An approach to improving radiation use efficiency (RUE) in wheat is to screen for variability in rates of leaf respiration in darkness (Rdark). We used a high-throughput system to quantify variation in Rdark among a diverse range of spring wheat genotypes (301 lines) grown in two countries (Mexico and Australia) and two seasons (2017 and 2018), and in doing so quantify the relative importance of genotype (G) and environment (E) in influencing variations in leaf Rdark. Through careful design, residual (unexplained) variation represented <10% of the total observed. Up to a third of the variation in Rdark (and related traits) was under genetic control. This suggests opportunities for breeders to use Rdark as a novel selection tool. In addition, E accounted for more than half of the total variation in area-based rates of Rdark. Here, the day of measurement was crucial, suggesting that day-to-day variations in the environment influence rates of Rdark measured at a common temperature. Overall, this study provides new insights into the role G and E play in determining variation in rates of leaf Rdark of one of the most important cereal crops, with implications for future improvements in carbon use efficiency and yield.

Low-temperature (LT) stress induces significant changes to plant cells including perturbations of various physio-biochemical and metabolic processes, which impact primary metabolism, respiratory rate, and the ATP production for biosynthesis and growth. Mitochondria from LT-tolerant species respond to LT through remodeling their composition that changes the structural and functional properties of the organelles. In this review, we discuss physiological aspects of mitochondrial respiration rate that are affected by LT, as well as, changes in the abundance of respiratory components under LT. The latter includes components of the phosphorylating and non-phosphorylating pathways and adjustments of mitochondrial membrane composition. Our objective is to provide a detailed overview of the often-contrasting reports of mitochondrial-specific changes and responses to LT and look for consensus themes to explain changes and draw more generally applicable observations about the LT response of plant respiration.

OBJECTIVE: Retinol-binding protein 4 (RBP4) has been identified as a novel adipokine mediating systemic insulin resistance, and elevated serum RBP4 indicates overt or impending insulin resistance in lean, obese, and type 2 diabetic subjects. As insulin resistance is present in nearly all patients with liver cirrhosis, we evaluated RBP4 in patients with chronic liver disease (CLD). RESEARCH DESIGN AND METHODS: Serum RBP4 was measured in 111 CLD patients. Ninety-nine age- and sex-matched healthy blood donors served as control subjects. RBP4 gene expression was also quantified in normal and cirrhotic rat liver. RESULTS: In CLD patients, serum RBP4 was significantly reduced compared with healthy control subjects and closely correlated with the stage of liver cirrhosis. CLD patients without cirrhosis showed normal RBP4 concentrations, which correlated with serum glucose and insulin secretion and inversely correlated with insulin sensitivity. In patients with Child A-C liver cirrhosis, however, RBP4 was not correlated with glucose metabolism or other adipokines, such as adiponectin or resistin, but closely linked to the hepatic biosynthetic capacity, fibrotic changes in liver histology, or clinical complications such as portal hypertension. In an animal model of experimental cirrhosis, hepatic RBP4 gene expression decreased in cirrhotic liver. CONCLUSIONS: RBP4 appears, unlike in obesity or type 2 diabetes, not to be a relevant systemic factor in the pathogenesis of insulin resistance in liver cirrhosis. Liver function has a tremendous impact on RBP4 levels, and future studies will need to take liver function into account when examining serum RBP4 levels.

Introduction: Activation of hepatic stellate cells (HSC), mainly triggert by transforming growth factor-β (TGF-β), is a key event in liver fibrogenesis accompanied by modulations of gene expression e.g. upregulation of contractile proteins known as markers of smooth muscle cell (SMC) differentiation. In SMCs the marker genes are principally regulated by binding of serum response factor (SRF) to unique sequences in their promoters, called CArG-boxes. In early chick embryonic cardiovascular development a functional co-association between SRF and the potent SMC marker cystein- and glycine-rich protein 2 (CRP2) was seen in the nucleus of proepicardial cells followed by a transition of CRP2 to the cytoskeleton during differentiation into SMCs [1]. Aim: We analysed the regulation and cellular distribution of CRP2 in activated HSC, which in liver is exclusively expressed in this cell type, as well as the presence of SRF in these cells. Methods: Endogenous CRP2 and SRF contents were detected in cell lysates from cultured rat HSC or cell lines (A10 cells, CFSC) by Western Blot analysis. Cells were treated with soluble TGF-β type II receptor (STR), TGF-β1, or different serum contents in medium. Regulation of CSRP2 (gene name of CRP2) was investigated by reporter gene constructs. Cellular distribution of CRP2 was detected by separation of activated HSC into 4 fractions (membrane, cytoplasm, nucleus, cytoskeleton). Results: CRP2 is upregulated by TGF-β in activated HSC and accumulates in the nucleus. These findings were confirmed by TGF-β mediated rapid increase of CRP2 content in vascular SMCs. Reporter gene constructs pointed out that evolutionary conserved CAGA-sites in between the proximal 1.2 kbp rat CSRP2 promoter are not relevant for this effect. Upregulation of CRP2 is also serum inducible, which was shown in transfection experiments in A10 cells. Another finding was a TGF-β regulated expression of SRF in activated HSC. Discussion: We found CRP2 and SRF increased in activated HSC by TGF-β. While SRF is an established transcription factor especially involved in SMC differentiation, the role and function of CRP2 in SMC or activated HSC is largely unknown. To date it is well accepted that CRP2 is associated to the cytoskeleton of SMC [2]. The localisation and accumulation of CRP2 in the nucleus of HSC during fibrogenesis indicates an additional function in gene regulation of SMC marker genes maybe together with SRF as described by Chang et al. [1].

The de novo evolution of genes and the novel proteins they encode has stimulated much interest in the contribution such innovations make to the diversity of life. Most research on this de novo evolution focuses on transcripts, so studies on the biochemical steps that can enable completely new proteins to evolve and the time required to do so have been lacking. Sunflower Preproalbumin with SFTI-1 (PawS1) is an unusual albumin precursor because in addition to producing albumin it also yields a potent, bicyclic protease-inhibitor called SunFlower Trypsin Inhibitor-1 (SFTI-1). Here, we show how this inhibitor peptide evolved stepwise over tens of millions of years. To trace the origin of the inhibitor peptide SFTI-1, we assembled seed transcriptomes for 110 sunflower relatives whose evolution could be resolved by a chronogram, which allowed dates to be estimated for the various stages of molecular evolution. A genetic insertion event in an albumin precursor gene ∼45 Ma introduced two additional cleavage sites for protein maturation and conferred duality upon PawS1-Like genes such that they also encode a small buried macrocycle. Expansion of this region, including two Cys residues, enlarged the peptide ∼34 Ma and made the buried peptides bicyclic. Functional specialization into a protease inhibitor occurred ∼23 Ma. These findings document the evolution of a novel peptide inside a benign region of a pre-existing protein. We illustrate how a novel peptide can evolve without de novo gene evolution and, critically, without affecting the function of what becomes the protein host.

Summary The rate with which crop yields per hectare increase each year is plateauing at the same time that human population growth and other factors increase food demand. Increasing yield potential () of crops is vital to address these challenges. In this review, we explore a component of that has yet to be optimised – that being improvements in the efficiency with which light energy is converted into biomass () via modifications to CO 2 fixed per unit quantum of light ( α ), efficiency of respiratory ATP production () and efficiency of ATP use (). For α , targets include changes in photoprotective machinery, ribulose bisphosphate carboxylase/oxygenase kinetics and photorespiratory pathways. There is also potential for to be increased via targeted changes to the expression of the alternative oxidase and mitochondrial uncoupling pathways. Similarly, there are possibilities to improve via changes to the ATP costs of phloem loading, nutrient uptake, futile cycles and/or protein/membrane turnover. Recently developed high‐throughput measurements of respiration can serve as a proxy for the cumulative energy cost of these processes. There are thus exciting opportunities to use our growing knowledge of factors influencing the efficiency of photosynthesis and respiration to create a step‐change in yield potential of globally important crops.

Measurements of respiratory properties have often been made at a single time point either during daytime using dark-adapted leaves or during nighttime. The influence of the day-night cycle on respiratory metabolism has received less attention but is crucial to understand photosynthesis and photorespiration. Here, we examined how CO2- and O2-based rates of leaf dark respiration (Rdark) differed between midday (after 30-min dark adaptation) and midnight in 8 C3 and C4 grasses. We used these data to calculate the respiratory quotient (RQ; ratio of CO2 release to O2 uptake), and assessed relationships between Rdark and leaf metabolome. Rdark was higher at midday than midnight, especially in C4 species. The day-night difference in Rdark was more evident when expressed on a CO2 than O2 basis, with the RQ being higher at midday than midnight in all species, except in rice (Oryza sativa). Metabolomic analyses showed little correlation of Rdark or RQ with leaf carbohydrates (sucrose, glucose, fructose, or starch) but strong multivariate relationships with other metabolites. The results suggest that rates of Rdark and differences in RQ were determined by several concurrent CO2-producing and O2-consuming metabolic pathways, not only the tricarboxylic acid cycle (organic acids utilization) but also the pentose phosphate pathway, galactose metabolism, and secondary metabolism. As such, Rdark was time-, type- (C3/C4) and species-dependent, due to the use of different substrates.

A new family of small plant peptides was recently described and found to be widespread throughout the Millereae and Heliantheae tribes of the sunflower family Asteraceae. These peptides originate from the post-translational processing of unusual seed-storage albumin genes, and have been termed PawS-derived peptides (PDPs). The prototypic family member is a 14-residue cyclic peptide with potent trypsin inhibitory activity named SunFlower Trypsin Inhibitor (SFTI-1). In this study we present the features of three new PDPs discovered in the seeds of the sunflower species Zinnia haageana by a combination of de novo transcriptomics and liquid chromatography-mass spectrometry. Two-dimensional solution NMR spectroscopy was used to elucidate their structural characteristics. All three Z. haageana peptides have well-defined folds with a head-to-tail cyclized peptide backbone and a single disulfide bond. Although two possess an anti-parallel β-sheet structure, like SFTI-1, the Z. haageana peptide PDP-21 has a more irregular backbone structure. Despite structural similarities with SFTI-1, PDP-20 was not able to inhibit trypsin, thus the functional roles of these peptides is yet to be discovered. Defining the structural features of the small cyclic peptides found in the sunflower family will be useful for guiding the exploitation of these peptides as scaffolds for grafting and protein engineering applications.

Das Gen des Leptinrezeptors liegt innerhalb des Chromosomenlokus 1p31. Ein Punktmutationspolymorphismus führt zu einem Aminosäureaustausch (Glutamin [Gln] > Arginin [Arg] an Position 223, die mit einem erhöhten Risiko der Insulinresistenz und der Fettsucht assoziiert ist. Jüngste Ergebnisse zeigen, dass die Aktivierung des Leptin-Signalweges fibrogene Mechanismen bei experimenteller toxischer Hepatitis stimuliert. Während von Leptinsignalwegen eine profibrogene Wirkung ausgeht, wirkt der nukleare Rezeptor PPARγ, der ebenfalls wie der Leptinrezeptor von Hepatischen Sternzellen synthetisiert wird und den Fettstoffwechsel beeinflusst, antifibrogen. Die Prolin-Alanin Mutation an Codon 12 (Pro12Ala) führt zu einer eingeschränkten Funktion des PPARγ2. Daher wird postuliert, dass die Punktmutationen in den Genen des Leptinrezeptors und des PPARγ2 sich nicht nur auf das metabolische Syndrom auswirken, sondern zusätzlich die Fibroseprogression bei chronischer Hepatitis C beeinflussen können.

The liver is the central organ of metabolism and at the same time constitutes the biggest gland of the mammalian organism. Breakdown of liver function after chronic damage caused by viral infection or toxic substances implicates severe consequences for the organism. Research on the causes and the pathogenesis of liver diseases therefore is of major importance. This work adresses two questions in relation to liver fibrosis. The first part of this work deals with the expression of EGFP as a transgenic hepatocyte specific surface marker, by means of which hepatic precursors can be isolated from in vitro differentiated murine embryonic stem cells to generate functional cells for toxicological screenings. After validation of the isolation method (MACS), a reporter construct with an albumin promotor driven expression of the surface-/selection-marker was cloned and the expression specificity was analysed in vitro and in vivo. The functional expression of the transgene and its correct subcellular localization was confirmed using the hepatoma cell line HEPA-1-6. However, transfected ES cells showed basal, albumin independent expression of the transgene and even after in vitro differentiation into hepatocyte like cells did not express sufficient amounts of EGFP protein. Analysis of a transgenic mouse model generated by oocyte injection with the same reporter construct revealed that even under in vivo conditions only marginal amounts of EGFP were produced in the liver. Using a targeting strategy which aimed to knock in the EGFP-selection marker into the mouse albumin locus did not lead to the identification of positive ES cell clones. In the second part of this work, the influence of the latent transforming growth factor beta binding protein 1 (LTBP1) on the „activation“ of hepatic stellate cells (HSC) was analysed. Following „activation“ HSC transdifferentiate towards a myofibroblastic phenotype and constitute the most important fibrogenic cell type during the pathogenesis of liver fibrosis. Using a ltbp1-deficient mouse model in which both known protein isoforms LTBP1-L and LTBP1-S are missing, revealed a new ltbp1 splice variant of which analogous isoforms also exist in rats and humans. Microarray analysis was used to identify known and so far unknown marker genes for activated HSC and more importantly demonstrated that ltbp1-deficient HSC have a reduced tendency to transdifferentiate in vitro. This observation was independently confirmed in vivo using an experimental model for the induction of liver fibrosis (ligation of the common bile duct). After 4 weeks of bile duct obstruction, ltbp1-/- mice showed markedly reduced signs of liver fibrosis, i.e. less Collagen I deposition by HSC in the affected liver parenchyma. The study therefore provides insight into the role of LTBP1 during the pathogenesis of liver fibrosis and points out the clinical relevance of LTBP1, which needs to be confirmed by subsequent analysis.

Oil palm is one of the most productive oil bearing crops grown in Southeast Asia. Due to the dwindling availability of agricultural land and increasing demand for high yielding oil palm seedlings, clonal propagation is vital to the oil palm industry. Most commonly, leaf explants are used for in vitro micropropagation of oil palm and to optimize this process it is important to unravel the physiological and molecular mechanisms underlying somatic embryo production from leaves. In this study, a proteomic approach was used to determine protein abundance of mature oil palm leaves. To do this, leaf proteins were extracted using TCA/acetone precipitation protocol and separated by 2DE. A total of 191 protein spots were observed on the 2D gels and 67 of the most abundant protein spots that were consistently observed were selected for further analysis with 35 successfully identified using MALDI TOF/TOF MS. The majority of proteins were classified as being involved in photosynthesis, metabolism, cellular biogenesis, stress response, and transport. This study provides the first proteomic assessment of oil palm leaves in this important oil crop and demonstrates the successful identification of selected proteins spots using the Malaysian Palm Oil Board (MPOB) Elaeis guineensis EST and NCBI-protein databases. The MS data have been deposited in the ProteomeXchange Consortium database with the data set identifier PXD001307.

Aims: Liver fibrosis and cirrhosis are a common consequence of the majority of chronic liver insults and represent a common and difficult clinical challenge of worldwide importance. Significant morbidity and mortality are associated with the development of liver fibrosis, and particularly cirrhosis. Thus, there is a considerable imperative to develop antifibrotic strategies that are applicable to liver fibrosis [1]. Biodegradable Poly(D,L-lactic-co-glycolic acid) (PLGA)-nanospheres were demonstrated to be a potent deliverer for peptides, proteins or DNA. They protect the embedded devices from enzymatic degradation and release them controlled during a longer time period [2]. Our aim is to administer antifibrotic devices by biodegradable nanospheres .