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.

Background: The application of microorganisms as bioestimulants in order to increase the yield and/or quality of agricultural products is becoming a widely used practice in many countries. In this work, five plant growth-promoting rhizobacteria (PGPR), isolated from cultivated rice paddy soils, were selected for their plant growth-promoting capacities (e.g., auxin synthesis, chitinase activity, phosphate solubilisation and siderophores production). Two different tomato cultivars were inoculated, Tres Cantos and cherry. Plants were grown under greenhouse conditions and different phenotypic characteristics were analysed at the time of harvesting. Results: Tres Cantos plants inoculated with PGPR produced less biomass but larger fruits. However, the photosynthetic rate was barely affected. Several antioxidant activities were upregulated in these plants, and no oxidative damage in terms of lipid peroxidation was observed. Finally, ripe fruits accumulated less sugar but, interestingly, more lycopene. By contrast, inoculation of cherry plants with PGPR had no effect on biomass, although photosynthesis was slightly affected, and the productivity was similar to the control plants. In addition, antioxidant activities were downregulated and a higher lipid peroxidation was detected. However, neither sugar nor lycopene accumulation was altered. Conclusion: These results support the use of microorganisms isolated from agricultural soils as interesting tools to manipulate the level of important bioactive molecules in plants. However, this effect seems to be very specific, even at the variety level, and deeper analyses are necessary to assess their use for specific applications.

Low phosphate availability is a major concern for agriculture. Plants develop a plethora of responses to improve phosphate acquisition, known as phosphate starvation responses (PSR). Among them, the induction of phosphoenolpyruvate carboxylase (PEPC) has been described in many plants. However, most studies have been conducted in the absence of phosphate, thus the real impact of PEPC in PSR is missing as there is no phosphate to take up. In this study, we used modified sorghum plants silenced in the main PEPC isozyme in roots, SbPPC3, and analyzed the role of PEPC in the presence of insoluble calcium phosphate (PCa), showing a phosphate starvation phenotype in silenced but not in WT plants. Interestingly, root exudation of citrate was not reduced in silenced plants, probably due to a higher citrate synthase activity, but it was reduced for succinate, another compound with phosphate solubilisation capacity. Finally, silenced plants accumulated less P in roots with PCa, leading to a reduced phosphate acquisition efficiency (PAE). Our results show, for the first time, the actual role of PEPC in phosphate solubilisation through succinate exudation, proposing PPC3 as a specific target to improve PAE in plants.

Phosphoenolpyruvate carboxylase (PEPC) plays central roles in photosynthesis, respiration, amino acid synthesis, and seed development. PEPC is regulated by different post-translational modifications. Between them, the phosphorylation by PEPC-kinase (PEPCk) is widely documented. In this work, we simultaneously silenced the three sorghum genes encoding PEPCk (SbPPCK1-3) by RNAi interference, obtaining 12 independent transgenic lines (Ppck1-12 lines), showing different degrees of SbPPCK1-3 silencing. Among them, two T2 homozygous lines (Ppck-2 and Ppck-4) were selected for further evaluation. Expression of SbPPCK1 was reduced by 65% and 83% in Ppck-2 and Ppck-4 illuminated leaves, respectively. Expression of SbPPCK2 was higher in roots and decreased by 50% in Ppck-2 and Ppck-4 in this tissue. Expression of SbPPCK3 was low and highly variable. Despite the incomplete gene silencing, it decreased the degree of phosphorylation of PEPC in illuminated leaves, P-deficient plants, and NaCl-treated plants. Both leaves and seeds of Ppck lines had altered metabolic profiles and a general decrease in amino acid content. In addition, Ppck lines showed delayed flowering, and 20% of Ppck-4 plants did not produce flowers at all. The total amount of seeds was lowered by 50% and 36% in Ppck-2 and Ppck-4 lines, respectively. The quality of seeds was lower in Ppck lines: lower amino acid content, including Lys, and higher phytate content. These data confirm the relevance of the phosphorylation of PEPC in sorghum development, stress responses, yield, and quality of seeds.