Mutations in CmVPS41 controlling resistance to cucumber mosaic virus display specific subcellular localization.

Resistance to cucumber mosaic virus (CMV) in melon (Cucumis melo L.) has been described in several exotic accessions and is controlled by a recessive resistance gene, cmv1, that encodes a vacuolar protein sorting 41 (CmVPS41). cmv1 prevents systemic infection by restricting the virus to the bundle sheath cells, preventing viral phloem entry. CmVPS41 from different resistant accessions carries two causal mutations, either a G85E change, found in Pat-81 and Freeman's cucumber, or L348R, found in PI161375, cultivar Songwhan Charmi (SC). Here, we analyzed the subcellular localization of CmVPS41 in Nicotiana benthamiana and found differential structures in resistant and susceptible accessions. Susceptible accessions showed nuclear and membrane spots and many transvacuolar strands, whereas the resistant accessions showed many intravacuolar invaginations. These specific structures colocalized with late endosomes. Artificial CmVPS41 carrying individual mutations causing resistance in the genetic background of CmVPS41 from the susceptible variety Piel de Sapo (PS) revealed that the structure most correlated with resistance was the absence of transvacuolar strands. Coexpression of CmVPS41 with viral movement proteins, the determinant of virulence, did not change these localizations; however, infiltration of CmVPS41 from either SC or PS accessions in CMV-infected N. benthamiana leaves showed a localization pattern closer to each other, with up to 30% cells showing some membrane spots in the CmVPS41SC and fewer transvacuolar strands (reduced from a mean of 4 to 1-2) with CmVPS41PS. Our results suggest that the distribution of CmVPS41PS in late endosomes includes transvacuolar strands that facilitate CMV infection and that CmVPS41 re-localizes during viral infection.

Three classes of hemoglobins are required for optimal vegetative and reproductive growth of Lotus japonicus: genetic and biochemical characterization of LjGlb2-1.

Legumes express two major types of hemoglobins, namely symbiotic (leghemoglobins) and non-symbiotic (phytoglobins), with the latter being categorized into three classes according to phylogeny and biochemistry. Using knockout mutants, we show that all three phytoglobin classes are required for optimal vegetative and reproductive development of Lotus japonicus. The mutants of two class 1 phytoglobins showed different phenotypes: Ljglb1-1 plants were smaller and had relatively more pods, whereas Ljglb1-2 plants had no distinctive vegetative phenotype and produced relatively fewer pods. Non-nodulated plants lacking LjGlb2-1 showed delayed growth and alterations in the leaf metabolome linked to amino acid processing, fermentative and respiratory pathways, and hormonal balance. The leaves of mutant plants accumulated salicylic acid and contained relatively less methyl jasmonic acid, suggesting crosstalk between LjGlb2-1 and the signaling pathways of both hormones. Based on the expression of LjGlb2-1 in leaves, the alterations of flowering and fruiting of nodulated Ljglb2-1 plants, the developmental and biochemical phenotypes of the mutant fed on ammonium nitrate, and the heme coordination and reactivity of the protein toward nitric oxide, we conclude that LjGlb2-1 is not a leghemoglobin but an unusual class 2 phytoglobin. For comparison, we have also characterized a close relative of LjGlb2-1 in Medicago truncatula, MtLb3, and conclude that this is an atypical leghemoglobin.

Unusually Fast bis-Histidyl Coordination in a Plant Hemoglobin.

The recently identified nonsymbiotic hemoglobin gene MtGlb1-2 of the legume Medicago truncatula possesses unique properties as it generates four alternative splice forms encoding proteins with one or two heme domains. Here we investigate the ligand binding kinetics of MtGlb1-2.1 and MtGlb1-2.4, bearing two hemes and one heme, respectively. Unexpectedly, the overall time-course of ligand rebinding was unusually fast. Thus, we complemented nanosecond laser flash photolysis kinetics with data collected with a hybrid femtosecond-nanosecond pump-probe setup. Most photodissociated ligands are rebound geminately within a few nanoseconds, which leads to rates of the bimolecular rebinding to pentacoordinate species in the 108 M-1s-1 range. Binding of the distal histidine to the heme competes with CO rebinding with extremely high rates (kh ~ 105 s-1). Histidine dissociation from the heme occurs with comparable rates, thus resulting in moderate equilibrium binding constants (KH ~ 1). The rate constants for ligation and deligation of distal histidine to the heme are the highest reported for any plant or vertebrate globin. The combination of microscopic rates results in unusually high overall ligand binding rate constants, a fact that contributes to explaining at the mechanistic level the extremely high reactivity of these proteins toward the physiological ligands oxygen, nitric oxide and nitrite.

Phytoglobins in the nuclei, cytoplasm and chloroplasts modulate nitric oxide signaling and interact with abscisic acid.

Symbiotic hemoglobins provide O2 to N2 -fixing bacteria within legume nodules, but the functions of non-symbiotic hemoglobins or phytoglobins (Glbs) are much less defined. Immunolabeling combined with confocal microscopy of the Glbs tagged at the C-terminus with green fluorescent protein was used to determine their subcellular localizations in Arabidopsis and Lotus japonicus. Recombinant proteins were used to examine nitric oxide (NO) scavenging in vitro and transgenic plants to show S-nitrosylation and other in vivo interactions with NO and abscisic acid (ABA) responses. We found that Glbs occur in the nuclei, chloroplasts and amyloplasts of both model plants, and also in the cytoplasm of Arabidopsis cells. The proteins show similar NO dioxygenase activities in vitro, are nitrosylated in Cys residues in vivo, and scavenge NO in the stomatal cells. The Cys/Ser mutation does not affect NO dioxygenase activity, and S-nitrosylation does not significantly consume NO. We demonstrate an interaction between Glbs and ABA on several grounds: Glb1 and Glb2 scavenge NO produced in stomatal guard cells following ABA supply; plants overexpressing Glb1 show higher constitutive expression of the ABA responsive genes Responsive to ABA (RAB18), Responsive to Dehydration (RD29A) and Highly ABA-Induced 2 (HAI2), and are more tolerant to dehydration; and ABA strongly upregulates class 1 Glbs. We conclude that Glbs modulate NO and interact with ABA in crucial physiological processes such as the plant's response to dessication.

Hemoglobins in the legume-Rhizobium symbiosis.

Legume nodules have two types of hemoglobins: symbiotic or leghemoglobins (Lbs) and nonsymbiotic or phytoglobins (Glbs). The latter are categorized into three phylogenetic classes differing in heme coordination and O2 affinity. This review is focused on the roles of Lbs and Glbs in the symbiosis of rhizobia with crop legumes and the model legumes for indeterminate (Medicago truncatula) and determinate (Lotus japonicus) nodulation. Only two hemoglobin functions are well established in nodules: Lbs deliver O2 to the bacteroids and act as O2 buffers, preventing nitrogenase inactivation; and Glb1-1 modulates nitric oxide concentration during symbiosis, from the early stage, avoiding the plant's defense response, to nodule senescence. Here, we critically examine early and recent results, update and correct the information on Lbs and Glbs with the latest genome versions, provide novel expression data and identify targets for future research. Crucial unresolved questions include the expression of multiple Lbs in nodules, their presence in the nuclei and in uninfected nodule cells, and, intriguingly, their expression in nonsymbiotic tissues. RNA-sequencing data analysis shows that Lbs are expressed as early as a few hours after inoculation and that their mRNAs are also detectable in roots and pods, which clearly suggests that these heme proteins play additional roles unrelated to nitrogen fixation. Likewise, issues awaiting investigation are the functions of other Glbs in nodules, the spatiotemporal expression profiles of Lbs and Glbs at the mRNA and protein levels, and the molecular mechanisms underlying their regulation during nodule development and in response to stress and hormones.

Genetics of nodulation in Aeschynomene evenia uncovers mechanisms of the rhizobium-legume symbiosis.

Among legumes (Fabaceae) capable of nitrogen-fixing nodulation, several Aeschynomene spp. use a unique symbiotic process that is independent of Nod factors and infection threads. They are also distinctive in developing root and stem nodules with photosynthetic bradyrhizobia. Despite the significance of these symbiotic features, their understanding remains limited. To overcome such limitations, we conduct genetic studies of nodulation in Aeschynomene evenia, supported by the development of a genome sequence for A. evenia and transcriptomic resources for 10 additional Aeschynomene spp. Comparative analysis of symbiotic genes substantiates singular mechanisms in the early and late nodulation steps. A forward genetic screen also shows that AeCRK, coding a receptor-like kinase, and the symbiotic signaling genes AePOLLUX, AeCCamK, AeCYCLOPS, AeNSP2, and AeNIN are required to trigger both root and stem nodulation. This work demonstrates the utility of the A. evenia model and provides a cornerstone to unravel mechanisms underlying the rhizobium-legume symbiosis.

A Plant Gene Encoding One-Heme and Two-Heme Hemoglobins With Extreme Reactivities Toward Diatomic Gases and Nitrite.

In plants, symbiotic hemoglobins act as carriers and buffers of O2 in nodules, whereas nonsymbiotic hemoglobins or phytoglobins (Glbs) are ubiquitous in tissues and may perform multiple, but still poorly defined, functions related to O2 and/or nitric oxide (NO). Here, we have identified a Glb gene of the model legume Medicago truncatula with unique properties. The gene, designated MtGlb1-2, generates four alternative splice forms encoding Glbs with one or two heme domains and 215-351 amino acid residues. This is more than double the size of any hemoglobin from plants or other organisms described so far. A combination of molecular, cellular, biochemical, and biophysical methods was used to characterize these novel proteins. RNA-sequencing showed that the four splice variants are expressed in plant tissues. MtGlb1-2 is transcriptionally activated by hypoxia and its expression is further enhanced by an NO source. The gene is preferentially expressed in the meristems and vascular bundles of roots and nodules. Two of the proteins, bearing one or two hemes, were characterized using mutants in the distal histidines of the hemes. The Glbs are extremely reactive toward the physiological ligands O2, NO, and nitrite. They show very high O2 affinities, NO dioxygenase activity (in the presence of O2), and nitrite reductase (NiR) activity (in the absence of O2) compared with the hemoglobins from vertebrates and other plants. We propose that these Glbs act as either NO scavengers or NO producers depending on the O2 tension in the plant tissue, being involved in the fast and fine tuning of NO concentration in the cytosol in response to sudden changes in O2 availability.

Corrigendum: A Plant Gene Encoding One-Heme and Two-Heme Hemoglobins With Extreme Reactivities Toward Diatomic Gases and Nitrite.

[This corrects the article DOI: 10.3389/fpls.2020.600336.].

Comparative neurocognitive effects of lithium and anticonvulsants in long-term stable bipolar patients.

BACKGROUND: The aim of choosing a mood-stabilizing drug (lithium or anticonvulsants) or a combination of them with minimal neurocognitive effects is to stimulate the development of criteria for a therapeutic adequacy, particularly in Bipolar Disorder (BD) patients who are clinically stabilized. METHOD: Three groups of BD patients were established according to their treatment: (i) lithium monotherapy (n=29); (ii) lithium together with one or more anticonvulsants (n=28); and (iii) one or more anticonvulsants (n=16). A group of healthy controls served as the control (n=25). The following tests were applied: Wechsler Adult Intelligence Scale, Trail Making Test, Wechsler Memory Scale, Rey Complex Figure Test, Stroop color-word test, Wisconsin Card Sorting Test, Tower of Hanoi, Frontal Assessment Battery, and Reading the Mind in the Eyes Test. RESULTS: Relative to healthy controls, BD patients showed the following: (i) those on lithium monotherapy, but not other BD groups, had preserved short-term auditory memory, long-term memory, and attention; (ii) those who took only anticonvulsants showed worse findings in short-term visual memory, working memory, and several executive functions; and (iii) all BD patients showed worse performance in processing speed, resistance to interference, and emotion recognition. LIMITATIONS: Medication alone cannot explain why all BD patients showed common cognitive deficits despite different pharmacological treatment. CONCLUSION: The impairment on some executive functions and emotion recognition is an inherent trait in BD patients, regardless of their pharmacological treatment. However, while memory, attention, and most of the executive functions are preserved in long-term stable BD patients, these cognitive functions are impaired in those who take anticonvulsants.

Morita-Baylis-Hillman reaction of lactams and lactones with alkyl halides and epoxides catalyzed by hydroxysulfides.

A new Morita-Baylis-Hillman reaction methodology in which alkyl halides and epoxides act as electrophiles in their reaction with lactones and lactams is shown. Catalysis is efficiently performed by hydroxy sulfides under basic conditions. The procedure works efficiently with many alkyl halides but fails with aldehydes with which a conventional acid-catalyzed procedure is used.