Learning plant biodiversity in nature: the use of the citizen–science platform iNaturalist as a collaborative tool in secondary education

Biodiversity is a concept of great scientific interest and social value studied in different subjects of the secondary education curriculum. Citizen–science programs may contribute to increasing the engagement of students when studying biodiversity. This work aimed to explore the use of the citizen–science platform iNaturalist as a complement of the elaboration of herbaria in an outdoor activity for 4th course 16-year-old students in the Basaula Reserve. The platform iNaturalist was chosen for its suitability to develop collaborative projects in an educational context. The Basaula project was created and 122 students were trained to record plant species in an outdoor activity. A total of 32 species were recorded, among them the most abundant were beech (Fagus sylvatica) and holm oak (Quercus ilex). The students positively evaluated their experience, highlighting its adequacy to record biodiversity data and make a virtual herbarium. Students valued the innovative character of iNaturalist and its usefulness for research but also the opportunity to integrate mobile devices in school education. We concluded that iNaturalist is a valuable tool to carry out collaborative projects dealing with biodiversity in secondary education.This research was funded by the Education Department of the Navarra Government, grant number CENEDUCA3/2019

Mechanisms of ammonium toxicity and the quest for tolerance

Ammonium sensitivity of plants is a worldwide problem, constraining crop production. Prolonged application of ammonium as the sole nitrogen source may result in physiological and morphological disorders that lead to decreased plant growth and toxicity. The main causes of ammonium toxicity/tolerance described until now include high ammonium assimilation by plants and/or low sensitivity to external pH acidification. The various ammonium transport-related components, especially the non-electrogenic influx of NH3 (related to the depletion of 15N) and the electrogenic influx of NH4+, may contribute to ammonium accumulation, and therefore to NH3 toxicity. However, this accumulation may be influenced by increasing K+ concentration in the root medium. Recently, new insights have been provided by “omics” studies, leading to a suggested involvement of GDP mannose-pyrophosphorylase in the response pathways of NH4+ stress. In this review, we highlight the cross-talk signaling between nitrate, auxins and NO, and the importance of the connection of the plants’ urea cycle to metabolism of polyamines. Overall, the tolerance and amelioration of ammonium toxicity are outlined to improve the yield of ammonium-grown plants. This review identifies future directions of research, focusing on the putative importance of aquaporins in ammonium influx, and on genes involved in ammonium sensitivity and tolerance.The authors acknowledge the support of research grants AGL2014-52396-P from the Spanish Ministry of Economy and Competitiveness (MINECO) and PTDC/BIA-BEC/099323/2008 and PTDC/BIA-ECS/122214/2010 from the Portuguese Fundação para a Ciência e Tecnologia (FCT). RE received a JAE-Doc-2011-046 fellow from the Spanish CSIC, co-financed by the European Social Fund

New insights on arabidopsis thaliana root adaption to ammonium nutrition by the use of a quantitative proteomic approach

Nitrogen is an essential element for plant nutrition. Nitrate and ammonium are the two major inorganic nitrogen forms available for plant growth. Plant preference for one or the other form depends on the interplay between plant genetic background and environmental variables. Ammonium-based fertilization has been shown less environmentally harmful compared to nitrate fertilization, because of reducing, among others, nitrate leaching and nitrous oxide emissions. However, ammonium nutrition may become a stressful situation for a wide range of plant species when the ion is present at high concentrations. Although studied for long time, there is still an important lack of knowledge to explain plant tolerance or sensitivity towards ammonium nutrition. In this context, we performed a comparative proteomic study in roots of Arabidopsis thaliana plants grown under exclusive ammonium or nitrate supply. We identified and quantified 68 proteins with differential abundance between both conditions. These proteins revealed new potential important players on root response to ammonium nutrition, such as H+ -consuming metabolic pathways to regulate pH homeostasis and specific secondary metabolic pathways like brassinosteroid and glucosinolate biosynthetic pathways

Depletion of the heaviest stable N isotope is associated with NH4+/NH3 toxicity in NH4+-fed plants

Open Access article - can be viewed online without a subscription at: http://dx.doi.org/doi:10.1186/1471-2229-11-83Background: In plants, nitrate (NO3 -) nutrition gives rise to a natural N isotopic signature (δ15N), which correlates with the δ15N of the N source. However, little is known about the relationship between the δ15N of the N source and the 14N/15N fractionation in plants under ammonium (NH4 +) nutrition. When NH4 + is the major N source, the two forms, NH4 + and NH3, are present in the nutrient solution. There is a 1.025 thermodynamic isotope effect between NH3 (g) and NH4 + (aq) which drives to a different δ15N. Nine plant species with different NH4 +-sensitivities were cultured hydroponically with NO3 - or NH4 + as the sole N sources, and plant growth and δ15N were determined. Short-term NH4 +/NH3 uptake experiments at pH 6.0 and 9.0 (which favours NH3 form) were carried out in order to support and substantiate our hypothesis. N source fractionation throughout the whole plant was interpreted on the basis of the relative transport of NH4 + and NH3. Results: Several NO3 --fed plants were consistently enriched in 15N, whereas plants under NH4 + nutrition were depleted of 15N. It was shown that more sensitive plants to NH4 + toxicity were the most depleted in 15N. In parallel, N-deficient pea and spinach plants fed with 15NH4 + showed an increased level of NH3 uptake at alkaline pH that was related to the 15N depletion of the plant. Tolerant to NH4 + pea plants or sensitive spinach plants showed similar trend on 15N depletion while slight differences in the time kinetics were observed during the initial stages. The use of RbNO3 as control discarded that the differences observed arise from pH detrimental effects. Conclusions: This article proposes that the negative values of δ15N in NH4 +-fed plants are originated from NH3 uptake by plants. Moreover, this depletion of the heavier N isotope is proportional to the NH4 +/NH3 toxicity in plants species. Therefore, we hypothesise that the low affinity transport system for NH4 + may have two components: one that transports N in the molecular form and is associated with fractionation and another that transports N in the ionic form and is not associated with fractionatio

Leaf δ15N as a physiological indicator of the responsiveness of N2-fixing alfalfa plants to elevated [CO2], temperature and low water availability

The contribution of carbon and nitrogen reserves to regrowth following shoot removal has been studied in the past. However, important gaps remain in understanding the effect of shoot cutting on nodule performance and its relevance during regrowth. In this study, isotopic labelling was conducted at root and canopy levels with both 15N2 and 13C-depleted CO2 on exclusively nitrogen-fixing alfalfa plants. As expected, our results indicate that the roots were the main sink organs before shoots were removed. Seven days after regrowth the carbon and nitrogen stored in the roots was invested in shoot biomass formation and partitioned to the nodules. The large depletion in nodule carbohydrate availability suggests that root-derived carbon compounds were delivered towards nodules in order to sustain respiratory activity. In addition to the limited carbohydrate availability, the upregulation of nodule peroxidases showed that oxidative stress was also involved during poor nodule performance. Fourteen days after cutting, and as a consequence of the stimulated photosynthetic and N2-fixing machinery, availability of Cnew and Nnew strongly diminished in the plants due to their replacement by C and N assimilated during the post-labelling period. In summary, our study indicated that during the first week of regrowth, root-derived C and N remobilization did not overcome C- and N-limitation in nodules and leaves. However, 14 days after cutting, leaf and nodule performance were re-established

Depletion of the heaviest stable N isotope is associated with NH4+/NH3 toxicity in NH4+-fed plants

Background: In plants, nitrate (NO3-) nutrition gives rise to a natural N isotopic signature (δ15N), which correlates with the δ15N of the N source. However, little is known about the relationship between the δ15N of the N source and the 14N/15N fractionation in plants under ammonium (NH4+) nutrition. When NH4 + is the major N source, the two forms, NH4 + and NH3, are present in the nutrient solution. There is a 1.025 thermodynamic isotope effect between NH3 (g) and NH4 + (aq) which drives to a different δ15N. Nine plant species with different NH4 +-sensitivities were cultured hydroponically with NO3 - or NH4 + as the sole N sources, and plant growth and δ15N were determined. Short-term NH4 +/NH3 uptake experiments at pH 6.0 and 9.0 (which favours NH3 form) were carried out in order to support and substantiate our hypothesis. N source fractionation throughout the whole plant was interpreted on the basis of the relative transport of NH4 + and NH3. Results: Several NO3 --fed plants were consistently enriched in 15N, whereas plants under NH4 + nutrition were depleted of 15N. It was shown that more sensitive plants to NH4 + toxicity were the most depleted in 15N. In parallel, N-deficient pea and spinach plants fed with 15NH4 + showed an increased level of NH3 uptake at alkaline pH that was related to the 15N depletion of the plant. Tolerant to NH4 + pea plants or sensitive spinach plants showed similar trend on 15N depletion while slight differences in the time kinetics were observed during the initial stages. The use of RbNO3 as control discarded that the differences observed arise from pH detrimental effects. Conclusions: This article proposes that the negative values of δ15N in NH4 +-fed plants are originated from NH3 uptake by plants. Moreover, this depletion of the heavier N isotope is proportional to the NH4 +/NH3 toxicity in plants species. Therefore, we hypothesise that the low affinity transport system for NH4 + may have two components: one that transports N in the molecular form and is associated with fractionation and another that transports N in the ionic form and is not associated with fractionation.This work was supported by the Spanish MICIIN (grant nos. AGL2006-12792-CO2- 01 and 02 and AGL2009- 13339-CO2-01 and 02 [to P.A.-T. and C.G.M.] and AGL2007-64432/AGR [to J.F.M.]), by the Portuguese FCT (PTDC/BIA- BEC/ 099323/2008) and by the Basque Government IT526-10

Nitrogen isotope signature evidences ammonium deprotonation as a common transport mechanism for the AMT-Mep-Rh protein superfamily

Ammonium is an important nitrogen (N) source for living organisms, a key metabolite for pH control, and a potent cytotoxic compound. Ammonium is transported by the widespread AMT-Mep-Rh membrane proteins, and despite their significance in physiological processes, the nature of substrate translocation (NH3/NH4+) by the distinct members of this family is still a matter of controversy. Using Saccharomyces cerevisiae cells expressing representative AMT-Mep-Rh ammonium carriers and taking advantage of the natural chemical-physical property of the N isotopic signature linked to NH4+/NH3 conversion, this study shows that only cells expressing AMT-Mep-Rh proteins were depleted in N-15 relative to N-14 when compared to the external ammonium source. We observed N-15 depletion over a wide range of external pH, indicating its independence of NH3 formation in solution. On the basis of inhibitor studies, ammonium transport by nonspecific cation channels did not show isotope fractionation but competition with K+. We propose that kinetic N isotope fractionation is a common feature of AMT-Mep-Rh-type proteins, which favor N-14 over N-15, owing to the dissociation of NH4+ into NH3+ H+ in the protein, leading to N-15 depletion in the cell and allowing NH3 passage or NH3/H+ cotransport. This deprotonation mechanism explains these proteins' essential functions in environments under a low NH4+/K+ ratio, allowing organisms to specifically scavenge NH4+. We show that N-15 isotope fractionation may be used in vivo not only to determine the molecular species being transported by ammonium transport proteins, but also to track ammonium toxicity and associated amino acids excretion.I. A. was supported by a postdoctoral fellowship from the Government of Navarra, Spain (Anabasid outgoing Programme, 2011) and by a postdoctoral fellowship from the Portuguese Fundaçao para a Ciencia e a Tecnologia (SFRH/BPD/90436/2012). A.M.M. is a senior research associate of the Belgian Fonds de la Recherche Scientifique Fonds de la Recherche Scientifique-FNRS (grants CDR J017617F, PDR T011515F, and ARC) and a WELBIO investigator, and M.B. is a scientific research worker supported by WELBIO. This work was also developed in the context of the following projects: PTDC/BIA-BEC/099323/2008 and PTDC/AGR-PRO/115888/2009 to cE3c and FCUL, UID/DTP/04138/2013 to iMed. ULisboa, and AGL2015-64582-C3-1-R and AGL2012-37815-C05-05 to UPNa

NH3 transport Determination through physiological and visual methods: Application in yeast as a model organism

Trabajo presentado en el XIII Congreso Hispano-Luso de Fisiología Vegetal, celebrado en Oeiras (Portugal) en julio de 2013.Ammonium toxicity is a global phenomenon with widely distinct thresholds across living organisms. In plants, prolonged application of ammonium as the only source of N results in physiological and morphological disorders that may lead to low plant growth and toxicity. The development of these symptoms may depend on the transport system that is dominant and the chemical form of reduced inorganic N that is transported - ion (NH4+) or molecule (NH3). However the relation between mechanisms of transport at the molecular level is, most of the time, difficult to highlight with experiments and protocols that can be performed in the high schools. In this work we propose the use of biological models (ex: Yeast) combined with classical techniques (pH indicators, physiological measurements), in order to approach complex biological processes (i.e. the distinct N systems of transport, NH4+ or NH3) to the students. The entrance of NH3 would acidify extracellular medium, and alkalinise cytosol. Thus, determinations such as pH measurement of culture media after yeast growth, culture of wild type vs mutants strains for MEP transporters (NH3 carriers in yeast) and the use of inhibitors of K+ channels (NH4+ carriers) are some of the tools used in a simplified biological system (yeast) during short-term experiments.Project supported by the Spanish Ministry of Science and Innovation [grant no. AGL2012-37815-C05-05 to P. A-T), Portuguese Foundation for the Science and the Technology, FCT, [grant nos. PTDC/AGRO-PRO/115888/2009; PTDC/BIA-ECS/122214/2010 to C. C.] and and by the Regional Government of Navarre [Anabasi+d programme 2011-2012, to P. A-T, C. C. and I. A.]. post-doctoral Fellowship from FCT: [grant no. SFRH/BPD/90436/2012 to I. A.].Peer Reviewe