Effect of elevated inorganic carbon on the cytosolic homeostasis of NO3- in the marine angiosperm Posidonia oceanica (L.) Delile

The marine angiosperm Posidonia oceanica is a mediterranean endemism of great ecological significance. As other marine plants, P. oceanica has adapted secondarily to the marine environment and develop anew different mechanisms to colonize it. Among others, this plant has developed a plasma membrane system for the direct uptake of bicarbonate. In this work we have developed both NO3- and Cl- selective microelectrodes for the continuous monitoring of the intracellular (cytosolic) NO3- and Cl-. In the light, leaf mesophyll cells show a cytosolic NO3- concentration of 5.7±0.2 mM (n=10), while in the dark cytosolic NO3- raises up to 8.7±1.1 mM; these values are in the range of concentrations quoted for Arabidopsis thaliana (Cookson et al., 2005). The enrichment of natural seawater (NSW) with 3 mM NaHCO3 caused a decrease of the cytosolic NO3- concentration of 1 mM and a decrease of the cytosolic concentration of Cl- of 3.5 mM. The saturation of NSW with 1000 µL CO2 L-1 produced a lower diminution of the cytosolic NO3- (0.3 mM). In the presence of 0.1 mM of the plasma membrane permeable inhibitor of the carbonic anhydrase (EZ) the diminution of cytosolic NO3- caused by the same concentration of CO2 was much lower, 0.1 mM. The addition of inorganic carbon, either HCO3- or CO2, has an effect on the cytosolic mechanisms for anionic homeostasis, one of which is the opening of the slow anion channels. These channels are permeable to NO3- and Cl- and could elicit the efflux of these ions. In P. oceanica, the response in the presence of EZ points out that the inorganic carbon species that cause the NO3-/Cl- efflux is HCO3-. This effect could contribute to plant biomass N dilution observed in elevated CO2. References: Cookson et al. 2005. Plant Physiology 138, 1097–1105.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

NO3- selective mini-electrodes as a tool to investigate the NO3- traffic in Chlamydomonas reinhardtii D.

Ion selective NO3- mini-electrodes were used to measure the external NO3- concentration in C. reinhardtii liquid cultures. Electrodes were prepared using glass capillaries (1.5 mm external diameter). Capillaries were cut in 10 cm long pieces, dehydrated for 45 minutes in an oven and silanized by addition of dimethyldichlorosilane in bencene 0.1% (V/V). Once silanized, the capillaries were baked again for 30 minutes. Once cold the capillaries were backfilled with the NO3- ionophore (Fluka: 72549), which contains PVC (5.75% w/w) dissolved in tetrahydrofurane. Then, the NO3- mini-electrodes were stored in dark in a desiccator until tetrahydrofurane gets evaporated. Before use, NO3- selective mini-electrodes were backfilled with 0.1 M NaNO3 and 0.1 M KCl and connected to a high-impedance differential amplifier (WPI FD223). Mini-electrodes were calibrated in N-free Beijerinck medium, which contains 0.1 mM Cl-. In those conditions, electrodes calibration slope was 54 mV/p NO3- in the range 1 - 1000 µM NO3-. The mini-electrodes were used to continuous monitoring of the external NO3- concentration in liquid culture of different C. reinhardtii strains, incubated in N-free Beijerinck medium supplemented with 100 µM NO3Na. Previous to the assays, strains were N starved for 6 days. In the light, wild type strain uptakes NO3- at a rate of 15 nmol NO3-·106 cells-1·h-1, in the dark this rate was one third of this figure. After 5 h, the external NO3- levelled off at 10 µM in the light and around 30 µM in the dark. C. reinhardtii cells cultured in the presence of 2 mM NO3NH4 do not show significant NO3- uptake nor a mutant strain, defective in nitrate transport and having an active nitrate reductase. However, a mutant strain lacking the nitrate reductase shows an enhanced NO3- uptake rate, compared with the value obtained for the wild type in the light.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

HCO3- enrichment causes cytosolic NO3- efflux in Posidonia oceanica leaf cells

Posidonia oceanica is a seagrass, the only group of vascular plants to colonize the marine environment. Seawater is an extreme yet stable environment, characterized by high salinity, alkaline pH and low availability of essential nutrients, such as nitrate or phosphate. In addition, in aquatic environments the supply of CO2 for the photosynthesis is limited by diffusion and therefore many aquatic plants use HCO3- as the inorganic carbon source for photosynthesis. Previous results have shown that Na+ -dependent transport systems operate on the plasma membrane of P. oceanica mesophyll leaf cells for the high-affinity NO3-, Pi or amino acids uptake. Also, a direct transport of HCO3- driven by H+ has been found in this species that provides inorganic carbon for photosynthesis and could be a significant component of a carbon concentrating mechanism in this species. Interestingly, this HCO3- direct uptake caused the efflux of chloride from the cytosol, probably through S-type anion channels, pointing that other anions could also be removed from the cytosol. This hypothesis could be relevant in the case of NO3-, since the decrease of cytosolic NO3- in response to HCO3- enrichment could limit N-assimilation. Here we analyse the effect of HCO3- increase on NO3- uptake and cytosolic homeostasis in P. oceanica. Enrichment of natural seawater with 3 mM HCO3- evokes the on-going decrease of cytosolic NO3-, from 5.7 ± 0.2 to 4.8 ± 0.7 mM after 40 min of treatment. The incubation of P. oceanica leaf pieces in 3 mM HCO3- NSW causes an initial increase of NO3- concentration in the medium. Maximum efflux (21 nmol NO3- gFM-1 min-1) occurs within the first minute of incubation. Then, external NO3- is depleted from the medium at lower net uptake rate than the value observed in non HCO3- -enriched natural seawater. These results fit the hypothesis that HCO3- enrichment causes the nitrogen loose and could impair nitrogen assimilation promoting N biomass impoverishment.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Spanish MINECO, projects BFU2017-85117-R and BIO2016-81957-RED

The Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis, and Stroke-like Episode Syndrome-associated Human Mitochondrial tRNALeu(UUR) Mutation Causes Aminoacylation Deficiency and Concomitant Reduced Association of mRNA with Ribosomes

The pathogenetic mechanism of the mitochondrial tRNALeu(UUR) A3243G transition associated with the mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome has been investigated in transmitochondrial cell lines constructed by transfer of mutant mitochondrial DNA (mtDNA)-carrying mitochondria from three genetically unrelated MELAS patients or of isogenic wild-type mtDNA-carrying organelles into human mtDNA-less cells. An in vivo footprinting analysis of the mtDNA segment within the tRNALeu(UUR) gene that binds the transcription termination factor failed to reveal any difference in occupancy of sites or qualitative interaction with the protein between mutant and wild-type mtDNAs. Cell lines nearly homoplasmic for the mutation exhibited a strong (70-75%) reduction in the level of aminoacylated tRNALeu(UUR) and a decrease in mitochondrial protein synthesis rate. The latter, however, did not show any significant correlation between synthesis defect of the individual polypeptides and number or proportion of UUR codons in their mRNAs, suggesting that another step, other than elongation, may be affected. Sedimentation analysis in sucrose gradient showed a reduction in size of the mitochondrial polysomes, while the distribution of the two rRNA components and of the mRNAs revealed decreased association of mRNA with ribosomes and, in the most affected cell line, pronounced degradation of the mRNA associated with slowly sedimenting structures. Therefore, several lines of evidence indicate that the protein synthesis defect in A3243G MELAS mutation-carrying cells is mainly due to a reduced association of mRNA with ribosomes, possibly as a consequence of the tRNALeu(UUR) aminoacylation defect

Na+-dependent NO3- uptake in leaf cells of the seagrass Posidonia oceanica (L.) Delile

Posidonia oceanica (L.) Delile is an endemic Mediterranean seagrass of recognized ecological significance and, as other seagrasses, this species has secondarily adapted to live in the marine environment. In this alkaline medium with a high Na+ concentration (0.5 M), the high inwardly directed electrochemical potential gradient for sodium is used in the seagrass Zostera marina to energize the uptake of nitrate1 and phosphate that usually occur at concentration below 10 µM. Here we summarize several evidences for the operation of a sodium-dependent high-affinity nitrate transport system at the plasma membrane of the mesophyll leaf cells of P. oceanica. Leaf cells of P. oceanica possess a H+-ATPase as a primary pump, exhibit a plasma membrane potential (Em) of -174 ± 10 mV and show reduced Na+ permeability. The addition of micromolar nitrate concentrations induces membrane depolarizations that show saturation kinetics. Curve fitting of the values renders a semisaturation constant (Km) of 21.3 ± 6.6 μM and a maximum depolarization (Dmax) of 7 ± 1 mV. In dark conditions, Dmax decreases by fifty percent but no significant effect is observed on the Km value. On the other hand, nitrate induced depolarizations show sodium dependence. The depolarizations induced by 100 µM NO3- in media containing increasing Na+ concentrations (from 0 to 250 mM) show saturation kinetics, rendering a Km value of 16 ± 5 mM Na+. Moreover, the depolarization induced by 100 µM NO3- is accompanied by a simultaneous increase of cytosolic sodium, measured by Na+-sensitive microelectrodes, of 0.4 ± 0.2 mM above the resting cytosolic sodium concentration (17 ± 2 mM). Finally, nitrate uptake rates, measured in depletion experiments, decreases by 50% and 80% in dark conditions and in the absence of Na+, respectively, compared with control conditions (0.5 M Na+ and light). All together, these results strongly suggest that NO3- uptake in P. oceanica leaf cells is mediated by a high-affinity nitrate carrier that uses Na+ as the driving ion. 1 Rubio et al. (2005). J. Exp. Bot, 412: 613-622. Project Funding: CTM 2011-30356. (MEC)Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

NRT2.5 a putative sodium dependent high affinity nitrate trasnporter of zostera marina l.

Seagrasses are the only group of vascular plants that recolonized the marine environment, possibly the most severe habitat shift ever accomplished by flowering plants. These plants have regained functions enabling them to thrive in liquid medium with an extremely high salinity (0.5 M Na+), high alkaline conditions (pH 8.2) and very low concentration of essential nutrients as NO3- or Pi. Despite this, seagrasses form one of the highest productive and widespread ecosystems of the planet (Larkum et al., 2006). Zostera marina was the first seagrass fully sequenced and its genome reveals important insights about this secondary adaption. Comparison with land plants indicates that less than 20 % of the genes families are specific in the genome of seagrasses. Thus, adaptation to marine environment seems to be due to molecular changes of the same family genes rather that the speciation of pre-existing genes. This appears to be the case of the high affinity nitrate transporter belonging to the NRT family. In contrast to terrestrial vascular plants, where NRT2 encode high affinity NO3- transporters that operate as H+ symporters, our electrophysiological analysis indicate that in Z. marina high affinity NO3- uptake is mediated by a Na+-dependent mechanism. A detailed analysis of the Z. marina genome indicates the presence of only one gene encoding for this type of transporter: Zosma70g00300.1. Phylogenetic analysis shows that this high affinity nitrate transporter is more related to NRT2.5 than to NTRT2.1, sharing a common ancestor with both, monocot and dicot plants. We have cloned Zosma70g00300.1 and the high-affinity nitrate transporter accessory protein NAR2 (Zosma63g00220.1) in order to characterize the specific transport mechanism mediated by these proteins in Z. marina. Thus, the putative Z. marina NRT2.5 transporter could have evolved to use Na+ as a driving ion, which might be an essential adaptation of seagrasses to colonize the marine environment.MICINN (BFU2017-85117-R; BIO2016-81957-REDT) Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

The range of the restriction map for a multiplicity variety in Hörmander algebras of entire functions

The final publication is available at Springer via http://dx.doi.org/10.1007/s00009-013-0318-5[EN] Characterizations of interpolating multiplicity varieties for Hörmander algebras Ap(C) and A0 p(C) of entire functions were obtained by Berenstein and Li (J Geom Anal 5(1):1–48, 1995) and Berenstein et al. (Can J Math 47(1):28–43, 1995) for a radial subharmonic weight p with the doubling property. In this note we consider the case when the multiplicity variety is not interpolating, we compare the range of the associated restriction map for two weights q ≤ p and investigate when the range of the restriction map on Ap(C) or A0 p(C) contains certain subspaces associated in a natural way with the smaller weight q.This research was partially supported by MEC and FEDER Project MTM2010-15200.Bonet Solves, JA.; Fernandez Rosell, C. (2014). The range of the restriction map for a multiplicity variety in Hörmander algebras of entire functions. Mediterranean Journal of Mathematics. 11(2):643-652. https://doi.org/10.1007/s00009-013-0318-5S643652112Berenstein, C.A., Gay, R.: Complex variables. An Introduction. Graduate Texts in Mathematics, vol. 125. Springer, New York (1991)Berenstein, C.A., Gay R.: Complex Analysis and Special Topics in Harmonic Analysis. Springer, New York (1995)Berenstein C.A., Li B.Q.: Interpolating varieties for spaces of meromorphic functions. J. Geom. Anal. 5(1), 1–48 (1995)Berenstein C.A., Li B.Q., Vidras A.: Geometric characterization of interpolating varieties for the (FN)-space $${A^{0}_p}$$ of entire functions. Can. J. Math. 47(1), 28–43 (1995)Berenstein C.A., Taylor B.A.: A new look at interpolation theory for entire functions of one variable. Adv. Math. 33(2), 109–143 (1979)Bonet J., Galbis A.: The range of non-surjective convolution operators on Beurling spaces. Glasg. Math. J. 38(1), 125–135 (1996)Bonet J., Galbis A., Momm S.: Nonradial Hörmander algebras of several variables and convolution operators. Trans. Am. Math. Soc. 353(6), 2275–2291 (2001)Braun R.W.: Weighted algebras of entire functions in which each closed ideal admits two algebraic generators. Mich. Math. J. 34(3), 441–450 (1987)Braun R.W., Meise R., Taylor B.A.: Ultradifferentiable functions and Fourier analysis. Results Math. 17(3–4), 206–237 (1990)Buckley J., Massaneda X., Ortega-Cerdà J.: Traces of functions in Fock spaces on lattices of critical density. Bull. Lond. Math. Soc. 44(2), 222–240 (2012)Hartmann A., Massaneda X.: On interpolating varieties for weighted spaces of entire functions. J. Geom. Anal. 10(4), 683–696 (2000)Massaneda X., Ortega-Cerdà J., Ounaïes M.: A geometric characterization of interpolation in $${\hat{\mathcal{E}}'(\mathbb{R})}$$ . Trans. Am. Math. Soc. 358, 3459–3472 (2006)Massaneda, X., Ortega-Cerdà, J., Ounaïes, M.: Traces of Hörmander algebras on discrete sequences. In: Analysis and mathematical physics. Trends in Mathematics, pp. 397–408. Birkhäuser, Basel (2009)Meise R.: Sequence space representations for (DFN)-algebras of entire functions modulo closed ideals. J. Reine Angew. Math. 363, 59–95 (1985)Meise R., Taylor B.A.: Sequence space representations for (FN)-algebras of entire functions modulo closed ideals. Studia Math. 85(3), 203–227 (1987)Meise, R., Vogt, D.: Introduction to functional analysis. Oxford Graduate Texts in Mathematics, vol. 2. The Clarendon Press Oxford University Press, New York (1997). Translated from the German by M. S. Ramanujan and revised by the authorsOunaïes M.: Geometric conditions for interpolation in weighted spaces of entire functions. J. Geom. Anal. 17(4), 701–715 (2007)Ounaïes M.: Interpolation by entire functions with growth conditions. Mich. Math. J. 56(1), 155–171 (2008)Ounaïes, M.: Interpolation dans les algèbres de Hörmander. Université Louis Pasteur. Institut de Recherche Mathématique Avancée (IRMA), Strasbourg (2008)Stromberg, K.R.: Introduction to classical real analysis. Wadsworth International, Belmont, California. Wadsworth International Mathematics Series (1981)Zioło P.: Geometric characterization of interpolation in the space of Fourier-Laplace transforms of ultradistributions of Roumieu type. Collect. Math. 62(2), 161–172 (2011

Smooth non linear high gain observers for a class of dynamical systems

High-gain observers are powerful tools for estimating the state of nonlinear systems. However, their design poses several challenges due to the need of dealing with phenomena such as peaking and chattering. To address these issues, we propose a differentiator operator design based on a non linear second order high-gain observer, which is suited to a class of dynamical systems. Our method includes a procedure to determine high gains in order to avoid chattering in the case of noise-free models, and cut-off frequency based gain design in the case of noisy measurements. Complementary, we suggest performing observability analyses to ensure a priori the feasibility of the estimation. The main strengths of our approach are its simplicity and robustness. We demonstrate the performance of the proposed method by applying it to two processes (chemical and biological).Xunta de Galicia | Ref. ED431F 2021/003MCIN/AEI/10.13039/501100011033 | Ref. RYC-2019-027537-

Analyzing interference between RGB-D cameras for human motion tracking

Multi-camera RGB-D systems are becoming popular as sensor setups in Computer Vision applications but they are prone to cause interference between them, compromising their accuracy. This paper extends previous works on the analysis of the noise introduced by interference with new and more realistic camera configurations and different brands of devices. As expected, the detected noise increases as distance and angle grows, becoming worse when interference is present. Finally, we evaluate the effectiveness of the proposed solutions of using DC vibration motors to mitigate them. The results of this study are being used to assess the effect of interference when applying these setups to human motion tracking.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Plan Propio de Investigación de la UMA. Junta de Andalucía, proyecto TEP2012-53

Effect of seawater inorganic carbon enrichment on NO3- transporter and reduction enzyme gene expression in Zostera marina leaves

In this work we have analysed the gene expression levels related to cytosolic nitrate homeostasis, including transporters and enzymes, in leaves of Zostera marina plants incubated in high HCO3- or in the absence of N. The expression levels of ZosmaNTR2, the only gene quoted as a high- affinity nitrate transporter belonging to NRT2 family in Z. marina genome, as well as its regulatory protein ZosmaNAR2, were induced in both conditions. Interestingly, expression levels of such genes were higher than the obtained for ZosmaCHL1 (ZosmaNPF6.3), which expression was lower in N-replete plants, but did not vary in response to HCO3-. These results suggest that the induction of the expression of the NO3- high-affinity transporter ZosmaNRT2 and its regulatory protein ZosmaNAR2 are the main response for the cytosolic NO3- decrease in both N-deficiency or in C-enrichment.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech