Orthogonal partial least squares discriminant analysis (OPLS-DA) of samples collected at different times during the circadian cycle from wild-type <i>Arabidopsis thaliana</i>.

<p>The data set contains 8 replicate samples of each 4 time points (1h, 7h into light and13h, 24h into the dark period) in the circadian cycle. (A-C) score plots of OPLS-DA analysis of 1D HR-MAS NMR spectra showing the variable responsible for the separation among samples measured 1 (■), 7 (●), 13 (♦) and 24 (★) hours during circadian cycle. Predictive component 1, 2 and 3 explained 70% of the total variance. (D) Loading plots of predictive component (PC) 1, 2 and 3 for all buckets containing assigned peaks (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163258#pone.0163258.t001" target="_blank">Table 1</a>). black, positive scores; Gray, negative scores. The metabolite on the vertical axis are arranged in accordance to their chemical shift order in the spectrum (ppm) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163258#pone.0163258.t001" target="_blank">Table 1</a>).</p

Changes in the levels of metabolites during circadian cycle in leaves of <i>Arabidopsis thaliana</i>.

<p>Wild-type <i>Arabidopsis thaliana</i> (Col-0) were grown in a 12-h-light/12-h-dark cycle as described in method section. The whole intact rosette leaf was harvested at different time points (as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163258#pone.0163258.g001" target="_blank">Fig 1</a>) during light and dark period. The results are given as the mean of 8 replicates ± standard error (* p < 0.05, **p < 0.01).</p

Metabolic Profiling of Intact <i>Arabidopsis thaliana</i> Leaves during Circadian Cycle Using ¹H High Resolution Magic Angle Spinning NMR

<div><p><i>Arabidopsis thaliana</i> is the most widely used model organism for research in plant biology. While significant advances in understanding plant growth and development have been made by focusing on the molecular genetics of <i>Arabidopsis</i>, extracting and understanding the functional framework of metabolism is challenging, both from a technical perspective due to losses and modification during extraction of metabolites from the leaves, and from the biological perspective, due to random variation obscuring how well the function is performed. The purpose of this work is to establish the <i>in vivo</i> metabolic profile directly from the <i>Arabidopsis thaliana</i> leaves without metabolite extraction, to reduce the complexity of the results by multivariate analysis, and to unravel the mitigation of cellular complexity by predominant functional periodicity. To achieve this, we use the circadian cycle that strongly influences metabolic and physiological processes and exerts control over the photosynthetic machinery. High resolution-magic angle spinning nuclear magnetic resonance (HR-MAS NMR) was applied to obtain the metabolic profile directly from intact <i>Arabidopsis</i> leaves. Combining one- and two-dimensional ¹H HR-MAS NMR allowed the identification of several metabolites including sugars and amino acids in intact leaves. Multivariate analysis on HR-MAS NMR spectra of leaves throughout the circadian cycle revealed modules of primary metabolites with significant and consistent variations of their molecular components at different time points of the circadian cycle. Since robust photosynthetic performance in plants relies on the functional periodicity of the circadian rhythm, our results show that HR-MAS NMR promises to be an important non-invasive method that can be used for metabolomics of the <i>Arabidopsis thaliana</i> mutants with altered physiology and photosynthetic efficiency.</p></div

Two-dimensional HR-MAS spectra of intact <i>Arabidopsis</i> leaf.

<p>A representative two-dimensional HR-MAS ¹H-¹H COSY spectrum obtained from intact leaf of <i>Arabidopsis thaliana</i> measured at 400 MHz DMX NMR spectrometer (Bruker, Germany) at a spinning speed of 4 kHz. The ¹H shifts were calibrated using TSP as an internal standard.</p

Changes in the levels of fumaric acid in the leaves of <i>Arabidopsis thaliana</i>.

<p>Fumaric acid was measured in intact leaves at different time points during: 12h light/12h dark period (A) and during continuous dark period (B). Results are mean of 4 replicates ± standard error (** p < 0.01, *** p < 0.001).</p

Time points of harvesting of <i>Arabidopsis</i> leaves during circadian cycle.

<p>Rosette leaves were collected at 15 time points from non-flowering <i>Arabidopsis</i> plants during growth stage 3.70–3.90.</p

Metabolite levels in leave of <i>Arabidopsis thaliana</i> at t = 7 hours.

<p>Results are mean ± standard error.</p