Interplay between MYZUS PERSICAE-INDUCED LIPASE 1 and OPDA signaling in limiting green peach aphid infestation on Arabidopsis thaliana.

MYZUS PERSICAE-INDUCED LIPASE1 (MPL1) encodes a lipase in Arabidopsis thaliana that is required for limiting infestation by the green peach aphid (GPA; Myzus persicae), an important phloem sap-consuming insect pest. Previously, we demonstrated that MPL1 expression was up-regulated in response to GPA infestation, and GPA fecundity was higher on the mpl1 mutant, compared with the wild-type (WT), and lower on 35S:MPL1 plants that constitutively expressed MPL1 from the 35S promoter. Here, we show that the MPL1 promoter is active in the phloem and expression of the MPL1 coding sequence from the phloem-specific SUC2 promoter in mpl1 is sufficient to restore resistance to GPA. The GPA infestation-associated up-regulation of MPL1 requires CYCLOPHILIN 20-3 (CYP20-3), which encodes a 12-oxo-phytodienoic acid (OPDA)-binding protein that is involved in OPDA signaling, and is required for limiting GPA infestation. OPDA promotes MPL1 expression to limit GPA fecundity, a process that requires CYP20-3 function. These results along with our observation that constitutive expression of MPL1 from the 35S promoter restores resistance to GPA in the cyp20-3 mutant, and MPL1 acts in a feedback loop to limit OPDA levels in GPA-infested plants, suggest that an interplay between MPL1, OPDA, and CYP20-3 contributes to resistance to GPA.

Biotin attachment domain-containing proteins mediate hydroxy fatty acid-dependent inhibition of acetyl CoA carboxylase.

Hundreds of naturally occurring specialized fatty acids (FAs) have potential as desirable chemical feedstocks if they could be produced at large scale by crop plants; however, transgenic expression of their biosynthetic genes has generally been accompanied by dramatic reductions in oil yield. For example, expression of castor (Ricinus communis) FA hydroxylase (FAH) in the Arabidopsis thaliana FA elongation mutant fae1 resulted in a 50% reduction of FA synthesis rate that was attributed to inhibition of acetyl-CoA carboxylase (ACCase) by an undefined mechanism. Here, we tested the hypothesis that the ricinoleic acid-dependent decrease in ACCase activity is mediated by biotin attachment domain-containing (BADC) proteins. BADCs are inactive homologs of biotin carboxy carrier protein that lack a biotin cofactor and can inhibit ACCase. Arabidopsis contains three BADC genes. To reduce expression levels of BADC1 and BADC3 in fae1/FAH plants, a homozygous badc1,3/fae1/FAH line was created. The rate of FA synthesis in badc1,3/fae1/FAH seeds doubled relative to fae1/FAH, restoring it to fae1 levels, increasing both native FA and HFA accumulation. Total FA per seed, seed oil content, and seed yield per plant all increased in badc1,3/fae1/FAH, to 5.8 µg, 37%, and 162 mg, respectively, relative to 4.9 µg, 33%, and 126 mg, respectively, for fae1/FAH. Transcript levels of FA synthesis-related genes, including those encoding ACCase subunits, did not significantly differ between badc1,3/fae1/FAH and fae1/FAH. These results demonstrate that BADC1 and BADC3 mediate ricinoleic acid-dependent inhibition of FA synthesis. We propose that BADC-mediated FAS inhibition as a general mechanism that limits FA accumulation in specialized FA-accumulating seeds.

Castor Stearoyl-ACP Desaturase Can Synthesize a Vicinal Diol by Dioxygenase Chemistry.

In previous work, we identified a triple mutant of the castor (Ricinus communis) stearoyl-Acyl Carrier Protein desaturase (T117R/G188L/D280K) that, in addition to introducing a double bond into stearate to produce oleate, performed an additional round of oxidation to convert oleate to a trans allylic alcohol acid. To determine the contributions of each mutation, in this work we generated individual castor desaturase mutants carrying residue changes corresponding to those in the triple mutant and investigated their catalytic activities. We observed that T117R, and to a lesser extent D280K, accumulated a novel product, namely erythro-9,10-dihydroxystearate, that we identified via its methyl ester through gas chromatography-mass spectrometry and comparison with authentic standards. The use of 18O2 labeling showed that the oxygens of both hydroxyl moieties originate from molecular oxygen rather than water. Incubation with an equimolar mixture of 18O2 and 16O2 demonstrated that both hydroxyl oxygens originate from a single molecule of O2, proving the product is the result of dioxygenase catalysis. Using prolonged incubation, we discovered that wild-type castor desaturase is also capable of forming erythro-9,10-dihydroxystearate, which presents a likely explanation for its accumulation to ∼0.7% in castor oil, the biosynthetic origin of which had remained enigmatic for decades. In summary, the findings presented here expand the documented constellation of di-iron enzyme catalysis to include a dioxygenase reactivity in which an unactivated alkene is converted to a vicinal diol.

Seedling Chloroplast Responses Induced by N-Linolenoylethanolamine Require Intact G-Protein Complexes.

In animals, several long-chain N-acylethanolamines (NAEs) have been identified as endocannabinoids and are autocrine signals that operate through cell surface G-protein-coupled cannabinoid receptors. Despite the occurrence of NAEs in land plants, including nonvascular plants, their precise signaling properties and molecular targets are not well defined. Here we show that the activity of N-linolenoylethanolamine (NAE 18:3) requires an intact G-protein complex. Specifically, genetic ablation of the Gßγ dimer or loss of the full set of atypical Gα subunits strongly attenuates an NAE-18:3-induced degreening of cotyledons in Arabidopsis (Arabidopsis thaliana) seedlings. This effect involves, at least in part, transcriptional regulation of chlorophyll biosynthesis and catabolism genes. In addition, there is feedforward transcriptional control of G-protein signaling components and G-protein interactors. These results are consistent with NAE 18:3 being a lipid signaling molecule in plants with a requirement for G-proteins to mediate signal transduction, a situation similar, but not identical, to the action of NAE endocannabinoids in animal systems.

Trehalose 6-Phosphate Positively Regulates Fatty Acid Synthesis by Stabilizing WRINKLED1.

WRINKLED1 (WRI1), the transcriptional activator of fatty acid synthesis, was recently identified as a target of KIN10, a catalytic α-subunit of the SUCROSE-NON-FERMENTING1-RELATED PROTEIN KINASE1 (SnRK1). We tested the hypothesis that trehalose 6-phosphate (T6P), a signal of cellular sucrose status, can regulate fatty acid synthesis by inhibiting SnRK1. Incubation of Brassica napus suspension cells in medium containing T6P, or overexpression of the Escherichia coli T6P synthase, OtsA, in Nicotiana benthamiana, significantly increased T6P levels, WRI1 levels, and fatty acid synthesis rates. T6P directly bound to purified recombinant KIN10 with an equilibrium dissociation constant (K d) of 32 ± 6 µM based on microscale thermophoresis. GEMINIVIRUS REP-INTERACTING KINASE1 (GRIK1) bound to KIN10 (K d 19 ± 3 µM) and activated it by phosphorylation. In the presence of T6P, the GRIK1-KIN10 association was weakened by more than 3-fold (K d 68 ± 9.8 µM), which reduced both the phosphorylation of KIN10 and its activity. T6P-dependent inhibition of SnRK1 activity was reduced in extracts of individual Arabidopsis thaliana grik1 and grik2 mutants relative to the wild type, while SnRK1 activity in grik1 grik2 extracts was enhanced by T6P. These results indicate that the T6P sensitivity of SnRK1 in vivo is GRIK1/GRIK2 dependent. Based on our findings, we propose a mechanistic model that links sugar signaling and fatty acid homeostasis.

WRINKLED1 Regulates BIOTIN ATTACHMENT DOMAIN-CONTAINING Proteins that Inhibit Fatty Acid Synthesis.

WRINKLED1 (WRI1) is a transcriptional activator that binds to a conserved sequence (designated as AW box) boxes in the promoters of many genes from central metabolism and fatty acid (FA) synthesis, resulting in their transcription. BIOTIN ATTACHMENT DOMAIN-CONTAINING (BADC) proteins lack a biotin-attachment domain and are therefore inactive, but in the presence of excess FA, BADC1 and BADC3 are primarily responsible for the observed long-term irreversible inhibition of ACETYL-COA CARBOXYLASE, and consequently FA synthesis. Here, we tested the interaction of WRI1 with BADC genes in Arabidopsis (Arabidopsis thaliana) and found purified WRI1 bound with high affinity to canonical AW boxes from the promoters of all three BADC genes. Consistent with this observation, both expression of BADC1, BADC2, and BADC3 genes and BADC1 protein levels were reduced in wri1-1 relative to the wild type, and elevated upon WRI1 overexpression. The double mutant badc1 badc2 phenocopied wri1-1 with respect to both reduction in root length and elevation of indole-3-acetic acid-Asp levels relative to the wild type. Overexpression of BADC1 in wri1-1 decreased indole-3-acetic acid-Asp content and partially rescued its short-root phenotype, demonstrating a role for BADCs in seedling establishment. That WRI1 positively regulates genes encoding both FA synthesis and BADC proteins (i.e. conditional inhibitors of FA synthesis), represents a coordinated mechanism to achieve lipid homeostasis in which plants couple the transcription of their FA synthetic capacity with their capacity to biochemically downregulate it.

Biotin Attachment Domain-Containing Proteins Irreversibly Inhibit Acetyl CoA Carboxylase.

The first committed step in fatty acid synthesis is mediated by acetyl-CoA carboxylase (ACCase), a biotin-dependent enzyme that carboxylates acetyl-CoA to produce malonyl-CoA. ACCase can be feedback regulated by short-term or long-term exposure to fatty acids in the form of Tween 80 (predominantly containing oleic acid), which results in reversible or irreversible ACCase inhibition, respectively. Biotin attachment domain-containing (BADC) proteins are inactive analogs of biotin carboxyl transfer proteins that lack biotin, and their incorporation into ACCase down-regulates its activity by displacing active (biotin-containing) biotin carboxyltransferase protein subunits. Arabidopsis (Arabidopsis thaliana) lines containing T-DNA insertions in BADC1, BADC2, and BADC3 were used to generate badc1 badc2 and badc1 badc3 double mutants. The badc1 badc3 mutant exhibited normal growth and development; however, ACCase activity was 26% higher in badc1 badc3 and its seeds contained 30.1% more fatty acids and 32.6% more triacylgycerol relative to wild-type plants. To assess whether BADC contributes to the irreversible phase of ACCase inhibition, cell suspension cultures were generated from the leaves of badc1 badc3 and wild-type plants and treated with 10 mm Tween 80. Reversible ACCase inhibition was similar in badc1 badc3 and wild-type cultures after 2 d of Tween 80 treatment, but irreversible inhibition was reduced by 50% in badc1 badc3 relative to wild-type plants following 4 d of Tween 80 treatment. In this study, we present evidence for two important homeostatic roles for BADC proteins in down-regulating ACCase activity: by acting during normal growth and development and by contributing to its long-term irreversible feedback inhibition resulting from the oversupply of fatty acids.

Thermal acclimation in American alligators: Effects of temperature regime on growth rate, mitochondrial function, and membrane composition.

We investigated the ability of juvenile American alligators (Alligator mississippiensis) to acclimate to temperature with respect to growth rate. We hypothesized that alligators would acclimate to cold temperature by increasing the metabolic capacity of skeletal muscles and the heart. Additionally, we hypothesized that lipid membranes in the thigh muscle and liver would respond to low temperature, either to maintain fluidity (via increased unsaturation) or to maintain enzyme reaction rates (via increased docosahexaenoic acid). Alligators were assigned to one of 3 temperature regimes beginning at 9 mo of age: constant warm (30°C), constant cold (20°C), and daily cycling for 12h at each temperature. Growth rate over the following 7 mo was highest in the cycling group, which we suggest occurred via high digestive function or feeding activity during warm periods and energy-saving during cold periods. The warm group also grew faster than the cold group. Heart and liver masses were proportional to body mass, while kidney was proportionately larger in the cold group compared to the warm animals. Whole-animal metabolic rate was higher in the warm and cycling groups compared to the cold group - even when controlling for body mass - when assayed at 30°C, but not at 20°C. Mitochondrial oxidative phosphorylation capacity in permeabilized fibers of thigh muscle and heart did not differ among treatments. Membrane fatty acid composition of the brain was largely unaffected by temperature treatment, but adjustments were made in the phospholipid headgroup composition that are consistent with homeoviscous adaptation. Thigh muscle cell membranes had elevated polyunsaturated fatty acids in the cold group relative to the cycling group, but this was not the case for thigh muscle mitochondrial membranes. Liver mitochondria from cold alligators had elevated docosahexaenoic acid, which might be important for maintenance of reaction rates of membrane-bound enzymes.

Lipoxygenase-derived 9-hydro(pero)xides of linoleoylethanolamide interact with ABA signaling to arrest root development during Arabidopsis seedling establishment.

Ethanolamide-conjugated fatty acid derivatives, also known as N-acylethanolamines (NAEs), occur at low levels (µg per g) in desiccated seeds, and endogenous amounts decline rapidly with seedling growth. Linoleoylethanolamide (NAE18:2) is the most abundant of these NAEs in seeds of almost all plants, including Arabidopsis thaliana. In Arabidopsis, NAE18:2 may be oxidized by lipoxygenase (LOX) or hydrolyzed by fatty acid amide hydrolase (FAAH) during normal seedling establishment, and this contributes to the normal progression of NAE depletion that is coincident with the depletion of abscisic acid (ABA). Here we provide biochemical, genetic and pharmacological evidence that a specific 9-LOX metabolite of NAE18:2 [9-hydro(pero)xy linoleoylethanolamide (9-NAE-H(P)OD)] has a potent negative influence on seedling root elongation, and acts synergistically with ABA to modulate the transition from embryo to seedling growth. Genetic analyses using mutants in ABA synthesis (aba1 and aba2), perception (pyr1, pyl1, pyl2, pyl4, pyl5 and pyl8) or transcriptional activation (abi3-1) indicated that arrest of root growth by 9-NAE-H(P)OD requires an intact ABA signaling pathway, and probably operates to increase ABA synthesis as part of a positive feedback loop to modulate seedling establishment in response to adverse environmental conditions. These results identify a specific, bioactive ethanolamide oxylipin metabolite of NAE18:2, different from those of ethanolamide-conjugated linolenic acid (NAE18:3), as well as a molecular explanation for its inhibitory action, emphasizing the oxidative metabolism of NAEs as an important feature of seedling development.

Ethanolamide oxylipins of linolenic acid can negatively regulate Arabidopsis seedling development.

N-Acylethanolamines (NAEs) are fatty-acid derivatives with potent biological activities in a wide range of eukaryotic organisms. Polyunsaturated NAEs are among the most abundant NAE types in seeds of Arabidopsis thaliana, and they can be metabolized by either fatty acid amide hydrolase (FAAH) or by lipoxygenase (LOX) to low levels during seedling establishment. Here, we identify and quantify endogenous oxylipin metabolites of N-linolenoylethanolamine (NAE 18:3) in Arabidopsis seedlings and show that their levels were higher in faah knockout seedlings. Quantification of oxylipin metabolites in lox mutants demonstrated altered partitioning of NAE 18:3 into 9- or 13-LOX pathways, and this was especially exaggerated when exogenous NAE was added to seedlings. When maintained at micromolar concentrations, NAE 18:3 specifically induced cotyledon bleaching of light-grown seedlings within a restricted stage of development. Comprehensive oxylipin profiling together with genetic and pharmacological interference with LOX activity suggested that both 9-hydroxy and 13-hydroxy linolenoylethanolamides, but not corresponding free fatty-acid metabolites, contributed to the reversible disruption of thylakoid membranes in chloroplasts of seedling cotyledons. We suggest that NAE oxylipins of linolenic acid represent a newly identified, endogenous set of bioactive compounds that may act in opposition to progression of normal seedling development and must be depleted for successful establishment.

The α/ß hydrolase CGI-58 and peroxisomal transport protein PXA1 coregulate lipid homeostasis and signaling in Arabidopsis.

COMPARATIVE GENE IDENTIFICATION-58 (CGI-58) is a key regulator of lipid metabolism and signaling in mammals, but its underlying mechanisms are unclear. Disruption of CGI-58 in either mammals or plants results in a significant increase in triacylglycerol (TAG), suggesting that CGI-58 activity is evolutionarily conserved. However, plants lack proteins that are important for CGI-58 activity in mammals. Here, we demonstrate that CGI-58 functions by interacting with the PEROXISOMAL ABC-TRANSPORTER1 (PXA1), a protein that transports a variety of substrates into peroxisomes for their subsequent metabolism by ß-oxidation, including fatty acids and lipophilic hormone precursors of the jasmonate and auxin biosynthetic pathways. We also show that mutant cgi-58 plants display changes in jasmonate biosynthesis, auxin signaling, and lipid metabolism consistent with reduced PXA1 activity in planta and that, based on the double mutant cgi-58 pxa1, PXA1 is epistatic to CGI-58 in all of these processes. However, CGI-58 was not required for the PXA1-dependent breakdown of TAG in germinated seeds. Collectively, the results reveal that CGI-58 positively regulates many aspects of PXA1 activity in plants and that these two proteins function to coregulate lipid metabolism and signaling, particularly in nonseed vegetative tissues. Similarities and differences of CGI-58 activity in plants versus animals are discussed.

Lipidomic Analysis of Endocannabinoid Signaling: Targeted Metabolite Identification and Quantification.

The endocannabinoids N-arachidonoylethanolamide (or anandamide, AEA) and 2-arachidonoylglycerol (2-AG) belong to the larger groups of N-acylethanolamines (NAEs) and monoacylglycerol (MAG) lipid classes, respectively. They are biologically active lipid molecules that activate G-protein-coupled cannabinoid receptors found in various organisms. After AEA and 2-AG were discovered in the 1990s, they have been extensively documented to have a broad range of physiological functions. Along with AEA, several NAEs, for example, N-palmitoylethanolamine (PEA), N-stearoylethanolamine (SEA), and N-oleoylethanolamine (OEA) are also present in tissues, usually at much larger concentrations than AEA. Any perturbation that involves the endocannabinoid pathway may subsequently alter basal level or metabolism of these lipid mediators. Further, the altered levels of these molecules often reflect pathological conditions associated with tissue damage. Robust and sensitive methodologies to analyze these lipid mediators are essential to understanding how they act as endocannabinoids. The recent advances in mass spectrometry allow researchers to develop lipidomics approaches and several methodologies have been proposed to quantify endocannabinoids in various biological systems.

N-Acylethanolamines: lipid metabolites with functions in plant growth and development.

Twenty years ago, N-acylethanolamines (NAEs) were considered by many lipid chemists to be biological 'artifacts' of tissue damage, and were, at best, thought to be minor lipohilic constituents of various organisms. However, that changed dramatically in 1993, when anandamide, an NAE of arachidonic acid (N-arachidonylethanolamine), was shown to bind to the human cannabinoid receptor (CB1) and activate intracellular signal cascades in mammalian neurons. Now NAEs of various types have been identified in diverse multicellular organisms, in which they display profound biological effects. Although targets of NAEs are still being uncovered, and probably vary among eukaryotic species, there appears to be remarkable conservation of the machinery that metabolizes these bioactive fatty acid conjugates of ethanolamine. This review focuses on the metabolism and functions of NAEs in higher plants, with specific reference to the formation, hydrolysis and oxidation of these potent lipid mediators. The discussion centers mostly on early seedling growth and development, for which NAE metabolism has received the most attention, but also considers other areas of plant development in which NAE metabolism has been implicated. Where appropriate, we indicate cross-kingdom conservation in NAE metabolic pathways and metabolites, and suggest areas where opportunities for further investigation appear most pressing.

Facilitation of Fusarium graminearum Infection by 9-Lipoxygenases in Arabidopsis and Wheat.

Fusarium graminearum causes Fusarium head blight, an important disease of wheat. F. graminearum can also cause disease in Arabidopsis thaliana. Here, we show that the Arabidopsis LOX1 and LOX5 genes, which encode 9-lipoxygenases (9-LOXs), are targeted during this interaction to facilitate infection. LOX1 and LOX5 expression were upregulated in F. graminearum-inoculated plants and loss of LOX1 or LOX5 function resulted in enhanced disease resistance in the corresponding mutant plants. The enhanced resistance to F. graminearum infection in the lox1 and lox5 mutants was accompanied by more robust induction of salicylic acid (SA) accumulation and signaling and attenuation of jasmonic acid (JA) signaling in response to infection. The lox1- and lox5-conferred resistance was diminished in plants expressing the SA-degrading salicylate hydroxylase or by the application of methyl-JA. Results presented here suggest that plant 9-LOXs are engaged during infection to control the balance between SA and JA signaling to facilitate infection. Furthermore, since silencing of TaLpx-1 encoding a 9-LOX with homology to LOX1 and LOX5, resulted in enhanced resistance against F. graminearum in wheat, we suggest that 9-LOXs have a conserved role as susceptibility factors in disease caused by this important fungus in Arabidopsis and wheat.

Lipoxygenase-mediated oxidation of polyunsaturated N-acylethanolamines in Arabidopsis.

N-acylethanolamines (NAEs) are bioactive fatty acid derivatives that occur in all eukaryotes. In plants, NAEs have potent negative growth-regulating properties, and fatty acid amide hydrolase (FAAH)-mediated hydrolysis is a primary catabolic pathway that operates during seedling establishment to deplete these compounds. Alternatively, polyunsaturated (PU)-NAEs may serve as substrates for lipid oxidation. In Arabidopsis, PU-NAEs (NAE 18:2 and NAE 18:3) were the most abundant NAE species in seeds, and their levels were depleted during seedling growth even in FAAH tDNA knock-out plants. Therefore, we hypothesized that lipoxygenase (LOX) participated in the metabolism of PU-NAEs through the formation of NAE-oxylipins. Comprehensive chromatographic and mass spectrometric methods were developed to identify NAE hydroperoxides and -hydroxides. Recombinant Arabidopsis LOX enzymes expressed in Escherichia coli utilized NAE 18:2 and NAE 18:3 as substrates with AtLOX1 and AtLOX5 exhibiting 9-LOX activity and AtLOX2, AtLOX3, AtLOX4, and AtLOX6 showing predominantly 13-LOX activity. Feeding experiments with exogenous PU-NAEs showed they were converted to hydroxide metabolites indicating that indeed Arabidopsis seedlings had the capacity for LOX-mediated metabolism of PU-NAEs in planta. Detectable levels of endogenous NAE-oxylipin metabolites were identified in FAAH fatty acid amide hydrolase seedlings but not in wild-type or FAAH overexpressors, suggesting that NAE hydroxide pools normally do not accumulate unless flux through hydrolysis is substantially reduced. These data suggest that Arabidopsis LOXs indeed compete with FAAH to metabolize PU-NAEs during seedling establishment. Identification of endogenous amide-conjugated oxylipins suggests potential significance of these metabolites in vivo, and FAAH mutants may offer opportunities to address this in the future.

An expanded role for the transcription factor WRINKLED1 in the biosynthesis of triacylglycerols during seed development.

The transcription factor WRINKLED1 (WRI1) is known as a master regulator of fatty acid synthesis in developing oilseeds of Arabidopsis thaliana and other species. WRI1 is known to directly stimulate the expression of many fatty acid biosynthetic enzymes and a few targets in the lower part of the glycolytic pathway. However, it remains unclear to what extent and how the conversion of sugars into fatty acid biosynthetic precursors is controlled by WRI1. To shortlist possible gene targets for future in-planta experimental validation, here we present a strategy that combines phylogenetic foot printing of cis-regulatory elements with additional layers of evidence. Upstream regions of protein-encoding genes in A. thaliana were searched for the previously described DNA-binding consensus for WRI1, the ASML1/WRI1 (AW)-box. For about 900 genes, AW-box sites were found to be conserved across orthologous upstream regions in 11 related species of the crucifer family. For 145 select potential target genes identified this way, affinity of upstream AW-box sequences to WRI1 was assayed by Microscale Thermophoresis. This allowed definition of a refined WRI1 DNA-binding consensus. We find that known WRI1 gene targets are predictable with good confidence when upstream AW-sites are phylogenetically conserved, specifically binding WRI1 in the in vitro assay, positioned in proximity to the transcriptional start site, and if the gene is co-expressed with WRI1 during seed development. When targets predicted in this way are mapped to central metabolism, a conserved regulatory blueprint emerges that infers concerted control of contiguous pathway sections in glycolysis and fatty acid biosynthesis by WRI1. Several of the newly predicted targets are in the upper glycolysis pathway and the pentose phosphate pathway. Of these, plastidic isoforms of fructokinase (FRK3) and of phosphoglucose isomerase (PGI1) are particularly corroborated by previously reported seed phenotypes of respective null mutations.

Hepatitis C virus NS3/4A inhibitors and other drug-like compounds as covalent binders of SARS-CoV-2 main protease.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), threatens global public health. The world needs rapid development of new antivirals and vaccines to control the current pandemic and to control the spread of the variants. Among the proteins synthesized by the SARS-CoV-2 genome, main protease (Mpro also known as 3CLpro) is a primary drug target, due to its essential role in maturation of the viral polyproteins. In this study, we provide crystallographic evidence, along with some binding assay data, that three clinically approved anti hepatitis C virus drugs and two other drug-like compounds covalently bind to the Mpro Cys145 catalytic residue in the active site. Also, molecular docking studies can provide additional insight for the design of new antiviral inhibitors for SARS-CoV-2 using these drugs as lead compounds. One might consider derivatives of these lead compounds with higher affinity to the Mpro as potential COVID-19 therapeutics for further testing and possibly clinical trials.

Mobilizing Vacuolar Sugar Increases Vegetative Triacylglycerol Accumulation.

Photosynthetically derived sugars provide carbon skeletons for metabolism and carbon signals that favor anabolism. The amount of sugar available for fatty acid (FA) and triacylglycerol (TAG) synthesis depends on sugar compartmentation, transport, and demands from competing pathways. We are exploring the influence of sugar partitioning between the vacuole and cytoplasm on FA synthesis in Arabidopsis by building on our previous finding that reduced leaf sugar export in the sucrose-proton symporter2 (suc2) mutant, in combination with impaired starch synthesis in the ADP-glucose pyrophosphorylase (adg1) mutant, accumulates higher sugar levels and increased total FA and TAG compared to the wild type parent. Here we sought to relocalize sugar from the vacuole to the cytoplasm to drive additional FA/TAG synthesis and growth. Arabidopsis suc2 adg1 was therefore crossed with tonoplast monosaccharide transporter mutants tmt1 and tmt2 and overexpression of the sucrose/proton cotransporter SUC4 in which tmt1 tmt2 impairs sugar transport to the vacuole from the cytoplasm and SUC4 overexpression enhances sugar transport in the reverse direction from the vacuole to the cytoplasm. A resulting homozygous suc2 adg1 tmt1 tmt2 SUC4 line was used to test the hypothesis that increased intracellular carbon supply in the form of sugars would increase both FA and TAG accumulation. The data shows that relative to suc2 adg1, suc2 adg1 tmt1 tmt2 SUC4 significantly increases leaf total FA content by 1.29-fold to 10.9% of dry weight and TAG by 2.4-fold to 2.88%, supporting the hypothesis that mobilizing vacuolar sugar is a valid strategy for increasing vegetative oil accumulation.

The Role of Sugar Signaling in Regulating Plant Fatty Acid Synthesis.

Photosynthates such as glucose, sucrose, and some of their derivatives play dual roles as metabolic intermediates and signaling molecules that influence plant cell metabolism. Such sugars provide substrates for de novo fatty acid (FA) biosynthesis. However, compared with the well-defined examples of sugar signaling in starch and anthocyanin synthesis, until recently relatively little was known about the role of signaling in regulating FA and lipid biosynthesis. Recent research progress shows that trehalose 6-phosphate and 2-oxoglutarate (2-OG) play direct signaling roles in the regulation of FA biosynthesis by modulating transcription factor stability and enzymatic activities involved in FA biosynthesis. Specifically, mechanistic links between sucrose non-fermenting-1-related protein kinase 1 (SnRK1)-mediated trehalose 6-phosphate (T6P) sensing and its regulation by phosphorylation of WRI1 stability, diacylglycerol acyltransferase 1 (DGAT1) enzyme activity, and of 2-OG-mediated relief of inhibition of acetyl-CoA carboxylase (ACCase) activity by protein PII are exemplified in detail in this review.