Metabolomic Responses of Guard Cells and Mesophyll Cells to Bicarbonate.

Anthropogenic CO2 presently at 400 ppm is expected to reach 550 ppm in 2050, an increment expected to affect plant growth and productivity. Paired stomatal guard cells (GCs) are the gate-way for water, CO2, and pathogen, while mesophyll cells (MCs) represent the bulk cell-type of green leaves mainly for photosynthesis. We used the two different cell types, i.e., GCs and MCs from canola (Brassica napus) to profile metabolomic changes upon increased CO2 through supplementation with bicarbonate (HCO3-). Two metabolomics platforms enabled quantification of 268 metabolites in a time-course study to reveal short-term responses. The HCO3- responsive metabolomes of the cell types differed in their responsiveness. The MCs demonstrated increased amino acids, phenylpropanoids, redox metabolites, auxins and cytokinins, all of which were decreased in GCs in response to HCO3-. In addition, the GCs showed differential increases of primary C-metabolites, N-metabolites (e.g., purines and amino acids), and defense-responsive pathways (e.g., alkaloids, phenolics, and flavonoids) as compared to the MCs, indicating differential C/N homeostasis in the cell-types. The metabolomics results provide insights into plant responses and crop productivity under future climatic changes where elevated CO2 conditions are to take center-stage

Stomatal movement in response to 1 mM HCO₃^- added at time 0 min.

<p>Data were obtained from 60 stomata in three independent experiments and presented as means ± SE. The asterisks indicate significantly different mean values at P <0.05.</p

Principal component analysis (PCA) and orthogonal partial least square discriminant (OPLS-DA) analysis of metabolite changes in GCs and MCs after HCO₃^- treatment.

<p>PCA was performed using four replicate data of relative metabolite abundances in the cell-types at 0, 5, 15, 30, 60, 120 mpi, and the generated PC1 and PC2 were plotted. PCA of two cell-types showing a clear separation of the two groups based on the 268 metabolites for the effect of treatments in (<b>A</b>) GCs, and (<b>B</b>) MCs. The effects of only <b>(C)</b> time and both (<b>D</b>) ‘treatment x time’ were displayed. In OPLS-DA, the metabolite changes as a result of interactions among ‘cell-type x treatment x time were displayed in <b>(E)</b> GCs and (<b>F</b>) MCs.</p

STEM analysis showing metabolite accumulation patterns across the time-course of HCO₃^- treatment of (A) MC and (B) GC.

<p>Numbers at the bottom-left indicate the metabolites with similar trends, and the star indicates significance (P < 0.05).</p

Responses of major metabolite groups in MCs and GCs upon HCO₃^- treatment in the time-course study.

<p>Green and red squares indicate decreased (<0.8) and increased (>1.2) fold changes, respectively. The asterisks indicate significant changes (P <0.05).</p

Summary of the increased and decreased metabolic pathways in (A) GCs and (B) MCs after HCO₃^- treatment.

<p>Summary of the increased and decreased metabolic pathways in (A) GCs and (B) MCs after HCO₃^- treatment.</p

Hierarchical cluster analysis (HCA) of mean values of metabolite contents from four biological replicates showing 268 metabolites common to the two cell-types depicting the data structure dependent on the cell-types and time course (0–120 mpi) of HCO₃^- treatment.

<p>Red and green indicate high and low concentrations of metabolites, respectively. Values were subjected to average linkage clustering (Euclidean distance). Outlined blocks in yellow show grouped metabolites in the two cell-types.</p