Leaf day respiration involves multiple carbon sources and depends on previous dark metabolism.

Day respiration (Rd) is the metabolic, nonphotorespiratory process by which illuminated leaves liberate CO2 during photosynthesis. Rd is used routinely in photosynthetic models and is thus critical for calculations. However, metabolic details associated with Rd are poorly known, and this can be problematic to predict how Rd changes with environmental conditions and relates to night respiration. It is often assumed that day respiratory CO2 release just reflects 'ordinary' catabolism (glycolysis and Krebs 'cycle'). Here, we carried out a pulse-chase experiment, whereby a 13CO2 pulse in the light was followed by a chase period in darkness and then in the light. We took advantage of nontargeted, isotope-assisted metabolomics to determine non-'ordinary' metabolism, detect carbon remobilisation and compare light and dark 13C utilisation. We found that several concurrent metabolic pathways ('ordinary' catabolism, oxidative pentose phosphates pathway, amino acid production, nucleotide biosynthesis and secondary metabolism) took place in the light and participated in net CO2 efflux associated with day respiration. Flux reconstruction from metabolomics leads to an underestimation of Rd, further suggesting the contribution of a variety of CO2-evolving processes. Also, the cornerstone of the Krebs 'cycle', citrate, is synthetised de novo from photosynthates mostly in darkness, and remobilised or synthesised from stored material in the light. Collectively, our data provides direct evidence that leaf day respiration (i) involves several CO2-producing reactions and (ii) is fed by different carbon sources, including stored carbon disconnected from current photosynthates.

d-amino acids metabolism reflects the evolutionary origin of higher plants and their adaptation to the environment.

d-amino acids are the d stereoisomers of the common l-amino acids found in proteins. Over the past two decades, the occurrence of d-amino acids in plants has been reported and circumstantial evidence for a role in various processes, including interaction with soil microorganisms or interference with cellular signalling, has been provided. However, examples are not numerous and d-amino acids can also be detrimental, some of them inhibiting growth and development. Thus, the persistence of d-amino acid metabolism in plants is rather surprising, and the evolutionary origins of d-amino acid metabolism are currently unclear. Systemic analysis of sequences associated with d-amino acid metabolism enzymes shows that they are not simply inherited from cyanobacterial metabolism. In fact, the history of plant d-amino acid metabolism enzymes likely involves multiple steps, cellular compartments, gene transfers and losses. Regardless of evolutionary steps, enzymes of d-amino acid metabolism, such as d-amino acid transferases or racemases, have been retained by higher plants and have not simply been eliminated, so it is likely that they fulfil important metabolic roles such as serine, folate or plastid peptidoglycan metabolism. We suggest that d-amino acid metabolism may have been critical to support metabolic functions required during the evolution of land plants.

Phloem Sap Composition: What Have We Learnt from Metabolomics?

Phloem sap transport is essential for plant nutrition and development since it mediates redistribution of nutrients, metabolites and signaling molecules. However, its biochemical composition is not so well-known because phloem sap sampling is difficult and does not always allow extensive chemical analysis. In the past years, efforts have been devoted to metabolomics analyses of phloem sap using either liquid chromatography or gas chromatography coupled with mass spectrometry. Phloem sap metabolomics is of importance to understand how metabolites can be exchanged between plant organs and how metabolite allocation may impact plant growth and development. Here, we provide an overview of our current knowledge of phloem sap metabolome and physiological information obtained therefrom. Although metabolomics analyses of phloem sap are still not numerous, they show that metabolites present in sap are not just sugars and amino acids but that many more metabolic pathways are represented. They further suggest that metabolite exchange between source and sink organs is a general phenomenon, offering opportunities for metabolic cycles at the whole-plant scale. Such cycles reflect metabolic interdependence of plant organs and shoot-root coordination of plant growth and development.

Carbon and Nitrogen Allocation between the Sink and Source Leaf Tissue in Response to the Excess Excitation Energy Conditions.

Plants are inevitably exposed to extreme climatic conditions that lead to a disturbed balance between the amount of absorbed energy and their ability to process it. Variegated leaves with photosynthetically active green leaf tissue (GL) and photosynthetically inactive white leaf tissue (WL) are an excellent model system to study source-sink interactions within the same leaf under the same microenvironmental conditions. We demonstrated that under excess excitation energy (EEE) conditions (high irradiance and lower temperature), regulated metabolic reprogramming in both leaf tissues allowed an increased consumption of reducing equivalents, as evidenced by preserved maximum efficiency of photosystem II (ФPSII) at the end of the experiment. GL of the EEE-treated plants employed two strategies: (i) the accumulation of flavonoid glycosides, especially cyanidin glycosides, as an alternative electron sink, and (ii) cell wall stiffening by cellulose, pectin, and lignin accumulation. On the other hand, WL increased the amount of free amino acids, mainly arginine, asparagine, branched-chain and aromatic amino acids, as well as kaempferol and quercetin glycosides. Thus, WL acts as an important energy escape valve that is required in order to maintain the successful performance of the GL sectors under EEE conditions. Finally, this role could be an adaptive value of variegation, as no consistent conclusions about its ecological benefits have been proposed so far.

Grain carbon isotope composition is a marker for allocation and harvest index in wheat.

The natural 13 C abundance (δ13 C) in plant leaves has been used for decades with great success in agronomy to monitor water-use efficiency and select modern cultivars adapted to dry conditions. However, in wheat, it is also important to find genotypes with high carbon allocation to spikes and grains, and thus with a high harvest index (HI) and/or low carbon losses via respiration. Finding isotope-based markers of carbon partitioning to grains would be extremely useful since isotope analyses are inexpensive and can be performed routinely at high throughput. Here, we took the advantage of a set of field trials made of more than 600 plots with several wheat cultivars and measured agronomic parameters as well as δ13 C values in leaves and grains. We find a linear relationship between the apparent isotope discrimination between leaves and grain (denoted as Δδcorr ), and the respiration use efficiency-to-HI ratio. It means that overall, efficient carbon allocation to grains is associated with a small isotopic difference between leaves and grains. This effect is explained by postphotosynthetic isotope fractionations, and we show that this can be modelled by equations describing the carbon isotope composition in grains along the wheat growth cycle. Our results show that 13 C natural abundance in grains could be useful to find genotypes with better carbon allocation properties and assist current wheat breeding technologies.

Nitrogen nutrition modifies the susceptibility of Arabidopsis thaliana to the necrotrophic fungus, Alternaria brassicicola.

The impact of the form of nitrogen (N) source (nitrate versus ammonium) on the susceptibility to Alternaria brassicicola, a necrotrophic fungus, has been examined in Arabidopsis thaliana at the rosette stage. Nitrate nutrition was found to increase fungal lesions considerably. There was a similar induction of defence gene expression following infection under both N nutritions, except for the phytoalexin deficient 3 gene, which was overexpressed with nitrate. Nitrate also led to a greater nitric oxide production occurring in planta during the saprophytic growth and lower nitrate reductase (NIA1) expression 7 days after inoculation. This suggests that nitrate reductase-dependent nitric oxide production had a dual role, whereby, despite its known role in the generic response to pathogens, it affected plant metabolism, and this facilitated fungal infection. In ammonium-grown plants, infection with A. brassicicola induced a stronger gene expression of ammonium transporters and significantly reduced the initially high ammonium content in the leaves. There was a significant interaction between N source and inoculation (presence versus absence of the fungus) on the total amino acid content, while N nutrition reconfigured the spectrum of major amino acids. Typically, a higher content of total amino acid, mainly due to a stronger increase in asparagine and glutamine, is observed under ammonium nutrition while, in nitrate-fed plants, glutamate was the only amino acid which content increased significantly after fungal inoculation. N nutrition thus appears to control fungal infection via a complex set of signalling and nutritional events, shedding light on how nitrate availability can modulate disease susceptibility.

Mitochondrial Complex I Disruption Causes Broad Reorchestration of Plant Lipidome Including Chloroplast Lipids.

Mitochondrial complex I (CI) plays a crucial role in oxidising NADH generated by the metabolism (including photorespiration) and thereby participates in the mitochondrial electron transfer chain feeding oxidative phosphorylation that generates ATP. However, CI mutations are not lethal in plants and cause moderate phenotypes, and therefore CI mutants are instrumental to examine consequences of mitochondrial homeostasis disturbance on plant cell metabolisms and signalling. To date, the consequences of CI disruption on the lipidome have not been examined. Yet, in principle, mitochondrial dysfunction should impact on lipid synthesis through chloroplasts (via changes in photorespiration, redox homeostasis, and N metabolism) and the endoplasmic reticulum (ER) (via perturbed mitochondrion-ER crosstalk). Here, we took advantage of lipidomics technology (by LC-MS), phospholipid quantitation by 31P-NMR, and total lipid quantitation to assess the impact of CI disruption on leaf, pollen, and seed lipids using three well-characterised CI mutants: CMSII in N. sylvestris and both ndufs4 and ndufs8 in Arabidopsis. Our results show multiple changes in cellular lipids, including galactolipids (chloroplastic), sphingolipids, and ceramides (synthesised by ER), suggesting that mitochondrial homeostasis is essential for the regulation of whole cellular lipidome via specific signalling pathways. In particular, the observed modifications in phospholipid and sphingolipid/ceramide molecular species suggest that CI activity controls phosphatidic acid-mediated signalling.

Experimental Evidence for Seed Metabolic Allometry in Barrel Medic (Medicago truncatula Gaertn.).

Seed size is often considered to be an important trait for seed quality, i.e., vigour and germination performance. It is believed that seed size reflects the quantity of reserve material and thus the C and N sources available for post-germinative processes. However, mechanisms linking seed size and quality are poorly documented. In particular, specific metabolic changes when seed size varies are not well-known. To gain insight into this aspect, we examined seed size and composition across different accessions of barrel medic (Medicago truncatula Gaertn.) from the genetic core collection. We conducted multi-elemental analyses and isotope measurements, as well as exact mass GC-MS metabolomics. There was a systematic increase in N content (+0.17% N mg-1) and a decrease in H content (-0.14% H mg-1) with seed size, reflecting lower lipid and higher S-poor protein quantity. There was also a decrease in 2H natural abundance (δ2H), due to the lower prevalence of 2H-enriched lipid hydrogen atoms that underwent isotopic exchange with water during seed development. Metabolomics showed that seed size correlates with free amino acid and hexoses content, and anticorrelates with amino acid degradation products, disaccharides, malic acid and free fatty acids. All accessions followed the same trend, with insignificant differences in metabolic properties between them. Our results show that there is no general, proportional increase in metabolite pools with seed size. Seed size appears to be determined by metabolic balance (between sugar and amino acid degradation vs. utilisation for storage), which is in turn likely determined by phloem source metabolite delivery during seed development.

Non-targeted 13 C metabolite analysis demonstrates broad re-orchestration of leaf metabolism when gas exchange conditions vary.

It is common practice to manipulate CO2 and O2 mole fraction during gas-exchange experiments to suppress or exacerbate photorespiration, or simply carry out CO2 response curves. In doing so, it is implicitly assumed that metabolic pathways other than carboxylation and oxygenation are altered minimally. In the past few years, targeted metabolic analyses have shown that this assumption is incorrect, with changes in the tricarboxylic acid cycle, anaplerosis (phosphoenolpyruvate carboxylation), and nitrogen or sulphur assimilation. However, this problem has never been tackled systematically using non-targeted analyses to embrace all possible affected metabolic pathways. Here, we exploited combined NMR, GC-MS, and LC-MS data and conducted non-targeted analyses on sunflower leaves sampled at different O2 /CO2 ratios in a gas exchange system. The statistical analysis of nearly 4,500 metabolic features not only confirms previous findings on anaplerosis or S assimilation, but also reveals significant changes in branched chain amino acids, phenylpropanoid metabolism, or adenosine turn-over. Noteworthy, all of these pathways involve CO2 assimilation or liberation and thus affect net CO2 exchange. We conclude that manipulating CO2 and O2 mole fraction has a broad effect on metabolism, and this must be taken into account to better understand variations in carboxylation (anaplerotic fixation) or apparent day respiration.

Arabidopsis seedlings display a remarkable resilience under severe mineral starvation using their metabolic plasticity to remain self-sufficient for weeks.

During the life cycle of plants, seedlings are considered vulnerable because they are at the interface between the highly stress tolerant seed embryos and the established plant, and must develop rapidly, often in a challenging environment, with limited access to nutrients and light. Using a simple experimental system, whereby the seedling stage of Arabidopsis is considerably prolonged by nutrient starvation, we analysed the physiology and metabolism of seedlings maintained in such conditions up to 4 weeks. Although development was arrested at the cotyledon stage, there was no sign of senescence and seedlings remained viable for weeks, yielding normal plants after transplantation. Photosynthetic activity compensated for respiratory carbon losses, and energy dissipation by photorespiration and alternative oxidase appeared important. Photosynthates were essentially stored as organic acids, while the pool of free amino acids remained stable. Seedlings lost the capacity to store lipids in cytosolic lipid droplets, but developed large plastoglobuli. Arabidopsis seedlings arrested in their development because of mineral starvation displayed therefore a remarkable resilience, using their metabolic and physiological plasticity to maintain a steady state for weeks, allowing resumption of development when favourable conditions ensue.

Exploiting the Genetic Diversity of Maize Using a Combined Metabolomic, Enzyme Activity Profiling, and Metabolic Modeling Approach to Link Leaf Physiology to Kernel Yield.

A combined metabolomic, biochemical, fluxomic, and metabolic modeling approach was developed using 19 genetically distant maize (Zea mays) lines from Europe and America. Considerable differences were detected between the lines when leaf metabolic profiles and activities of the main enzymes involved in primary metabolism were compared. During grain filling, the leaf metabolic composition appeared to be a reliable marker, allowing a classification matching the genetic diversity of the lines. During the same period, there was a significant correlation between the genetic distance of the lines and the activities of enzymes involved in carbon metabolism, notably glycolysis. Although large differences were observed in terms of leaf metabolic fluxes, these variations were not tightly linked to the genome structure of the lines. Both correlation studies and metabolic network analyses allowed the description of a maize ideotype with a high grain yield potential. Such an ideotype is characterized by low accumulation of soluble amino acids and carbohydrates in the leaves and high activity of enzymes involved in the C4 photosynthetic pathway and in the biosynthesis of amino acids derived from glutamate. Chlorogenates appear to be important markers that can be used to select for maize lines that produce larger kernels.

Exploring natural diversity of Medicago truncatula reveals physiotypes and loci associated with the response of seedling performance to nitrate supply.

Seedling pre-emergence is a critical phase of development for successful crop establishment because of its susceptibility to environmental conditions. In a context of reduced use of inorganic fertilizers, the genetic bases of the response of seedlings to nitrate supply received little attention. This issue is important even in legumes where nitrate absorption starts early after germination, before nodule development. Natural variation of traits characterizing seedling growth in the absence or presence of nitrate was investigated in a core collection of 192 accessions of Medicago truncatula. Plasticity indexes to the absence of nitrate were calculated. The genetic determinism of the traits was dissected by genome-wide association study (GWAS). The absence of nitrate affected seed biomass mobilization and root/shoot length ratio. However, the large range of genetic variability revealed different seedling performances within natural diversity. A principal component analysis (PCA) carried out with plasticity indexes highlighted four physiotypes of accessions differing in relationships between seedling elongation and seed biomass partitioning traits in response to the absence of nitrate. Finally, GWAS revealed 45 associations with single or combined traits corresponding to coordinates of accessions on PCA, as well as two clusters of genes encoding sugar transporters and glutathione transferases surrounding loci associated with seedling elongation traits.

Net photosynthetic CO2 assimilation: more than just CO2 and O2 reduction cycles.

Net photosynthetic assimilation in C3 plants is mostly viewed as a simple balance between CO2 fixation by Rubisco-catalyzed carboxylation and CO2 production by photorespiration (and to a lower extent, by day respiration) that can be easily manipulated during gas exchange experiments using the CO2  : O2 ratio of the environment. However, it now becomes clear that it is not so simple, because the photosynthetic response to gaseous conditions involves 'ancillary' metabolisms, even in the short-term. That is, carbon and nitrogen utilization by pathways other than the Calvin cycle and the photorespiratory cycle, as well as rapid signaling events, can influence the observed rate of net photosynthesis. The potential impact of such ancillary metabolisms is assessed as well as how it must be taken into account to avoid misinterpretation of photosynthetic CO2 response curves or low O2 effects in C3 leaves.

Contrast echocardiogram in diagnosing rare type of persistent left superior vena cava.

We report a rare case of persistent left superior vena cava (PLSVC) with direct drainage into the left atrium in a 3-yearsold boy who had been electively admitted for hypospadias repair, when he was noticed to have finger clubbing and mild hypoxia but was otherwise asymptomatic. The diagnosis of PLSVC can be made without an invasive tool as direct drainage of PLSVC into the left atrium be visualised using transthoracic echocardiography (TTE) by injecting agitated saline into the left arm.

Interventions for children with dyslexia: A review on current intervention methods.

INTRODUCTION: Dyslexia is a neurobiological impairment that primarily affects reading ability. It is commonly known as a reading disorder which is likely to be present at birth and is generally identified at pre-school level. Dyslexia is manifested through difficulties with accurate word recognition and also by poor performance in reading and writing. METHOD: The main objective of this paper is to review the various methods or treatments that are used to manage the literacy and cognitive abilities for children with dyslexia particularly in Malaysia. The articles were obtained from online databases such as PubMed, Ebscohost and Medline during the time frame of six years starting from 2000 until 2016. An initial count of 300 articles were generated but only 13 articles met the inclusive criteria. RESULTS: There are a few types of interventions such as the multisensory method, the phonological intervention, and the cognitive training method which can be used to improve literacy and cognitive deficits among children with dyslexia. In Malaysia, most of the treatments are focused on the aspects of language such as word mastery, alphabet identification and writing skills. The cognitive training were carried out to improve specific domain such as visuospatial skills, memory skills and psychomotor skills. CONCLUSION: There is yet no studies which has employed the comprehensive method of combining the intervention of cognitive functions and linguistics-literacy deficits. It is imperative that researchers in Malaysia go beyond literacy skills and take into consideration the underlying cognitive functions which contribute to the specific reading and writing difficulties of Malaysian children with dyslexia.

Nitrate transporters: an overview in legumes.

MAIN CONCLUSION: The nitrate transporters, belonging to NPF and NRT2 families, play critical roles in nitrate signaling, root growth and nodule development in legumes. Nitrate plays an essential role during plant development as nutrient and also as signal molecule, in both cases working via the activity of nitrate transporters. To date, few studies on NRT2 or NPF nitrate transporters in legumes have been reported, and most of those concern Lotus japonicus and Medicago truncatula. A molecular characterization led to the identification of 4 putative LjNRT2 and 37 putative LjNPF gene sequences in L. japonicus. In M. truncatula, the NRT2 family is composed of 3 putative members. Using the new genome annotation of M. truncatula (Mt4.0), we identified, for this review, 97 putative MtNPF sequences, including 32 new sequences relative to previous studies. Functional characterization has been published for only two MtNPF genes, encoding nitrate transporters of M. truncatula. Both transporters have a role in root system development via abscisic acid signaling: MtNPF6.8 acts as a nitrate sensor during the cell elongation of the primary root, while MtNPF1.7 contributes to the cellular organization of the root tip and nodule formation. An in silico expression study of MtNPF genes confirmed that NPF genes are expressed in nodules, as previously shown for L. japonicus, suggesting a role for the corresponding proteins in nitrate transport, or signal perception in nodules. This review summarizes our knowledge of legume nitrate transporters and discusses new roles for these proteins based on recent discoveries.

Regulation of Primary Metabolism in Response to Low Oxygen Availability as Revealed by Carbon and Nitrogen Isotope Redistribution.

Based on enzyme activity assays and metabolic responses to waterlogging of the legume Lotus japonicus, it was previously suggested that, during hypoxia, the tricarboxylic acid cycle switches to a noncyclic operation mode. Hypotheses were postulated to explain the alternative metabolic pathways involved, but as yet, a direct analysis of the relative redistribution of label through the corresponding pathways was not made. Here, we describe the use of stable isotope-labeling experiments for studying metabolism under hypoxia using wild-type roots of the crop legume soybean (Glycine max). [(13)C]Pyruvate labeling was performed to compare metabolism through the tricarboxylic acid cycle, fermentation, alanine metabolism, and the γ-aminobutyric acid shunt, while [(13)C]glutamate and [(15)N]ammonium labeling were performed to address the metabolism via glutamate to succinate. Following these labelings, the time course for the redistribution of the (13)C/(15)N label throughout the metabolic network was evaluated with gas chromatography-time of flight-mass spectrometry. Our combined labeling data suggest the inhibition of the tricarboxylic acid cycle enzyme succinate dehydrogenase, also known as complex II of the mitochondrial electron transport chain, providing support for the bifurcation of the cycle and the down-regulation of the rate of respiration measured during hypoxic stress. Moreover, up-regulation of the γ-aminobutyric acid shunt and alanine metabolism explained the accumulation of succinate and alanine during hypoxia.

Response of Dapsone in Refractory Chronic Immune Thrombocytopenia.

This was a prospective study. A total number of 19 patients with chronic ITP with platelet count <50×108/L, treated with dapsone at a dose of 100 mg/day for nine months and followed up to 12 months were included in this study. Among them 13 patients (76.5%) were responded well shortly to dapsone which persisted during Dapsone therapy. Response rate of Dapsone was declining after Dapsone therapy. Response persisted in 6 patients (35.3%) after stoppage of dapsone up to last follow up at 1 year. Mean platelets count during Dapsone therapy was 168.35×108/L. Shorter duration of thrombocypenic patients were responded well. Except one all patients experienced mild to moderate anaemia. Mean fall of Haemoglobin was 1.4gm/dl during dapsone therapy observed which come back to normal shortly after stoppage of dapsone. Only 2 patients were suffered from moderate anaemia. Dapsone syndrome and methemoglobinamia caused permanent discontinuation of therapy in 2 patients (10.5%) which was revert back to normal shortly after stoppage of drug. This study demonstrates that dapsone is an effective, inexpensive and well tolerated treatment for chronic, refractory ITP.