A robust circadian rhythm of metabolites in Arabidopsis thaliana mutants with enhanced growth characteristics

Abstract

Climate change and the rising food demand provide a need for smart crops that yield more biomass. Recently, two Arabidopsis thaliana mutants with enhanced growth characteristics, VP16-02-003 and the VP16-05-014, were obtained by genome-wide reprogramming of gene expression, which led to the identification of novel biomarkers of these enhanced growth phenotypes. Since the circadian cycle strongly influences metabolic and physiological processes and exerts control over the photosynthetic machinery responsible for enhanced growth, in this study, we investigate the influences of the circadian clock on the metabolic rhythm of eighteen key biomarkers for the larger rosette surface area phenotype. The metabolic profile was studied in intact leaves at seven different time points throughout the circadian cycle using high-resolution magic angle spinning (HR-MAS) NMR. The results show that the circadian rhythm of biomarker metabolites are remarkably robust across wild-type Col-0 and VP16-02-003 and the VP16-05-014 mutants, with widely different metabolite levels of both mutants compared to Col-0 throughout the circadian cycle. Our analysis reveals that robustness is achieved through functional independence between the circadian clock and primary metabolic processes.

Fig 1. Schematic overview of the Arabidopsis thaliana circadian clock.

A) The central oscillator consists of three interlocked feedback loops. External signals like light and temperature can influence the input pathways for the central oscillator. In turn, the central oscillator activates output pathways depending on the time of the day. B) Model of the central oscillator containing three interlocked transcriptional-translation feedback loops.

Fig 2

Changes in concentration of free amino acids (A), proteins (B), soluble sugars (C) and starch (D) throughout the circadian cycle for Arabidopsis thaliana Col-0 (●), VP16-02-003 (◼) and VP16-05-014 (▲) in mg/g fresh weight. The results are given as mean ± SEM (n = 6). * p < 0.05 Col-0 vs VP16-02-003, ^† p < 0.05 Col-0 vs VP16-05-014.

Fig 3

One-dimensional ¹H CPMG NMR spectra for Arabidopsis thaliana Col-0 (bottom panels), VP16-02-003 (middle panels) and VP16-05-014 (top panels) obtained from intact rosette leaf harvested at t = 8 hours into the light/dark cycle. Main metabolites have been assigned in the spectra.

Fig 4. Orthogonal partial least square-discriminant analysis (OPLS-DA) score plots of the metabolite profiles derived from the intact leaves of Arabidopsis thaliana wild-type Col-0 (●), VP16-02-003 (■) and VP16-05-014 (▲) at the seven different time-points of harvesting throughout the light/dark cycle.

There is a clear separation between the wild-type and the mutants at the different time-points.

Fig 5

Metabolite concentrations during the circadian cycle of fumaric acid (A), malic acid (B), lactic acid (C), fructose (D), glucose (E), myo-inositol (F), choline (G) and betaine (H) in Arabidopsis thaliana Col-0 (●), VP16-02-003 (◼) and VP16-05-014 (▲). Means ± SEM of 6 biological replicates is shown. Concentrations are expressed relative to the concentration of Col-0 at t = 0 hours. * p < 0.05 Col-0 vs VP16-02-003, ^† p < 0.05 Col-0 vs VP16-05-014.

Fig 6

Concentration of the free amino acids L-alanine (A), 𝛃-alanine (B), L-asparagine (C), L-aspartic acid (D), L-glutamic acid (E), L-glutamine (F), L-glycine (G), L-lysine (H), L-phenylalanine (I) and L-tyrosine (J) throughout the circadian cycle in Arabidopsis thaliana Col-0 (●), VP16-02-003 (◼) and VP16-05-014 (▲). Means ± SEM of 6 biological replicates is shown. Concentrations are expressed relative to the concentration of Col-0 at t = 0 hours. * p < 0.05 Col-0 vs VP16-02-003, ^† p < 0.05 Col-0 vs VP16-05-014.

Fig 7. Pathway view of the central carbon metabolism of primary metabolites for the VP16-02-003 and VP16-05-014 mutant in comparison to Col-0.

Colours indicate higher levels (green) or lower levels (red) of the primary metabolite. Dashed lines indicate multiple conversion steps.