Plant hemoglobins can be maintained in functional form by reduced flavins in the nuclei and confer differential tolerance to nitro-oxidative stress

39 Pags., 1 Tabl., 5 Figs., with Supplementary Supporting Information (1 Suppl. Tabl., 5 Suppl. Figs.). The definitive version is available at: http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1365-313XThe heme of bacteria, plant and animal hemoglobins (Hbs) must be in the ferrous state to bind O2 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 O2 affinity of Glb1-1 (50 pm) was the highest known for any Hb, and the protein may function as an O2 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.This work was funded by MINECO-FEDER (grant AGL2011-24524) and Gobierno de Aragón-FSE (group A53). M.S. was supported by a predoctoral contract from Junta de Ampliación de Estudios-CSIC.Peer reviewe

Thiol synthetases of legumes: immunogold localization and differential gene regulation by phytohormones

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

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

Heme catabolism mediated by heme oxygenase in uninfected interstitial cells enables efficient symbiotic nitrogen fixation in Lotus japonicus nodules

18 Pags.- 8 Figs. © 2023 The Authors. New Phytologist.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.This work was supported by the Ministry of Science and Technology of the People’s Republic of China (2021YFA0910800), National Natural Science Foundation of China (31870220), the Foundation of Hubei Hongshan Laboratory (2022hszd014), HZAU-AGIS Cooperation Fund (SZYJY2022005), and MCIN/AEI/10.13039/ 501100011033 of Spain (grant PID2020-113985GB-I00).Peer reviewe

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

El ciclo de las xantofilas en plantas sometidas a estrés nutricional o climático

Beca de apoyo a la investigación de Ibercaja ; Beca para estancia en el extranjero de la European Science Foundation (ESF).Peer reviewe

Photosynthetic changes of "Prunus avium L" grafted on different rootstocks in relation to mineral deficiencies

ISHS Acta Horticulturae 448: III International Symposium on Mineral Nutrition of Deciduous Fruit Trees. 5 Pag., 2 Tabl., 1 Fig. The definitive version is available at: http://www.actahort.org/The performance of three different rootstock (Adara (Prunus cerasifera L.), SL 64 (Prunus mahaleb), and Colt (Prunus avium x Prunus pseudocerasus)) as nutrient suppliers has been studied for sweet cherry cultivars (Prunus avium L.). The results obtained suggested that, depending on the rootstock, the trees are submitted to different degrees of stress. According to the data obtained, it could be expected a different photosynthetic pattern of these trees for the same sweet cherry cultivar. Fluorescence measurements as well as photosynthetic pigment composition has been assessed to know the photosynthetic capacity of the trees. The highest Fv/Fm ratio was found in leaves from trees grafted over the rootstock Adara. An increase of the zeaxanthin (Z) content could be observed in the less adequate rootstock variety.Peer reviewe

Summer-drought constrains the phenology and growth of two coexisting Mediterranean oaks with contrasting leaf habit: implications for their persistence and reproduction

13 páginas, 9 figuras, 5 tablas.-- El PDF es la versión post-print.This study analyses how coexisting evergreen and deciduous oaks adjust their phenology to cope with the stressful Mediterranean summer conditions. We test the hypothesis that the vegetative and reproductive growth of the winter deciduous (Quercus faginea Lam.) is more affected by summer drought than that of the evergreen [Quercus ilex L. subsp. ballota (Desf.) Samp.]. First, we assessed the complete aboveground phenology of both species during two consecutive years. Shoot and litter production and bud, acorn and secondary growth were monitored monthly. Second, we identified several parameters affected by summer conditions: apical bud size, individual leaf area (LA), leaf mass per area (LMA) and acorn yield in both species, and leaf-fall in Q. faginea; and analysed their variation over 10 years. Q. ilex performed up to 25% of shoot growth and most leaf development during summer, whereas Q. faginea completed most of both phenophases during spring. Secondary growth was arrested in summer under drought conditions. Approximately, 30–40% of bud and 40–50% of acorn growth was undertaken during summer in both species. Summer drought related to differences in LA, LMA and leaf senescence, but not to acorn yield. Both species had similar year-to-year patterns of acorn production, though yields were always lower in Q. faginea. Bud size decreased severely in both species during extremely dry years. In Q. ilex, bud size tended to alternate between years of large and small buds, and these patterns were followed by opposite trends in stem length. In Q. faginea, bud size was more stable through time. Q. ilex was more phenologically active during summer than Q. faginea, indicating a higher tolerance to drought. Furthermore, bud and fruit growth (the only two phenophases that both species performed during summer) were more severely affected by summer drought in Q. faginea than in the evergreen. The differential effects of summer drought on key phenophases for the persistence (bud growth) and colonization ability (fruit production) of both species may have consequences for their coexistence.This study was possible thanks to the collaboration within the GLOBIMED network (Ministerio de Educación y Ciencia, Spain) and it was supported by the MEC-CICyT projects AGF96-0399, CGL2007-66066-C04/BOS and CGL2008- 04847-C02-01, DGA projects P-038/96 and GA-LC-011/2008, and INIA projects RTA2005-00100-C02-00 and SUM2006-00025-00-00. JJC acknowledges the support of the ‘‘Fundación Aragón I+D’’. SP and RM were funded by MEC by a postdoc (SEUI-FECYT) and a Juan de la Cierva contract, respectively. JA was funded by DGA.Peer reviewe

Effects of irrigation on water salinization in semi-arid environments. A case study in Las Bardenas, Spain

[ES] Se estudia la exportación de sales y sedimentos en una cuenca de regadío de ambiente semiárido en Bardenas (Zaragoza). Los resultados muestran un incremento de sedimentos en el agua de salida de cuenca respecto a la de entrada, correspondiendo mayoritariamente a sales disueltas y muy poco a material en suspensión. Durante el periodo riego la concentración de sales es más baja que en los días sin riego, debido a una mayor dilución, si bien las pérdidas totales son superiores en fase de riego en relación con un caudal de retorno mucho más elevado.[EN] In this work, we analyse the salt and sediment load from an irrigation area located in a semi-arid environment in the Ebro Valley (Bardenas, Zaragoza). The results show higher solute and sediment load in the waters exiting the irrigation area compared to the inputs waters. Changes in the suspended load are very small, and most of the mercase corresponds to soluble salts. During the period of irrigation, solute concentration is lower than during the non-irrigation periods because of higher dilution. However, the total load output during the irrigation periods is greater because the irrigation flows and returns are also higher.This study has been supported by: Water resources management in a changing envirornnent: the impact of sediment on sustainabilitv” (WARMICE, ENV 4-CT98-0789), (Comisión Europea, Dirección General XII) La identificación de fuentes de sedimento y áreas generadoras de escorrentía en relación con los cambios de uso del suelo” CICYT (REN2000-1709-C04-O1/GLO). La recuperación del espacio agrícola como estrategia de gestión integrada del territorio en áreas de montaña: El ejemplo de los Altos Valles del Aragón y del Gállego” DGA(P049/2000). Los retornos salinos del sistema de riego Bardenas 1 y su contribución a la salirilzación de las aguas superficiales” CICYT (Nat 90-1077 - C02-02). “Impacto ambiental del regadío de Bardenas 1. Fuentes de contaminación y transporte de nutrientes en los efluentes de riego CICYT (HID. 96-1306).Peer reviewe