Is plastidic glutamine synthetase essential for C-3 plants? A tale of photorespiratory mutants, ammonium tolerance and conifers

[EN] Agriculture faces the considerable challenge of having to adapt to a progressively changing climate (including the increase in CO2 levels and temperatures); environmental impact must be reduced while at the same time crop yields need to be maintained or increased to ensure food security. Under this scenario, increasing plants' nitrogen (N) use efficiency and minimizing the energy losses associated with photorespiration are two goals of crop breeding that are long sought after. The plastidic glutamine synthetase (GS2) enzyme stands at the crossroads of N assimilation and photorespiration, and is therefore a key candidate for the improvement of crop performance. The GS2 enzyme has long been considered essential for angiosperm survival under photorespiratory conditions. Surprisingly, in Arabidopsis GS2 is not essential for plant survival, and its absence confers tolerance towards ammonium stress, which is in conflict with the idea that NH4+ accumulation is one of the main causes of ammonium stress. Altogether, it appears that the 'textbook' view of this enzyme must be revisited, especially regarding the degree to which it is essential for plant growth under photorespiratory conditions, and the role of NH4+ assimilation during ammonium stress. In this article we open the debate on whether more or less GS2 is a desirable trait for plant productivity.This research was funded by the Basque Government (IT932-16), the Spanish State Research Agency (AEI) (PID2020-113385RB-I00 and RTI2018-093571-B-100 co-funded by FEDER, EU), Junta de Andalucia (P20_00036 PAIDI 2020/FEDER, UE) and the project US-1256179 grant from Junta de Andalucia, FEDER and Universidad de Sevilla

Pine has two glutamine synthetase paralogs, GS1b.1 and GS1b.2, exhibiting distinct biochemical properties

The enzyme glutamine synthetase (EC 6.3.1.2) is mainly responsible for the incorporation of inorganic nitrogen into organic molecules in plants. In the present work, a pine (Pinus pinaster) GS1 (PpGS1b.2) gene was identified, showing a high sequence identity with the GS1b.1 gene previously characterized in conifers. Phylogenetic analysis revealed that the presence of PpGS1b.2 is restricted to the genera Pinus and Picea and is not found in other conifers. Gene expression data suggest a putative role of PpGS1b.2 in plant development, similar to other GS1b genes from angiosperms, suggesting evolutionary convergence. The characterization of GS1b.1 and GS1b.2 at the structural, physicochemical, and kinetic levels has shown differences even though they have high sequence homology. GS1b.2 had a lower optimum pH (6 vs. 6.5) and was less thermally stable than GS1b.1. GS1b.2 exhibited positive cooperativity for glutamate and substrate inhibition for ammonium. However, GS1b.1 exhibited substrate inhibition behavior for glutamate and ATP. Alterations in the kinetic characteristics produced by site-directed mutagenesis carried out in this work strongly suggest an implication of amino acids at positions 264 and 267 in the active center of pine GS1b.1 and GS1b.2 being involved in affinity toward ammonium. Therefore, the amino acid differences between GS1b.1 and GS1b.2 would support the functioning of both enzymes to meet distinct plant needsFunding for open access charge: Universidad de Malaga/CBUA

Emerging insights into nitrogen assimilation in gymnosperms

Gymnosperms are a heterogeneous and ancient group of seed plants that includes conifers, ginkgos, cycads and gnetophytes. Molecular studies on extant gymnosperms have been constrained by some discouraging features for experimental research such as their long life cycles, large sizes, complex megagenomes and abundant phenolic compounds in their woody tissues. However, the development of high-throughput sequencing and refined multiomics technologies in the last few years has allowed to explore the molecular basis of essential processes in this ancient lineage of plants. Nitrogen is one of the main limiting factors determining vascular development and biomass production in woody plants. Therefore, nitrogen uptake, metabolism, storage and recycling are essential processes for fundamental gymnosperm biology. Here, recent progress in the molecular regulation of nitrogen assimilation in gymnosperms is reviewed and some future perspectives on this topic are outlined.This research was fnancially supported by Ministry of Science and Innovation (BIO2015-73512-JIN, RTI2018-094041-B-I00 and PID2021-125040OB-I00) and by Junta de Andalucía (P20-00036 PAIDI 2020/FEDER, UE). JMVM was supported by a Grant from the Spanish Ministry of Education (FPU17/03517). Funding for open access publishing: Universidad Málaga/CBUA

Efecto combinado de la nutrición nitrogenada y concentración de CO2 en biomasa y perfiles de expresión génica en Pinus pinaster

Carbon dioxide (CO2) in high concentration is beneficial for crop development, but due to the reduction of photorespiration in C3 plants, there is less reducing power available for nitrate (NO3-) reduction and later nitrogen assimilation compared to ammonium (NH4+) nutrition. To overcome this problem, research is focusing on NH4+ nutrition, because its assimilation is less expensive in terms of energy (Bloom, 2015). Knowing how plants manage NH4+ toxicity by gene expression and enzymatic pathways activation, will result in a novel set of molecular mechanisms that could be applied in crops in the near future, solving the problem of photorespiration reduction and nitrogen use efficiency (South et al., 2018). In this work we studied the growth of Pinus pinaster (Aiton) seedlings in 400 and 720 ppm CO2 concentrations and under NO3- and NH4+ nutrition, due to the tolerance that conifers have for NH4+ (Marino et al., 2022). Results show that during the early development, the combination of high CO2 concentration and NH4+ leads to an increase in biomass of the seedling and growth rates. To further investigate the set of genes differentially regulated in these conditions of nitrogen nutrition and CO2 concentration, we analysed the data from RNA-seq experiments from different organs of 2 months seedlings growth in 400 and 720 ppm of CO2 and different nitrogen nutrition (8 mM NH4+ or 8 mM NO3-).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech