.Plant Systematics and Evolution https://doi.org/10.3389/fpls.2021.774818

Surface-induced dissociation (SID) was first introduced as an ion activation method for tandem mass spectrometry in the laboratory of Graham Cooks at Purdue University. Early studies focused on small molecules that could be ionized and fragmented by the instruments available at that time. It was predicted that SID would someday be applied to much more massive ions and that the large collision target (the surface) would be advantageous for dissociation of very large ions. This chapter describes the development of SID from its early days until the present, closing with examples that illustrate the benefits of using SID to cleave large noncovalent protein complexes.

Kinases play central roles in signaling cascades, relaying information from the outside to the inside of mammalian cells. De novo designed protein switches capable of interfacing with tyrosine kinase signaling pathways would open new avenues for controlling cellular behavior, but, so far, no such systems have been described. Here we describe the de novo design of two classes of protein switch that link phosphorylation by tyrosine and serine kinases to protein-protein association. In the first class, protein-protein association is required for phosphorylation by the kinase, while in the second class, kinase activity drives protein-protein association. We design systems that couple protein binding to kinase activity on the immunoreceptor tyrosine-based activation motif central to T-cell signaling, and kinase activity to reconstitution of green fluorescent protein fluorescence from fragments and the inhibition of the protease calpain. The designed switches are reversible and function in vitro and in cells with up to 40-fold activation of switching by phosphorylation.