Stress response to CO2 deprivation by Arabidopsis thaliana in plant cultures

Abstract

After being the standard plant propagation protocol for decades, cultures of Arabidopsis thaliana sealed with Parafilm remain common today out of practicality, habit, or necessity (as in co-cultures with microorganisms). Regardless of concerns over the aeration of these cultures, no investigation has explored the CO2 transport inside these cultures and its effect on the plants. Thereby, it was impossible to assess whether Parafilm-seals used today or in thousands of older papers in the literature constitute a treatment, and whether this treatment could potentially affect the study of other treatments.For the first time we report the CO2 concentrations in Parafilm-sealed cultures of A. thaliana with a 1 minute temporal resolution, and the transcriptome comparison with aerated cultures. The data show significant CO2 deprivation to the plants, a drastic suppression of photosynthesis, respiration, starch accumulation, chlorophyll biosynthesis, and an increased accumulation of reactive oxygen species. Most importantly, CO2 deprivation occurs as soon as the cotyledons emerge. Gene expression analysis indicates a significant alteration of 35% of the pathways when compared to aerated cultures, especially in stress response and secondary metabolism processes. On the other hand, the observed increase in the production of glucosinolates and flavonoids suggests intriguing possibilities for CO2 deprivation as an organic biofortification treatment in high-value crops.

Fig 1. CO₂ deprivation in Petri dish cultures of A. thaliana.

A. Time evolution of the CO₂ concentrations in Parafilm-sealed (purple) and Micropore-sealed (orange) Petri dishes containing 15 A. thaliana (Col-0) plants, compared to room concentration (grey). B. CO₂ concentrations averaged over the light periods (empty points) and dark periods (filled points). C. CO₂ concentration (top plot) and CO₂ exchange rates (bottom plot) at day 10 in Parafilm-sealed and Micropore sealed dishes. The Parafilm-sealed dishes experience severe suppression of both photosynthetic and respiratory rates, as compared to Micropore-sealed dishes. White and grey background shadings indicate light and dark periods, respectively. Different number of asterisks indicate three stages of development namely cotyledon emergence, true leaves emergence, and rosette emergence. D. CO₂ concentration (top plot) and CO₂ exchange rates (bottom plot) at day 6 in Parafilm-sealed dishes. The mild depletion of CO₂ seen in the top plot, is still accompanied by severe suppression of photosynthetic CO₂ uptake and respiration (bottom plot). E. Photographs of Parafilm-sealed, Micropore-sealed, and aerated plates. F. Dry biomass. Different numbers of asterisks above the histograms indicate a significant difference (P<0.05, Students t-test) between the treatments. G. Hue distribution for the leaves of A. thaliana cultures in Parafilm-sealed, Micropore-sealed, and aerated plates.Probability density indicate the probability density function of the hue angle variable (obtained using the “ksdensity” function in Matlab with a bandwidth of 0.8).

Fig 2. Gene expression analysis.

A. Count and regulation of Differentially Expressed Genes (DEGs) in Parafilm-sealed plants compared to aerated plants. B. Gene Ontology enrichment analysis results for biological process. Panther analysis was made using Panther term enrichment tool version 11. C. Number of pathways significantly altered by culture in Parafilm-sealed dishes, compared to aerated conditions. Nonparametric multivariate analysis was performed to identify the Differentially Expressed (DE) gene categories for different pathways.

Fig 3. Plant molecular and metabolic phenotypes.

A. DEGs in pathways related to sugar metabolism, indicating upregulation of glycolysis and sucrose metabolism and downregulation of starch degradation. B. Accumulation of starch in leaves (whole plant colorimetric assay and 10x micrographs) in aerated (left), Micropore-sealed (middle), and Parafilm-sealed (right) plants. C. Time-resolved starch accumulation quantification during the light period in aerated (green squares), Micropore-sealed (orange circles), and Parafilm-sealed (purple triangles) plants. Lines are guides to the eye. D. DEGs on pathways related to chlorophylls metabolism, showing downregulation of the chlorophyll biosynthesis pathways. E. Chlorophyll concentration in Micropore-sealed and Parafilm-sealed dishes (% of the aerated plants). Error bars represent standard error (95% CI, n = 3 replicates /10 plants each). F-G. Number and regulation of DEGs associated with abiotic or biotic stresses. H. DEGs in pathways associated with the synthesis of glucosinolates and flavonoids. I. Concentration (% of aerated plants) of flavonoids, glucosinolates and phenolic compounds. Error bars represent standard error (95% CI, n = 3 replicates /10 plants each).