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

Date Published: June 25, 2019

Publisher: Public Library of Science

Author(s): Dieuwertje Augustijn, Huub J. M. de Groot, A. Alia, Nicholas Simon Foulkes.


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.

Partial Text

Smart crops with high biomass yield and with a reduced need for fertilizer and pesticides can help to meet the increasing demand for agricultural products [1,2]. Such smart crops can be developed by genome editing tools including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) [3,4]. In a recent study, zinc finger artificial transcription factors (ZF-ATFs) were used to obtain Arabidopsis thaliana mutants with enhanced growth characteristics [5]. The transcriptional activator protein VP16 from the herpes simplex virus was fused to an array of three zinc fingers (3F) to provide an artificial gene construct, denoted 3F-VP16. Arabidopsis thaliana plants were transformed with the 3F-VP16 construct under the control of the RPS5A promotor. The 3F motif has ~1000 binding sites in the nuclear Arabidopsis thaliana genome of 130 Mbp. This could lead to drastic changes in genome-wide expression patterns and provide access to rare phenotypes of plants [4,6]. Indeed two Arabidopsis thaliana mutants, VP16-02-003 and VP16-05-014, with enhanced growth characteristics were obtained using such genome interrogation with ZF-ATFs [5].

In this study, we investigated the circadian rhythm of the eighteen earlier identified biomarker for the enhanced growth characteristics phenotype of the VP16-02-003 and VP16-05-014 mutant. HR-MAS NMR was used to obtain the metabolic profile throughout the light/dark cycle for Arabidopsis thaliana wild-type Col-0 and the VP16-02-003 and VP16-05-014 mutant. The metabolic rhythm of the eighteen biomarkers was not altered in both mutants as compared to Col-0, while the concentrations of metabolites differ significantly throughout the whole light/dark cycle. Since the clock functional periodicity is independent of the cellular complexity and growth-defence trade-off, the results contribute to converging evidence that it may not be necessary to—upstream—alter the circadian clock when the goal is to achieve enhanced growth characteristics, and that—downstream—phenotypic engineering of sinks and bottlenecks leading to growth may be more effective in a multifactorial context that can be altered by whole genome reprogramming.




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