Daniel Marino

Exploring ammonium tolerance in a large panel of Arabidopsis thaliana natural accessions

Summary Ammonium nutrition is toxic to many plants. Arabidopsis displays high intraspecific variability in ammonium tolerance (shoot biomass), and ammonium accumulation seems to be an important player in this variability.

PGPRs and nitrogen-fixing legumes: a perfect team for efficient Cd phytoremediation?

Cadmium (Cd) is a toxic, biologically non-essential and highly mobile metal that has become an increasingly important environmental hazard to both wildlife and humans. In contrast to conventional remediation technologies, phytoremediation based on legume–rhizobia symbiosis has emerged as an inexpensive decontamination alternative which also revitalize contaminated soils due to the role of legumes in nitrogen cycling. In recent years, there is a growing interest in understanding symbiotic legume–rhizobia relationship and its interactions with Cd. The aim of the present review is to provide a comprehensive picture of the main effects of Cd in N2-fixing leguminous plants and the benefits of exploiting this symbiosis together with plant growth promoting rhizobacteria to boost an efficient reclamation of Cd-contaminated soils.

Split-root systems applied to the study of the legume-rhizobial symbiosis： What have we learned？

Split-root system (SRS) approaches allow the differential treatment of separate and independent root systems, while sharing a common aerial part. As such, SRS is a useful tool for the discrimination of systemic (shoot origin) versus local (root/nodule origin) regulation mechanisms. This type of approach is particularly useful when studying the complex regulatory mechanisms governing the symbiosis established between legumes and Rhizobium bacteria. The current work provides an overview of the main insights gained from the application of SRS approaches to understand how nodule number (nodulation autoregulation) and nitrogen fixation are controlled both under non-stressful conditions and in response to a variety of stresses. Nodule number appears to be mainly controlled at the systemic level through a signal which is produced by nodule/root tissue, translocated to the shoot, and transmitted back to the root system, involving shoot Leu-rich repeat receptor-like kinases. In contrast, both local and systemic mechanisms have been shown to operate for the regulation of nitrogenase activity in nodules. Under drought and heavy metal stress, the regulation is mostly local, whereas the application of exogenous nitrogen seems to exert a regulation of nitrogen fixation both at the local and systemic levels.

Quantitative proteomics reveals the importance of nitrogen source to control glucosinolate metabolism in Arabidopsis thaliana and Brassica oleracea

Highlight A quantitative proteomic approach demonstrates how ammonium nutrition induces glucosinolate biosynthetic and catabolic pathways in Arabidopsis and broccoli.

Nitrogen Source and External Medium pH Interaction Differentially Affects Root and Shoot Metabolism in Arabidopsis

Ammonium nutrition often represents an important growth-limiting stress in plants. Some of the symptoms that plants present under ammonium nutrition have been associated with pH deregulation, in fact external medium pH control is known to improve plants ammonium tolerance. However, the way plant cell metabolism adjusts to these changes is not completely understood. Thus, in this work we focused on how Arabidopsis thaliana shoot and root respond to different nutritional regimes by varying the nitrogen source (NO3- and NH4+), concentration (2 and 10 mM) and pH of the external medium (5.7 and 6.7) to gain a deeper understanding of cell metabolic adaptation upon altering these environmental factors. The results obtained evidence changes in the response of ammonium assimilation machinery and of the anaplerotic enzymes associated to Tricarboxylic Acids (TCA) cycle in function of the plant organ, the nitrogen source and the degree of ammonium stress. A greater stress severity at pH 5.7 was related to NH4+ accumulation; this could not be circumvented in spite of the stimulation of glutamine synthetase, glutamate dehydrogenase, and TCA cycle anaplerotic enzymes. Moreover, this study suggests specific functions for different gln and gdh isoforms based on the nutritional regime. Overall, NH4+ accumulation triggering ammonium stress appears to bear no relation to nitrogen assimilation impairment.

A proteomic approach reveals new actors of nodule response to drought in split-root grown pea plants

Drought is considered the more harmful abiotic stress resulting in crops yield loss. Legumes in symbiosis with rhizobia are able to fix atmospheric nitrogen. Biological nitrogen fixation (SNF) is a very sensitive process to drought and limits legumes agricultural productivity. Several factors are known to regulate SNF including oxygen availability to bacteroids, carbon and nitrogen metabolisms; but the signaling pathways leading to SNF inhibition are largely unknown. In this work, we have performed a proteomic approach of pea plants grown in split-root system where one half of the root was well-irrigated and the other was subjected to drought. Water stress locally provoked nodule water potential decrease that led to SNF local inhibition. The proteomic approach revealed 11 and 7 nodule proteins regulated by drought encoded by Pisum sativum and Rhizobium leguminosarum genomes respectively. Among these 18 proteins, 3 proteins related to flavonoid metabolism, 2 to sulfur metabolism and 3 RNA-binding proteins were identified. These proteins could be molecular targets for future studies focused on the improvement of legumes tolerance to drought. Moreover, this work also provides new hints for the deciphering of SNF regulation machinery in nodules.

CO2 enrichment modulates ammonium nutrition in tomato adjusting carbon and nitrogen metabolism to stomatal conductance

Ammonium (NH4(+)) toxicity typically occurs in plants exposed to high environmental NH4(+) concentration. NH4(+) assimilating capacity may act as a biochemical mechanism avoiding its toxic accumulation but requires a fine tuning between nitrogen assimilating enzymes and carbon anaplerotic routes. In this work, we hypothesized that extra C supply, exposing tomato plants cv. Agora Hybrid F1 to elevated atmospheric CO2, could improve photosynthetic process and thus ameliorate NH4(+) assimilation and tolerance. Plants were grown under nitrate (NO3(-)) or NH4(+) as N source (5-15mM), under two atmospheric CO2 levels, 400 and 800ppm. Growth and gas exchange parameters, (15)N isotopic signature, C and N metabolites and enzymatic activities were determined. Plants under 7.5mM N equally grew independently of the N source, while higher ammonium supply resulted toxic for growth. However, specific stomatal closure occurred in 7.5mM NH4(+)-fed plants under elevated CO2 improving water use efficiency (WUE) but compromising plant N status. Elevated CO2 annulled the induction of TCA anaplerotic enzymes observed at non-toxic NH4(+) nutrition under ambient CO2. Finally, CO2 enrichment benefited tomato growth under both nutritions, and although it did not alleviate tomato NH4(+) tolerance it did differentially regulate plant metabolism in N-source and -dose dependent manner.

Physiological Responses of N2-Fixing Legumes to Water Limitation

A significant decline in the content of water in soils provokes a water deficit at the plant level. In plant physiology, water deficit can be defined as the water content of a tissue or cell below the highest water content under the optimum hydrated state. The basis of the fundamental mechanism involved in stress tolerance, although intensively explored, is still matter of debate. Cell growth is the physiological process first affected as cell water content decreases when plants encounter mild water-deficit levels, followed by an inhibition of cell wall and protein biosynthesis. Although stomatal conductance and photosynthesis are affected in more intense water-deficit stages, most research efforts have focused on the study of these processes. In legume plants grown under symbiotic conditions, one of the primary effects of water deficit is a decline in the rates of symbiotic nitrogen fixation (SNF). The causes of this inhibition, which occurs even before a measurable decline in the rates of photosynthesis, have been explored in detail in the last decades, although the molecular mechanism involved are yet not fully understood. In the present chapter, we summarize our current understanding of the factors involved in the regulation of SNF in different legume species, including crops such as soybean (Glycine max), alfalfa (Medicago sativa), bean (Phaseolus vulgaris), and pea (Pisum sativum) but also model legumes like Medicago truncatula. Finally, an overview of the available resources and applications of molecular system-based approaches for understanding the complex responses of legumes to drought stress is provided.

Dimethyl pyrazol-based nitrification inhibitors effect on nitrifying and denitrifying bacteria to mitigate N 2 O emission

Nitrous oxide (N2O) emissions have been increasing as a result of intensive nitrogen (N) fertilisation. Soil nitrification and denitrification are the main sources of N2O, and the use of ammonium-based fertilisers combined with nitrification inhibitors (NIs) could be useful in mitigating N2O emissions from agricultural systems. In this work we looked at the N2O mitigation capacity of two dimethylpyrazol-based NIs, 3,4-dimethylpyrazole phosphate (DMPP) and 2-(N-3,4-dimethyl-1H-pyrazol-1-yl) succinic acid isomeric mixture (DMPSA), on soil nitrifying and denitrifying microbial populations under two contrasting soil water contents (40% and 80% soil water filled pore space; WFPS). Our results show that DMPP and DMPSA are equally efficient at reducing N2O emissions under 40% WFPS conditions by inhibiting bacterial ammonia oxidation. In contrast, at 80% WFPS DMPSA was less efficient than DMPP at reducing N2O emissions. Interestingly, at 80% WFPS, where lowered oxygen availability limits nitrification, both DMPP and DMPSA not only inhibited nitrification but also stimulated N2O reduction to molecular nitrogen (N2) via nitrous oxide reductase activity (Nos activity). Therefore, in this work we observed that DMP-based NIs stimulated the reduction of N2O to N2 by nitrous oxide reductase during the denitrification process.

Mild ammonium stress increases chlorophyll content in Arabidopsis thaliana

Nitrate (NO3−) and ammonium (NH4+) are the main forms of nitrogen available in the soil for plants. Excessive NH4+ accumulation in tissues is toxic for plants and exclusive NH4+-based nutrition enhances this effect. Ammonium toxicity syndrome commonly includes growth impairment, ion imbalance and chlorosis among others. In this work, we observed high intraspecific variability in chlorophyll content in 47 Arabidopsis thaliana natural accessions grown under 1 mM NH4+ or 1 mM NO3− as N-source. Interestingly, chlorophyll content increased in every accession upon ammonium nutrition. Moreover, this increase was independent of ammonium tolerance capacity. Thus, chlorosis seems to be an exclusive effect of severe ammonium toxicity while mild ammonium stress induces chlorophyll accumulation.