Proteogenic Dipeptides Are Characterized by Diel Fluctuations and Target of Rapamycin Complex-Signaling Dependency in the Model Plant Arabidopsis thaliana

Abstract

As autotrophic organisms, plants capture light energy to convert carbon dioxide into ATP, nicotinamide adenine dinucleotide phosphate (NADPH), and sugars, which are essential for the biosynthesis of building blocks, storage, and growth. At night, metabolism and growth can be sustained by mobilizing carbon (C) reserves. In response to changing environmental conditions, such as light-dark cycles, the small-molecule regulation of enzymatic activities is critical for reprogramming cellular metabolism. We have recently demonstrated that proteogenic dipeptides, protein degradation products, act as metabolic switches at the interface of proteostasis and central metabolism in both plants and yeast. Dipeptides accumulate in response to the environmental changes and act via direct binding and regulation of critical enzymatic activities, enabling C flux distribution. Here, we provide evidence pointing to the involvement of dipeptides in the metabolic rewiring characteristics for the day-night cycle in plants. Specifically, we measured the abundance of 13 amino acids and 179 dipeptides over short- (SD) and long-day (LD) diel cycles, each with different light intensities. Of the measured dipeptides, 38 and eight were characterized by day-night oscillation in SD and LD, respectively, reaching maximum accumulation at the end of the day and then gradually falling in the night. Not only the number of dipeptides, but also the amplitude of the oscillation was higher in SD compared with LD conditions. Notably, rhythmic dipeptides were enriched in the glucogenic amino acids that can be converted into glucose. Considering the known role of Target of Rapamycin (TOR) signaling in regulating both autophagy and metabolism, we subsequently investigated whether diurnal fluctuations of dipeptides levels are dependent on the TOR Complex (TORC). The Raptor1b mutant (raptor1b), known for the substantial reduction of TOR kinase activity, was characterized by the augmented accumulation of dipeptides, which is especially pronounced under LD conditions. We were particularly intrigued by the group of 16 dipeptides, which, based on their oscillation under SD conditions and accumulation in raptor1b, can be associated with limited C availability or photoperiod. By mining existing protein-metabolite interaction data, we delineated putative protein interactors for a representative dipeptide Pro-Gln. The obtained list included enzymes of C and amino acid metabolism, which are also linked to the TORC-mediated metabolic network. Based on the obtained results, we speculate that the diurnal accumulation of dipeptides contributes to its metabolic adaptation in response to changes in C availability. We hypothesize that dipeptides would act as alternative respiratory substrates and by directly modulating the activity of the focal enzymes.

Rewiring of the protein–protein–metabolite interactome during the diauxic shift in yeast

“…Against this background we speculate that in the early stationary phase CP functions as stand-alone 20S proteasome degrading unstructured, aggregated proteins that accumulate in the ageing yeast cells. (iii) Lastly, and on par with our previous report from plants [ 12 ], we demonstrate a co-elution, and hence a putative interaction, between the proline-containing, Pro-X, dipeptides, and various enzymes of the central carbon metabolism specifically in the early stationary phase. Intriguingly the appearance of Pro-X dipeptides in the protein complexes is independent of the cellular concentrations pointing to the binding being dependent on, e.g.…”

“…Enzymes co-fractionating with P-X include glyceraldehyde-3-phosphate dehydrogenase (TDH1, TDH2 and TDH3), fructose 1,6-bisphosphate aldolase (FBA1), phosphoglycerate mutase (GPM1), 3-phosphoglycerate kinase (PGK1) and citrate synthases (CIT1 and CIT2) (Table S21 + S22). Interestingly, a similar list of putative interaction partners of proline-containing dipeptides has been reported in Arabidopsis thaliana [ 12 ], pointing to the putative conserved role of proline-containing dipeptides in the regulation of central carbon metabolism.…”

“…Dipeptides originate from protein degradation, and, although they are always present in the cell, they accumulate in conditions associated with high rates of protein clearance, such as in response to stress [ 15 , 36 , 45 , 58 , 60 , 71 , 73 ]. We, for instance, found that in plants, dipeptide levels increase in response to heat and dark stresses in an autophagy-dependent manner [ 60 ], and that specific dipeptides display diurnal oscillation in response to the change in a plant carbon status, downstream of TOR signalling [ 12 ]. In comparison, yeast accumulate dipeptides in response to glucose depletion prior to the diauxic shift transition [ 36 ].…”

A 1-aminocyclopropane-1-carboxylic-acid (ACC) dipeptide elicits ethylene responses through ACC-oxidase mediated substrate promiscuity

“…Although currently unknown, it is possible that conjugation of ACC with other amino acids exist, to form dipeptides. Several dipeptides have been characterized in plants, many of which have signaling or metabolic roles (Calderan-Rodrigues et al, 2021 ; Moreno et al, 2021 ). Here, we found that the synthetic ACC dipeptide is able to evoke an ethylene response in Arabidopsis ( Figure 1 ), independent of ACC signaling ( Figure 3 ).…”

“…Whether ACC also occurs as a dipeptide remains uninvestigated. Several dipeptide molecules have been discovered in plants, but these are in large part synthesized via protein catabolism (Calderan-Rodrigues et al, 2021 ; Thirumalaikumar et al, 2021 ). Formation of NPAA dipeptides however, must occur via dedicated enzymes, as they cannot be incorporated into peptides via mRNA translation.…”