Root phosphoenolpyruvate carboxylase activity is essential for Sorghum bicolor tolerance to ammonium nutrition

Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is an enzyme family with pivotal roles in plant carbon and nitrogen metabolism. A main role for non-photosynthetic PEPC is as anaplerotic enzyme to load tricarboxylic acid (TCA) cycle with carbon skeletons that compensate the intermediates diverted for biomolecule synthesis such as amino acids. When plants are grown under ammonium (NH₄⁺) nutrition, the excessive uptake of NH₄⁺ often provokes a stress situation. When plants face NH₄⁺ stress, N assimilation is greatly induced and thus, requires the supply of carbon skeletons coming from TCA cycle. In this work, we addressed the importance of root PEPC and TCA cycle for sorghum (Sorghum bicolor L. Moench), a C4 cereal crop, grown under ammonium nutrition. To do so, we used RNAi sorghum lines that display a decrease expression of SbPPC3 (Ppc3 lines), the main root PEPC isoform, and reduced root PEPC activity. SbPPC3 silencing provoked ammonium hypersensitivity, meaning lower biomass accumulation in Ppc3 respect to WT plants when growing under ammonium nutrition. The silenced plants presented a deregulation of primary metabolism as highlighted by the accumulation of NH₄⁺ in the root and the alteration of normal TCA functioning, which was evidenced by the accumulation of organic acids in the root under ammonium nutrition. Altogether, our work evidences the importance of non-photosynthetic PEPC, and root TCA cycle, in sorghum to deal with high external NH₄⁺ availability.