Photosynthetic Gas Exchange in Land Plants at the Leaf Level.

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, what to pay attention to while making measurements, and provide some guidelines on how to analyze and interpret the data obtained.

A guide to photosynthetic gas exchange measurements: Fundamental principles, best practice and potential pitfalls.

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.

Oxonium Ion-Guided Optimization of Ion Mobility-Assisted Glycoproteomics on the timsTOF Pro.

Spatial separation of ions in the gas phase, providing information about their size as collisional cross-sections, can readily be achieved through ion mobility. The timsTOF 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 fragmentation spectra, both beneficial for glycoproteomics analyses of complex samples. The glycan moieties enlarge the size of glycopeptides compared with nonmodified peptides, yielding a clear cluster in the mobilogram 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 interest. 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 glycopeptide 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 separation 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, phosphomannose, 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.

Accurate and Automated High-Coverage Identification of Chemically Cross-Linked Peptides with MaxLynx.

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/.

De novo design of tyrosine and serine kinase-driven protein switches.

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.

Cellular mRNA triggers structural transformation of Ebola virus matrix protein VP40 to its essential regulatory form.

The Ebola virus matrix protein VP40 forms distinct structures linked to distinct functions in the virus life cycle. Dimeric VP40 is a structural protein associated with virus assembly, while octameric, ring-shaped VP40 is associated with transcriptional control. In this study, we show that suitable nucleic acid is sufficient to trigger a dynamic transformation of VP40 dimer into the octameric ring. Deep sequencing reveals a binding preference of the VP40 ring for the 3' untranslated region of cellular mRNA and a guanine- and adenine-rich binding motif. Complementary analyses of the nucleic-acid-induced VP40 ring by native mass spectrometry, electron microscopy, and X-ray crystal structures at 1.8 and 1.4 Å resolution reveal the stoichiometry of RNA binding, as well as an interface involving a key guanine nucleotide. The host factor-induced structural transformation of protein structure in response to specific RNA triggers in the Ebola virus life cycle presents unique opportunities for therapeutic inhibition.

Optimization can provide the fundamental link between leaf photosynthesis, gas exchange and water relations.

Tight coordination in the photosynthetic, gas exchange and water supply capacities of leaves is a globally conserved trend across land plants. Strong selective constraints on leaf carbon gain create the opportunity to use quantitative optimization theory to understand the connected evolution of leaf photosynthesis and water relations. We developed an analytical optimization model that maximizes the long-term rate of leaf carbon gain, given the carbon costs in building and maintaining stomata, leaf hydraulics and osmotic pressure. Our model demonstrates that selection for optimal gain should drive coordination between key photosynthetic, gas exchange and water relations traits. It also provides predictions of adaptation to drought and the relative costs of key leaf functional traits. Our results show that optimization in terms of carbon gain, given the carbon costs of physiological traits, successfully unites leaf photosynthesis and water relations and provides a quantitative framework to consider leaf functional evolution and adaptation.

Photosynthetic Gas Exchange in Land Plants at the Leaf Level.

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.

Estimation of Photorespiratory Fluxes by Gas Exchange.

Photorespiratory fluxes can be easily estimated by photosynthetic gas exchange using an infrared gas analyzer and applying the Farquhar, von Caemmerer, and Berry (Farquhar et al. Planta 149:78-90, 1980) photosynthesis model. For a more direct measurement of photorespiratory CO2 release from glycine decarboxylation, infrared gas analysis can be coupled to membrane-inlet mass spectrometry, capable of separating the total CO2 concentration into its 12CO2 and 13CO2 components in a continuous online fashion. This chapter discusses how to calculate rates of photorespiration from Rubisco kinetics and describes in detail a method for measuring the CO2 release from glycine decarboxylation using 13CO2.

Over-expression of bacterial gamma-glutamylcysteine synthetase (GSH1) in plastids affects photosynthesis, growth and sulphur metabolism in poplar (Populus tremula x Populus alba) dependent on the resulting gamma-glutamylcysteine and glutathione levels.

We compared three transgenic poplar lines over-expressing the bacterial gamma-glutamylcysteine synthetase (GSH1) targeted to plastids. Lines Lggs6 and Lggs12 have two copies, while line Lggs20 has three copies of the transgene. The three lines differ in their expression levels of the transgene and in the accumulation of gamma-glutamylcysteine (gamma-EC) and glutathione (GSH) in leaves, roots and phloem exudates. The lowest transgene expression level was observed in line Lggs6 which showed an increased growth, an enhanced rate of photosynthesis and a decreased excitation pressure (1-qP). The latter typically represents a lower reduction state of the plastoquinone pool, and thereby facilitates electron flow along the electron transport chain. Line Lggs12 showed the highest transgene expression level, highest gamma-EC accumulation in leaves and highest GSH enrichment in phloem exudates and roots. This line also exhibited a reduced growth, and after a prolonged growth of 4.5 months, symptoms of leaf injury. Decreased maximum quantum yield (F(v)/F(m)) indicated down-regulation of photosystem II reaction centre (PSII RC), which correlates with decreased PSII RC protein D1 (PsbA) and diminished light-harvesting complex (Lhcb1). Potential effects of changes in chloroplastic and cytosolic GSH contents on photosynthesis, growth and the whole-plant sulphur nutrition are discussed for each line.