Isotopic labelling reveals the efficient adaptation of wheat root TCA cycle flux modes to match carbon demand under ammonium nutrition

Izargi Vega-Mas,Carmen González-Murua,Daniel Marino,M Begoña González-Moro,Inmaculada Coleto,Caroline Cukier,Anis M Limami

doi:10.1038/s41598-019-45393-8

Abstract

Proper carbon (C) supply is essential for nitrogen (N) assimilation especially when plants are grown under ammonium (NH4+) nutrition. However, how C and N metabolic fluxes adapt to achieve so remains uncertain. In this work, roots of wheat (Triticum aestivum L.) plants grown under exclusive NH4+ or nitrate (NO3−) supply were incubated with isotope-labelled substrates (15NH4+, 15NO3−, or [13C]Pyruvate) to follow the incorporation of 15N or 13C into amino acids and organic acids. Roots of plants adapted to ammonium nutrition presented higher capacity to incorporate both 15NH4+ and 15NO3− into amino acids, thanks to the previous induction of the NH4+ assimilative machinery. The 15N label was firstly incorporated into [15N]Gln vía glutamine synthetase; ultimately leading to [15N]Asn accumulation as an optimal NH4+ storage. The provision of [13C]Pyruvate led to [13C]Citrate and [13C]Malate accumulation and to rapid [13C]2-OG consumption for amino acid synthesis and highlighted the importance of the anaplerotic routes associated to tricarboxylic acid (TCA) cycle. Taken together, our results indicate that root adaptation to ammonium nutrition allowed efficient assimilation of N thanks to the promotion of TCA cycle open flux modes in order to sustain C skeleton availability for effective NH4+ detoxification into amino acids.

Highlights

Metabolic networks are generally considered as more complex in plants compared to other organisms
Ammonium nutrition led to an increased NH4+, amino acids, protein and C contents in the root compared to nitrate nutrition (Table 1)
Gln and Ala were the major amino acids in wheat roots, their contents being superior in RA with respect to RN (Fig. S2; RN and RA stand for root of plants grown for six weeks under nitrate or ammonium nutrition, respectively)

Summary

Introduction

Metabolic networks are generally considered as more complex in plants compared to other organisms. The root is the first organ facing high NH4+ concentrations in the medium and acts as a physiological barrier to prevent its transport to the more sensitive shoot, where the excess of NH4+ can impair photosynthetic apparatus[5,6] In this line, the metabolic adjustment of roots to exclusive ammonium nutrition has even been shown to determine the capacity of the plants to cope with ammonium stress in many species including wheat and tomato[15,16]. Proper carbon (C) supply to maintain ammonium assimilation in the roots is considered as a key aspect to deal with an excess of NH4+ 18. There is still a need to understand the in vivo adaptation of C and N metabolic fluxes when plants grow under ammonium nutrition compared to nitrate nutrition

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Results

Conclusion