RESUMO
Legume nodules express multiple leghemoglobins (Lbs) and non-symbiotic hemoglobins (Glbs), but how they are regulated is unclear. Here, we study the regulation of all Lbs and Glbs of Lotus japonicus in different physiologically relevant conditions and mutant backgrounds. We quantified hemoglobin expression, localized reactive oxygen species (ROS) and nitric oxide (NO) in nodules, and deployed mutants deficient in Lbs and in the transcription factors NLP4 (associated with nitrate sensitivity) and NAC094 (associated with senescence). Expression of Lbs and class 2 Glbs was suppressed by nitrate, whereas expression of class 1 and 3 Glbs was positively correlated with external nitrate concentrations. Nitrate-responsive elements were found in the promoters of several hemoglobin genes. Mutant nodules without Lbs showed accumulation of ROS and NO and alterations of antioxidants and senescence markers. NO accumulation occurred by a nitrate-independent pathway, probably due to the virtual disappearance of Glb1-1 and the deficiency of Lbs. We conclude that hemoglobins are regulated in a gene-specific manner during nodule development and in response to nitrate and dark stress. Mutant analyses reveal that nodules lacking Lbs experience nitro-oxidative stress and that there is compensation of expression between Lb1 and Lb2. They also show modulation of hemoglobin expression by NLP4 and NAC094.
Assuntos
Lotus , Nitratos , Nitratos/metabolismo , Lotus/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Hemoglobinas/genética , Hemoglobinas/metabolismo , Leghemoglobina/metabolismo , Óxido Nítrico/metabolismo , Simbiose , Nódulos Radiculares de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
Human mitochondria contain a molybdoprotein capable of reducing amidoximes using cytochrome b5/cytochrome b5 reductase (Cb/CbR). This 'amidoxime reducing component' (ARC) also reduces nitrite to nitric oxide (NO). In the plant kingdom, distinct functions have been suggested for ARCs. Thus, the single ARC of Chlamydomonas reinhardtii (crARC) reduces nitrite to NO by taking electrons from nitrate reductase (NR). Therefore, it was proposed that a dual NR/crARC system can generate NO under physiological conditions and the crARC was renamed to 'NO-forming nitrite reductase' (NOFNiR). In contrast to this, the two ARC enzymes from Arabidopsis thaliana were not found to produce NO in vitro at physiological nitrite concentrations, suggesting a different, as yet unknown, function in vascular plants. Here, we have investigated the two ARCs of Lotus japonicus (LjARCs) to shed light on this controversy and to examine, for the first time, the distribution of ARCs in plant tissues. The LjARCs are localized in the cytosol and their activities and catalytic efficiencies, which are much higher than those of A. thaliana, are consistent with a role as NOFNiR. LjARCs are prone to S-nitrosylation in vitro by S-nitrosoglutathione and this post-translational modification drastically inhibits their activities. The enzymes are mainly expressed in flowers, seeds and pods, but are absent in nodules. LjARCs are active with NR and Cb/CbR as electron-transferring systems. However, the LjNR mRNA levels in seeds and pods are negligible, whereas our proteomic analyses show that pods contain the two ARCs, Cb and CbR. We conclude that LjARCs may play a role as NOFNiR by receiving electrons from the Cb/CbR system but do not act in combination with NR.
Assuntos
Lotus , Óxido Nítrico , Nitrito Redutases , Proteínas de Plantas , Lotus/genética , Lotus/enzimologia , Lotus/metabolismo , Óxido Nítrico/metabolismo , Nitrito Redutases/metabolismo , Nitrito Redutases/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Regulação da Expressão Gênica de Plantas , Nitritos/metabolismo , OxirreduçãoRESUMO
Legume nodules produce large quantities of heme required for the synthesis of leghemoglobin (Lb) and other hemoproteins. Despite the crucial function of Lb in nitrogen fixation and the toxicity of free heme, the mechanisms of heme homeostasis remain elusive. Biochemical, cellular, and genetic approaches were used to study the role of heme oxygenases (HOs) in heme degradation in the model legume Lotus japonicus. Heme and biliverdin were quantified and localized, HOs were characterized, and knockout LORE1 and CRISPR/Cas9 mutants for LjHO1 were generated and phenotyped. We show that LjHO1, but not the LjHO2 isoform, is responsible for heme catabolism in nodules and identify biliverdin as the in vivo product of the enzyme in senescing green nodules. Spatiotemporal expression analysis revealed that LjHO1 expression and biliverdin production are restricted to the plastids of uninfected interstitial cells. The nodules of ho1 mutants showed decreased nitrogen fixation, and the development of brown, rather than green, nodules during senescence. Increased superoxide production was observed in ho1 nodules, underscoring the importance of LjHO1 in antioxidant defense. We conclude that LjHO1 plays an essential role in degradation of Lb heme, uncovering a novel function of nodule plastids and uninfected interstitial cells in nitrogen fixation.
Assuntos
Lotus , Fixação de Nitrogênio , Fixação de Nitrogênio/genética , Lotus/metabolismo , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase (Desciclizante)/metabolismo , Biliverdina/metabolismo , Leghemoglobina/genética , Simbiose/genética , Nódulos Radiculares de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
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.
Assuntos
Lotus , Medicago truncatula , Hemoglobinas/genética , Leghemoglobina , Lotus/genética , SimbioseRESUMO
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.
Assuntos
Heme/química , Hemoglobinas/química , Medicago truncatula/química , Proteínas de Plantas/química , Histidina/química , Ligação ProteicaRESUMO
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.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Cloroplastos/metabolismo , Citoplasma/metabolismo , Hemoglobinas/genética , Óxido Nítrico/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , Hemoglobinas/metabolismo , Lotus/genética , Lotus/metabolismo , Microscopia Imunoeletrônica , Estômatos de Plantas/genética , Estômatos de Plantas/metabolismo , Estômatos de Plantas/ultraestrutura , Plantas Geneticamente Modificadas , Ligação Proteica , Transdução de SinaisRESUMO
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.
Assuntos
Lotus , Rhizobium , Hemoglobinas/metabolismo , Lotus/metabolismo , Fixação de Nitrogênio , Filogenia , Nódulos Radiculares de Plantas/metabolismo , SimbioseRESUMO
Nitric oxide (NO) is a signaling molecule with multiple functions in plants. Given its critical importance and reactivity as a gaseous free radical, we have examined NO production in legume nodules using electron paramagnetic resonance (EPR) spectroscopy and the specific fluorescent dye 4,5-diaminofluorescein diacetate. Also, in this context, we critically assess previous and current views of NO production and detection in nodules. EPR of intact nodules revealed that nitrosyl-leghemoglobin (Lb2+NO) was absent from bean or soybean nodules regardless of nitrate supply, but accumulated in soybean nodules treated with nitrate that were defective in nitrite or nitric oxide reductases or that were exposed to ambient temperature. Consequently, bacteroids are a major source of NO, denitrification enzymes are required for NO homeostasis, and Lb2+NO is not responsible for the inhibition of nitrogen fixation by nitrate. Further, we noted that Lb2+NO is artifactually generated in nodule extracts or in intact nodules not analyzed immediately after detachment. The fluorescent probe detected NO formation in bean and soybean nodule infected cells and in soybean nodule parenchyma. The NO signal was slightly decreased by inhibitors of nitrate reductase but not by those of nitric oxide synthase, which could indicate a minor contribution of plant nitrate reductase and supports the existence of nitrate- and arginine-independent pathways for NO production. Together, our data indicate that EPR and fluorometric methods are complementary to draw reliable conclusions about NO production in plants.
Assuntos
Fabaceae/metabolismo , Leghemoglobina/metabolismo , Óxido Nítrico/metabolismo , Fixação de Nitrogênio , Espectroscopia de Ressonância de Spin Eletrônica , Corantes Fluorescentes , Nódulos Radiculares de Plantas/metabolismo , SimbioseRESUMO
Protein tyrosine (Tyr) nitration is a post-translational modification yielding 3-nitrotyrosine (NO2 -Tyr). Formation of NO2 -Tyr is generally considered as a marker of nitro-oxidative stress and is involved in some human pathophysiological disorders, but has been poorly studied in plants. Leghemoglobin (Lb) is an abundant hemeprotein of legume nodules that plays an essential role as an O2 transporter. Liquid chromatography coupled to tandem mass spectrometry was used for a targeted search and quantification of NO2 -Tyr in Lb. For all Lbs examined, Tyr30, located in the distal heme pocket, is the major target of nitration. Lower amounts were found for NO2 -Tyr25 and NO2 -Tyr133. Nitrated Lb and other as yet unidentified nitrated proteins were also detected in nodules of plants not receiving NO3- and were found to decrease during senescence. This demonstrates formation of nitric oxide (ËNO) and NO2- by alternative means to nitrate reductase, probably via a ËNO synthase-like enzyme, and strongly suggests that nitrated proteins perform biological functions and are not merely metabolic byproducts. In vitro assays with purified Lb revealed that Tyr nitration requires NO2- + H2 O2 and that peroxynitrite is not an efficient inducer of nitration, probably because Lb isomerizes it to NO3-. Nitrated Lb is formed via oxoferryl Lb, which generates nitrogen dioxide and tyrosyl radicals. This mechanism is distinctly different from that involved in heme nitration. Formation of NO2 -Tyr in Lb is a consequence of active metabolism in functional nodules, where Lb may act as a sink of toxic peroxynitrite and may play a protective role in the symbiosis.
Assuntos
Glycine max/metabolismo , Leghemoglobina/metabolismo , Óxido Nítrico/metabolismo , Phaseolus/metabolismo , Processamento de Proteína Pós-Traducional , Tirosina/metabolismo , Heme/metabolismo , Peróxido de Hidrogênio/metabolismo , Nitratos/metabolismo , Nitritos/metabolismo , Dióxido de Nitrogênio/metabolismo , Estresse Oxidativo/genética , Ácido Peroxinitroso/metabolismo , Glycine max/genética , Tirosina/análogos & derivadosRESUMO
Globins constitute a superfamily of proteins widespread in all kingdoms of life, where they fulfill multiple functions, such as efficient O(2) transport and modulation of nitric oxide bioactivity. In plants, the most abundant Hbs are the symbiotic leghemoglobins (Lbs) that scavenge O(2) and facilitate its diffusion to the N(2)-fixing bacteroids in nodules. The biosynthesis of Lbs during nodule formation has been studied in detail, whereas little is known about the green derivatives of Lbs generated during nodule senescence. Here we characterize modified forms of Lbs, termed Lba(m), Lbc(m), and Lbd(m), of soybean nodules. These green Lbs have identical globins to the parent red Lbs but their hemes are nitrated. By combining UV-visible, MS, NMR, and resonance Raman spectroscopies with reconstitution experiments of the apoprotein with protoheme or mesoheme, we show that the nitro group is on the 4-vinyl. In vitro nitration of Lba with excess nitrite produced several isomers of nitrated heme, one of which is identical to those found in vivo. The use of antioxidants, metal chelators, and heme ligands reveals that nitration is contingent upon the binding of nitrite to heme Fe, and that the reactive nitrogen species involved derives from nitrous acid and is most probably the nitronium cation. The identification of these green Lbs provides conclusive evidence that highly oxidizing and nitrating species are produced in nodules leading to nitrosative stress. These findings are consistent with a previous report showing that the modified Lbs are more abundant in senescing nodules and have aberrant O(2) binding.
Assuntos
Fabaceae/química , Heme/química , Leghemoglobina/química , Espécies Reativas de Nitrogênio/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Espectrofotometria UltravioletaRESUMO
The heme of bacteria, plant and animal hemoglobins (Hbs) must be in the ferrous state to bind O(2) and other physiological ligands. Here we have characterized the full set of non-symbiotic (class 1 and 2) and 'truncated' (class 3) Hbs of Lotus japonicus. Class 1 Hbs are hexacoordinate, but class 2 and 3 Hbs are pentacoordinate. Three of the globins, Glb1-1, Glb2 and Glb3-1, are nodule-enhanced proteins. The O(2) affinity of Glb1-1 (50 pm) was the highest known for any Hb, and the protein may function as an O(2) scavenger. The five globins were reduced by free flavins, which transfer electrons from NAD(P)H to the heme iron under aerobic and anaerobic conditions. Class 1 Hbs were reduced at very fast rates by FAD, class 2 Hbs at slower rates by both FMN and FAD, and class 3 Hbs at intermediate rates by FMN. The members of the three globin classes were immunolocalized predominantly in the nuclei. Flavins were quantified in legume nodules and nuclei, and their concentrations were sufficient to maintain Hbs in their functional state. All Hbs, except Glb1-1, were expressed in a flavohemoglobin-deficient yeast mutant and found to confer tolerance to oxidative stress induced by methyl viologen, copper or low temperature, indicating an anti-oxidative role for the hemes. However, only Glb1-2 and Glb2 afforded protection against nitrosative stress induced by S-nitrosoglutathione. Because this compound is specifically involved in transnitrosylation reactions with thiol groups, our results suggest a contribution of the single cysteine residues of both proteins in the stress response.
Assuntos
Núcleo Celular/metabolismo , Flavinas/metabolismo , Hemoglobinas/metabolismo , Estresse Oxidativo , Proteínas de Plantas/metabolismo , Lotus/metabolismoRESUMO
⢠Aluminum (Al) toxicity is a major limiting factor of crop production on acid soils, but the implication of oxidative stress in this process is controversial. A multidisciplinary approach was used here to address this question in the forage legume Lotus corniculatus. ⢠Plants were treated with low Al concentrations in hydroponic culture, and physiological and biochemical parameters, together with semiquantitative metabolic and proteomic profiles, were determined. ⢠The exposure of plants to 10 µM Al inhibited root and leaf growth, but had no effect on the production of reactive oxygen species or lipid peroxides. By contrast, exposure to 20 µM Al elicited the production of superoxide radicals, peroxide and malondialdehyde. In response to Al, there was a progressive replacement of the superoxide dismutase isoforms in the cytosol, a loss of ascorbate and consistent changes in amino acids, sugars and associated enzymes. ⢠We conclude that oxidative stress is not a causative factor of Al toxicity. The increased contents in roots of two powerful Al chelators, malic and 2-isopropylmalic acids, together with the induction of an Al-activated malate transporter gene, strongly suggest that both organic acids are implicated in Al detoxification. The effects of Al on key proteins involved in cytoskeleton dynamics, protein turnover, transport, methylation reactions, redox control and stress responses underscore a metabolic dysfunction, which affects multiple cellular compartments, particularly in plants exposed to 20 µM Al.
Assuntos
Alumínio/toxicidade , Lotus/efeitos dos fármacos , Lotus/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Antioxidantes/metabolismo , Ácidos Carboxílicos/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas/genética , Lotus/genética , Lotus/crescimento & desenvolvimento , Metabolômica , Fenômenos Fisiológicos da Nutrição/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Isoformas de Proteínas/metabolismo , Proteoma/metabolismo , Proteômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genéticaRESUMO
In plants and other organisms, glutathione (GSH) biosynthesis is catalysed sequentially by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS). In legumes, homoglutathione (hGSH) can replace GSH and is synthesized by γECS and a specific homoglutathione synthetase (hGSHS). The subcellular localization of the enzymes was examined by electron microscopy in several legumes and gene expression was analysed in Lotus japonicus plants treated for 1-48 h with 50 µM of hormones. Immunogold localization studies revealed that γECS is confined to chloroplasts and plastids, whereas hGSHS is also in the cytosol. Addition of hormones caused differential expression of thiol synthetases in roots. After 24-48 h, abscisic and salicylic acids downregulated GSHS whereas jasmonic acid upregulated it. Cytokinins and polyamines activated GSHS but not γECS or hGSHS. Jasmonic acid elicited a coordinated response of the three genes and auxin induced both hGSHS expression and activity. Results show that the thiol biosynthetic pathway is compartmentalized in legumes. Moreover, the similar response profiles of the GSH and hGSH contents in roots of non-nodulated and nodulated plants to the various hormonal treatments indicate that thiol homeostasis is independent of the nitrogen source of the plants. The differential regulation of the three mRNA levels, hGSHS activity, and thiol contents by hormones indicates a fine control of thiol biosynthesis at multiple levels and strongly suggests that GSH and hGSH play distinct roles in plant development and stress responses.
Assuntos
Fabaceae/enzimologia , Regulação Enzimológica da Expressão Gênica , Ligases/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Compostos de Sulfidrila/metabolismo , Fabaceae/química , Fabaceae/genética , Regulação da Expressão Gênica de Plantas , Imuno-Histoquímica , Ligases/química , Ligases/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Raízes de Plantas/química , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Transporte ProteicoRESUMO
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.
RESUMO
[This corrects the article DOI: 10.3389/fpls.2020.600336.].
RESUMO
This study assesses how different phases of shoot growth underlie seasonal change in leaf and stem dry matter content (LDMC and SDMC, respectively) of 12 woody Mediterranean species. The relationship between LDMC and nonstructural carbohydrate (NSC) concentrations is also explored and the seasonal vs interspecies variability of LDMC compared. LDMC, SDMC and shoot elongation rate (SER) were measured on a monthly basis for a minimum of 12 months. Bud growth rate (BGR) and NSC concentrations were also assessed in several of the study species. LDMC and SDMC decreased during shoot elongation in spring and increased in summer, showing a significant negative correlation with SER, but were unrelated to BGR. Half of the species analysed showed a positive relationship between LDMC and NSC. Seasonal fluctuations of LDMC within species were higher than interspecies differences, and species ranking was significantly affected by the month of sampling, except during winter months. Seasonal changes in LDMC and SDMC are mainly related to shoot elongation phenology, and NSC sink-source relationships between old and growing organs can explain this relationship in some species. Owing to the high seasonal variability in LDMC, it is recommended that samples for comparative purposes should be collected as close to the winter as possible.
Assuntos
Metabolismo dos Carboidratos , Plantas/metabolismo , Estações do Ano , Desenvolvimento Vegetal , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Caules de Planta/crescimento & desenvolvimento , Caules de Planta/metabolismo , Especificidade da EspécieRESUMO
The biosynthesis of phytochelatins and homophytochelatins has been studied in nodulated plants of the model legume Lotus (Lotus japonicus). In the first 6 to 24 h of treatment with cadmium (Cd), roots started to synthesize elevated amounts of both polypeptides, with a concomitant increase of glutathione and a decrease of homoglutathione, indicating the presence of active phytochelatin synthase (PCS) genes. Screening of transformation-competent artificial chromosome libraries allowed identification of a cluster of three genes, LjPCS1, LjPCS2, and LjPCS3, which were mapped at 69.0 cM on chromosome 1. The genes differ in exon-intron composition and responsiveness to Cd. Gene structures and phylogenetic analysis of the three protein products, LjPCS1-8R, LjPCS2-7N, and LjPCS3-7N, are consistent with two sequential gene duplication events during evolution of vascular plants. Two sites for alternative splicing in the primary transcripts were identified. One of them, involving intron 2 of the LjPCS2 gene, was confirmed by the finding of the two predicted mRNAs, encoding LjPCS2-7R in roots and LjPCS2-7N in nodules. The amino acid sequences of LjPCS2-7R (or LjPCS2-7N) and LjPCS3-7N share 90% identity, but have only 43% to 59% identity with respect to the typical PCS1 enzymes of Lotus and other plants. The unusual LjPCS2-7N and LjPCS3-7N proteins conferred Cd tolerance when expressed in yeast (Saccharomyces cerevisiae) cells, whereas the alternatively spliced form, LjPCS2-7R, differing only in a five-amino acid motif (GRKWK) did not. These results unveil complex regulatory mechanisms of PCS expression in legume tissues in response to heavy metals and probably to other developmental and environmental factors.
Assuntos
Aminoaciltransferases/metabolismo , Cádmio/farmacologia , Lotus/enzimologia , Processamento Alternativo , Sequência de Aminoácidos , Aminoaciltransferases/genética , Mapeamento Cromossômico , Duplicação Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genoma de Planta , Lotus/efeitos dos fármacos , Lotus/genética , Dados de Sequência Molecular , Família Multigênica , Filogenia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Isoformas de Proteínas/metabolismo , Saccharomyces cerevisiae/genética , Alinhamento de SequênciaRESUMO
BACKGROUND AND AIMS: The objective of this study was to test whether the mean values of several root morphological variables were related to the ability to develop root-borne shoots and/or shoot-borne roots in a wide range of vascular plants. METHODS: A comparative study was carried out on the 123 most common plant species from eroded lands in north-east Spain. After careful excavations in the field, measurements were taken of the maximum root depth, absolute and relative basal root diameter, specific root length (SRL), and the root depth/root lateral spread ratio on at least three individuals per species. Shoot-rooting and root-sprouting were observed in a large number of individuals in many eroded and sedimentary environments. The effect of life history and phylogeny on shoot-rooting and root-sprouting abilities was also analysed. KEY RESULTS: The species with coarse and deep tap-roots tended to be root-sprouting and those with fine, fasciculate and long main roots (which generally spread laterally), tended to be shoot-rooting. Phylogeny had an important influence on root system morphology and shoot-rooting and root-sprouting capacities. However, the above relations stood after applying analyses based on phylogenetically independent contrasts (PICs). CONCLUSIONS: The main morphological features of the root system of the study species are related to their ability to sprout from their roots and form roots from their shoots. According to the results, such abilities might only be functionally viable in restricted root system morphologies and ecological strategies.