Arabidopsis thaliana 2,3-bisphosphoglycerate-independent phosphoglycerate mutase 2 activity requires serine 82 phosphorylation.

Phosphoglycerate mutases (PGAMs) catalyse the reversible isomerisation of 3-phosphoglycerate and 2-phosphoglycerate, a step of glycolysis. PGAMs can be sub-divided into 2,3-bisphosphoglycerate-dependent (dPGAM) and -independent (iPGAM) enzymes. In plants, phosphoglycerate isomerisation is carried out by cytosolic iPGAM. Despite its crucial role in catabolism, little is known about post-translational modifications of plant iPGAM. In Arabidopsis thaliana, phosphoproteomics analyses have previously identified an iPGAM phosphopeptide where serine 82 is phosphorylated. Here, we show that this phosphopeptide is less abundant in dark-adapted compared to illuminated Arabidopsis leaves. In silico comparison of iPGAM protein sequences and 3D structural modelling of AtiPGAM2 based on non-plant iPGAM enzymes suggest a role for phosphorylated serine in the catalytic reaction mechanism. This is confirmed by the activity (or the lack thereof) of mutated recombinant Arabidopsis iPGAM2 forms, affected in different steps of the reaction mechanism. We thus propose that the occurrence of the S82-phosphopeptide reflects iPGAM2 steady-state catalysis. Based on this assumption, the metabolic consequences of a higher iPGAM activity in illuminated versus darkened leaves are discussed.

Corrigendum: An Ethylene-Protected Achilles' Heel of Etiolated Seedlings for Arthropod Deterrence.

[This corrects the article DOI: 10.3389/fpls.2016.01246.].

The complex world of plant protease inhibitors: Insights into a Kunitz-type cysteine protease inhibitor of Arabidopsis thaliana.

Plants have evolved an intricate regulatory network of proteases and corresponding protease inhibitors (PI), which operate in various biological pathways and serve diverse spatiotemporal functions during the sedentary life of a plant. Intricacy of the regulatory network can be anticipated from the observation that, depending on the developmental stage and environmental cue(s), either a single PI or multiple PIs regulate the activity of a given protease. On the other hand, the same PI often interacts with different targets at different places, necessitating another level of fine control to be added in planta. Here, it is reported on how the activity of a papain-like cysteine protease dubbed RD21 (RESPONSIVE TO DESICCATION 21) is differentially regulated by serpin and Kunitz PIs over plant development and how this mechanism contributes to defenses against herbivorous arthropods and microbial pests.

Direct assessment of the metabolic origin of carbon atoms in glutamate from illuminated leaves using 13 C-NMR.

Glutamate (Glu) is the cornerstone of nitrogen assimilation and photorespiration in illuminated leaves. Despite this crucial role, our knowledge of the flux to Glu de novo synthesis is rather limited. Here, we used isotopic labelling with 13 CO2 and 13 C-NMR analyses to examine the labelling pattern and the appearance of multi-labelled species of Glu molecules to trace the origin of C-atoms found in Glu. We also compared this with 13 C-labelling patterns in Ala and Asp, which reflect citrate (and thus Glu) precursors, that is, pyruvate and oxaloacetate. Glu appeared to be less 13 C-labelled than Asp and Ala, showing that the Glu pool was mostly formed by 'old' carbon atoms. There were modest differences in intramolecular 13 C-13 C couplings between Glu C-2 and Asp C-3, showing that oxaloacetate metabolism to Glu biosynthesis did not involve C-atom redistribution by the Krebs cycle. The apparent carbon allocation increased with carbon net photosynthesis. However, when expressed relative to CO2 fixation, it was clearly higher at low CO2 while it did not change in 2% O2 , as compared to standard conditions. We conclude that Glu production from current photosynthetic carbon represents a small flux that is controlled by the gaseous environment, typically upregulated at low CO2 .

Serpin1 and WSCP differentially regulate the activity of the cysteine protease RD21 during plant development in Arabidopsis thaliana.

Proteolytic enzymes (proteases) participate in a vast range of physiological processes, ranging from nutrient digestion to blood coagulation, thrombosis, and beyond. In plants, proteases are implicated in host recognition and pathogen infection, induced defense (immunity), and the deterrence of insect pests. Because proteases irreversibly cleave peptide bonds of protein substrates, their activity must be tightly controlled in time and space. Here, we report an example of how nature evolved alternative mechanisms to fine-tune the activity of a cysteine protease dubbed RD21 (RESPONSIVE TO DESICCATION-21). One mechanism in the model plant Arabidopsis thaliana studied here comprises irreversible inhibition of RD21's activity by Serpin1, whereas the other mechanism is a result of the reversible inhibition of RD21 activity by a Kunitz protease inhibitor named water-soluble chlorophyll-binding protein (WSCP). Activity profiling, complex isolation, and homology modeling data revealed unique interactions of RD21 with Serpin1 and WSCP, respectively. Expression studies identified only partial overlaps in Serpin1 and WSCP accumulation that explain how RD21 contributes to the innate immunity of mature plants and arthropod deterrence of seedlings undergoing skotomorphogenesis and greening.

An Ethylene-Protected Achilles' Heel of Etiolated Seedlings for Arthropod Deterrence.

A small family of Kunitz protease inhibitors exists in Arabidopsis thaliana, a member of which (encoded by At1g72290) accomplishes highly specific roles during plant development. Arabidopsis Kunitz-protease inhibitor 1 (Kunitz-PI;1), as we dubbed this protein here, is operative as cysteine PI. Activity measurements revealed that despite the presence of the conserved Kunitz-motif the bacterially expressed Kunitz-PI;1 was unable to inhibit serine proteases such as trypsin and chymotrypsin, but very efficiently inhibited the cysteine protease RESPONSIVE TO DESICCATION 21. Western blotting and cytolocalization studies using mono-specific antibodies recalled Kunitz-PI;1 protein expression in flowers, young siliques and etiolated seedlings. In dark-grown seedlings, maximum Kunitz-PI;1 promoter activity was detected in the apical hook region and apical parts of the hypocotyls. Immunolocalization confirmed Kunitz-PI;1 expression in these organs and tissues. No transmitting tract (NTT) and HECATE 1 (HEC1), two transcription factors previously implicated in the formation of the female reproductive tract in flowers of Arabidopsis, were identified to regulate Kunitz-PI;1 expression in the dark and during greening, with NTT acting negatively and HEC1 acting positively. Laboratory feeding experiments with isopod crustaceans such as Porcellio scaber (woodlouse) and Armadillidium vulgare (pillbug) pinpointed the apical hook as ethylene-protected Achilles' heel of etiolated seedlings. Because exogenous application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and mechanical stress (wounding) strongly up-regulated HEC1-dependent Kunitz-PI;1 gene expression, our results identify a new circuit controlling herbivore deterrence of etiolated plants in which Kunitz-PI;1 is involved.

Jasmonic acid protects etiolated seedlings of Arabidopsis thaliana against herbivorous arthropods.

Seed predators can cause mass ingestion of larger seed populations. As well, herbivorous arthropods attempt to attack etiolated seedlings and chose the apical hook for ingestion, aimed at dropping the cotyledons for later consumption. Etiolated seedlings, as we show here, have established an efficient mechanism of protecting their Achilles' heel against these predators, however. Evidence is provided for a role of jasmonic acid (JA) in this largely uncharacterized plant-herbivore interaction during skotomorphogenesis and that this comprises the temporally and spatially tightly controlled synthesis of a cysteine protease inhibitors of the Kunitz family. Interestingly, the same Kunitz protease inhibitor was found to be expressed in flowers of Arabidopsis where endogenous JA levels are high for fertility. Because both the apical hook and inflorescences were preferred isopod targets in JA-deficient plants that could be rescued by exogenously administered JA, our data identify a JA-dependent mechanism of plant arthropod deterrence that is recalled in different organs and at quite different times of plant development.

Kinetic commitment in the catalysis of glutamine synthesis by GS1 from Arabidopsis using 14N/15N and solvent isotope effects.

Glutamine synthetase (GS, EC 6.3.1.2) catalyzes the production of glutamine from glutamate, ammonium and ATP. Although being essential in plants for N assimilation and recycling, kinetic commitments and transition states of the reaction have not been clearly established yet. Here, we examined 12C/13C, 14N/15N and H2O/D2O isotope effects in Arabidopsis GS1 catalysis and compared to the prokaryotic (Escherichia coli) enzyme. A14N/15N isotope effect (15V/K ≈ 1.015, with respect to substrate NH4+) was observed in the prokaryotic enzyme, indicating that ammonium utilization (deprotonation and/or amidation) was partially rate-limiting. In the plant enzyme, the isotope effect was inverse (15V/K = 0.965), suggesting that the reaction intermediate is involved in an amidation-deamidation equilibrium favoring 15N. There was no 12C/13C kinetic isotope effect (13V/K = 1.000), suggesting that the amidation step of the catalytic cycle involves a transition state with minimal alteration of overall force constants at the C-5 carbon. Surprisingly, the solvent isotope effect was found to be inverse, that is, with a higher turn-over rate in heavy water (DV ≈ 0.5), showing that restructuration of the active site due to displacement of H2O by D2O facilitates the processing of intermediates.

A Brachypodium UDP-Glycosyltransferase Confers Root Tolerance to Deoxynivalenol and Resistance to Fusarium Infection.

Fusarium head blight (FHB) is a cereal disease caused by Fusarium graminearum, a fungus able to produce type B trichothecenes on cereals, including deoxynivalenol (DON), which is harmful for humans and animals. Resistance to FHB is quantitative, and the mechanisms underlying resistance are poorly understood. Resistance has been related to the ability to conjugate DON into a glucosylated form, deoxynivalenol-3-O-glucose (D3G), by secondary metabolism UDP-glucosyltransferases (UGTs). However, functional analyses have never been performed within a single host species. Here, using the model cereal species Brachypodium distachyon, we show that the Bradi5g03300 UGT converts DON into D3G in planta. We present evidence that a mutation in Bradi5g03300 increases root sensitivity to DON and the susceptibility of spikes to F. graminearum, while overexpression confers increased root tolerance to the mycotoxin and spike resistance to the fungus. The dynamics of expression and conjugation suggest that the speed of DON conjugation rather than the increase of D3G per se is a critical factor explaining the higher resistance of the overexpressing lines. A detached glumes assay showed that overexpression but not mutation of the Bradi5g03300 gene alters primary infection by F. graminearum, highlighting the involvement of DON in early steps of infection. Together, these results indicate that early and efficient UGT-mediated conjugation of DON is necessary and sufficient to establish resistance to primary infection by F. graminearum and highlight a novel strategy to promote FHB resistance in cereals.

In vivo stoichiometry of photorespiratory metabolism.

Photorespiration is a major light-dependent metabolic pathway that consumes oxygen and produces carbon dioxide. In the metabolic step responsible for carbon dioxide production, two molecules of glycine (equivalent to two molecules of O2) are converted into one molecule of serine and one molecule of CO2. Here, we use quantitative isotopic techniques to determine the stoichiometry of this reaction in sunflower leaves, and thereby the O2/CO2 stoichiometry of photorespiration. We find that the effective O2/CO2 stoichiometric coefficient at the leaf level is very close to 2 under normal photorespiratory conditions, in line with expectations, but increases slightly at high rates of photorespiration. The net metabolic impact of this imbalance is likely to be modest.

A Kunitz-type protease inhibitor regulates programmed cell death during flower development in Arabidopsis thaliana.

Flower development and fertilization are tightly controlled in Arabidopsis thaliana. In order to permit the fertilization of a maximum amount of ovules as well as proper embryo and seed development, a subtle balance between pollen tube growth inside the transmitting tract and pollen tube exit from the septum is needed. Both processes depend on a type of programmed cell death that is still poorly understood. Here, it is shown that a Kunitz protease inhibitor related to water-soluble chlorophyll proteins of Brassicaceae (AtWSCP, encoded by At1g72290) is involved in controlling cell death during flower development in A. thaliana. Genetic, biochemical, and cell biology approaches revealed that WSCP physically interacts with RD21 (RESPONSIVE TO DESICCATION) and that this interaction in turn inhibits the activity of RD21 as a pro-death protein. The regulatory circuit identified depends on the restricted expression of WSCP in the transmitting tract and the septum epidermis. In a respective Atwscp knock-out mutant, flowers exhibited precocious cell death in the transmitting tract and unnatural death of septum epidermis cells. As a consequence, apical-basal pollen tube growth, fertilization of ovules, as well as embryo development and seed formation were perturbed. Together, the data identify a unique mechanism of cell death regulation that fine-tunes pollen tube growth.

Water-soluble chlorophyll protein is involved in herbivore resistance activation during greening of Arabidopsis thaliana.

Water-soluble chlorophyll proteins (WSCPs) constitute a small family of unusual chlorophyll (Chl)-binding proteins that possess a Kunitz-type protease inhibitor domain. In Arabidopsis thaliana, a WSCP has been identified, named AtWSCP, that forms complexes with Chl and the Chl precursor chlorophyllide (Chlide) in vitro. AtWSCP exhibits a quite unexpected expression pattern for a Chl binding protein and accumulated to high levels in the apical hook of etiolated plants. AtWSCP expression was negatively light-regulated. Transgenic expression of AtWSCP fused to green fluorescent protein (GFP) revealed that AtWSCP is localized to cell walls/apoplastic spaces. Biochemical assays identified AtWSCP as interacting with RD21 (responsive to desiccation 21), a granulin domain-containing cysteine protease implicated in stress responses and defense. Reconstitution experiments showed tight interactions between RD21 and WSCP that were relieved upon Chlide binding. Laboratory feeding experiments with two herbivorous isopod crustaceans, Porcellio scaber (woodlouse) and Armadillidium vulgare (pillbug), identified the apical hook as Achilles' heel of etiolated plants and that this was protected by RD21 during greening. Because Chlide is formed in the apical hook during seedling emergence from the soil, our data suggest an unprecedented mechanism of herbivore resistance activation that is triggered by light and involves AtWSCP.

Experimental evidence for a hydride transfer mechanism in plant glycolate oxidase catalysis.

In plants, glycolate oxidase is involved in the photorespiratory cycle, one of the major fluxes at the global scale. To clarify both the nature of the mechanism and possible differences in glycolate oxidase enzyme chemistry from C3 and C4 plant species, we analyzed kinetic parameters of purified recombinant C3 (Arabidopsis thaliana) and C4 (Zea mays) plant enzymes and compared isotope effects using natural and deuterated glycolate in either natural or deuterated solvent. The (12)C/(13)C isotope effect was also investigated for each plant glycolate oxidase protein by measuring the (13)C natural abundance in glycolate using natural or deuterated glycolate as a substrate. Our results suggest that several elemental steps were associated with an hydrogen/deuterium isotope effect and that glycolate α-deprotonation itself was only partially rate-limiting. Calculations of commitment factors from observed kinetic isotope effect values support a hydride transfer mechanism. No significant differences were seen between C3 and C4 enzymes.

Photosynthetic activity influences cellulose biosynthesis and phosphorylation of proteins involved therein in Arabidopsis leaves.

Cellulose is one of the most important organic compounds in terrestrial ecosystems and represents a major plant structural polymer. However, knowledge of the regulation of cellulose biosynthesis is still rather limited. Recent studies have shown that the phosphorylation of cellulose synthases (CESAs) may represent a key regulatory event in cellulose production. However, the impact of environmental conditions on the carbon flux of cellulose deposition and on phosphorylation levels of CESAs has not been fully elucidated. Here, we took advantage of gas exchange measurements, isotopic techniques, metabolomics, and quantitative phosphoproteomics to investigate the regulation of cellulose production in Arabidopsis rosette leaves in different photosynthetic contexts (different CO2 mole fractions) or upon light/dark transition. We show that the carbon flux to cellulose production increased with photosynthesis, but not proportionally. The phosphorylation level of several phosphopeptides associated with CESA1 and 3, and several enzymes of sugar metabolism was higher in the light and/or increased with photosynthesis. By contrast, a phosphopeptide (Ser126) associated with CESA5 seemed to be more phosphorylated in the dark. Our data suggest that photosynthetic activity affects cellulose deposition through the control of both sucrose metabolism and cellulose synthesis complexes themselves by protein phosphorylation.

Photosynthetic control of Arabidopsis leaf cytoplasmic translation initiation by protein phosphorylation.

Photosynthetic CO2 assimilation is the carbon source for plant anabolism, including amino acid production and protein synthesis. The biosynthesis of leaf proteins is known for decades to correlate with photosynthetic activity but the mechanisms controlling this effect are not documented. The cornerstone of the regulation of protein synthesis is believed to be translation initiation, which involves multiple phosphorylation events in Eukaryotes. We took advantage of phosphoproteomic methods applied to Arabidopsis thaliana rosettes harvested under controlled photosynthetic gas-exchange conditions to characterize the phosphorylation pattern of ribosomal proteins (RPs) and eukaryotic initiation factors (eIFs). The analyses detected 14 and 11 new RP and eIF phosphorylation sites, respectively, revealed significant CO2-dependent and/or light/dark phosphorylation patterns and showed concerted changes in 13 eIF phosphorylation sites and 9 ribosomal phosphorylation sites. In addition to the well-recognized role of the ribosomal small subunit protein RPS6, our data indicate the involvement of eIF3, eIF4A, eIF4B, eIF4G and eIF5 phosphorylation in controlling translation initiation when photosynthesis varies. The response of protein biosynthesis to the photosynthetic input thus appears to be the result of a complex regulation network involving both stimulating (e.g. RPS6, eIF4B phosphorylation) and inhibiting (e.g. eIF4G phosphorylation) molecular events.

A new anaplerotic respiratory pathway involving lysine biosynthesis in isocitrate dehydrogenase-deficient Arabidopsis mutants.

The cornerstone of carbon (C) and nitrogen (N) metabolic interactions - respiration - is presently not well understood in plant cells: the source of the key intermediate 2-oxoglutarate (2OG), to which reduced N is combined to yield glutamate and glutamine, remains somewhat unclear. We took advantage of combined mutations of NAD- and NADP-dependent isocitrate dehydrogenase activity and investigated the associated metabolic effects in Arabidopsis leaves (the major site of N assimilation in this genus), using metabolomics and (13)C-labelling techniques. We show that a substantial reduction in leaf isocitrate dehydrogenase activity did not lead to changes in the respiration efflux rate but respiratory metabolism was reorchestrated: 2OG production was supplemented by a metabolic bypass involving both lysine synthesis and degradation. Although the recycling of lysine has long been considered important in sustaining respiration, we show here that lysine neosynthesis itself participates in an alternative respiratory pathway. Lys metabolism thus contributes to explaining the metabolic flexibility of plant leaves and the effect (or the lack thereof) of respiratory mutations.

Short-term effects of CO(2) and O(2) on citrate metabolism in illuminated leaves.

Although there is now a considerable literature on the inhibition of leaf respiration (CO(2) evolution) by light, little is known about the effect of other environmental conditions on day respiratory metabolism. In particular, CO(2) and O(2) mole fractions are assumed to cause changes in the tricarboxylic acid pathway (TCAP) but the amplitude and even the direction of such changes are still a matter of debate. Here, we took advantage of isotopic techniques, new simple equations and instant freeze sampling to follow respiratory metabolism in illuminated cocklebur leaves (Xanthium strumarium L.) under different CO(2) /O(2) conditions. Gas exchange coupled to online isotopic analysis showed that CO(2) evolved by leaves in the light came from 'old' carbon skeletons and there was a slight decrease in (13) C natural abundance when [CO(2) ] increased. This suggested the involvement of enzymatic steps fractionating more strongly against (13) C and thus increasingly limiting for the metabolic respiratory flux as [CO(2) ] increased. Isotopic labelling with (13) C(2) -2,4-citrate lead to (13) C-enriched Glu and 2-oxoglutarate (2OG), clearly demonstrating poor metabolism of citrate by the TCAP. There was a clear relationship between the ribulose-1,5-bisphosphate oxygenation-to-carboxylation ratio (v(o) /v(c) ) and the (13) C commitment to 2OG, demonstrating that 2OG and Glu synthesis via the TCAP is positively influenced by photorespiration.

Respiratory carbon fluxes in leaves.

Leaf respiration is a major metabolic process that drives energy production and growth. Earlier works in this field were focused on the measurement of respiration rates in relation to carbohydrate content, photosynthesis, enzymatic activities or nitrogen content. Recently, several studies have shed light on the mechanisms describing the regulation of respiration in the light and in the dark and on associated metabolic flux patterns. This review will highlight advances made into characterizing respiratory fluxes and provide a discussion of metabolic respiration dynamics in relation to important biological functions.

Experimental evidence of phosphoenolpyruvate resynthesis from pyruvate in illuminated leaves.

Day respiration is the cornerstone of nitrogen assimilation since it provides carbon skeletons to primary metabolism for glutamate (Glu) and glutamine synthesis. However, recent studies have suggested that the tricarboxylic acid pathway is rate limiting and mitochondrial pyruvate dehydrogenation is partly inhibited in the light. Pyruvate may serve as a carbon source for amino acid (e.g. alanine) or fatty acid synthesis, but pyruvate metabolism is not well documented, and neither is the possible resynthesis of phosphoenolpyruvate (PEP). Here, we examined the capacity of pyruvate to convert back to PEP using (13)C and (2)H labeling in illuminated cocklebur (Xanthium strumarium) leaves. We show that the intramolecular labeling pattern in Glu, 2-oxoglutarate, and malate after (13)C-3-pyruvate feeding was consistent with (13)C redistribution from PEP via the PEP-carboxylase reaction. Furthermore, the deuterium loss in Glu after (2)H(3)-(13)C-3-pyruvate feeding suggests that conversion to PEP and back to pyruvate washed out (2)H atoms to the solvent. Our results demonstrate that in cocklebur leaves, PEP resynthesis occurred as a flux from pyruvate, approximately 0.5‰ of the net CO(2) assimilation rate. This is likely to involve pyruvate inorganic phosphate dikinase and the fundamental importance of this flux for PEP and inorganic phosphate homeostasis is discussed.