Effects of low temperature plasmas and plasma activated waters on Arabidopsis thaliana germination and growth

Abstract

Two plasma devices at atmospheric pressure (air dielectric barrier discharge and helium plasma jet) have been used to study the early germination of Arabidopsis thaliana seeds during the first days. Then, plasma activated waters are used during the later stage of plant development and growth until 42 days. The effects on both testa and endospserm ruptures during the germination stage are significant in the case of air plasma due to its higher energy and efficiency of producing reactive oxygen species than the case of helium plasma. The latter has shown distinct effects only for testa rupture. Analysis of germination stimulations are based on specific stainings for reactive oxygen species production, peroxidase activity and also membrane permeability tests. Furthermore, scanning electron microscopy (SEM) has shown a smoother seed surface for air plasma treated seeds that can explain the plasma induced-germination. During the growth stage, plants were watered using 4 kinds of water (tap and deionized waters activated or not by the low temperature plasma jet). With regards to other water kinds, the characterization of the tap water has shown a larger conductivity, acidity and concentration of reactive nitrogen and oxygen species. Only the tap water activated by the plasma jet has shown a significant effect on the plant growth. This effect could be correlated to reactive nitrogen species such as nitrite/nitrate species present in plasma activated tap water.

Fig 1. Different plasma devices.

A. Schematic view of the helium plasma jet device on the right side and picture during the generation of plasma activated water inside a glass beaker using the helium plasma jet and a magnetic stirrer. B. Schematic view of the floating electrode-dielectric barrier discharges (FE-DBD) in air showing the powered electrode covered by glass (in blue colour) with seeds in ambient air plasma above the lamella (right side) and picture displaying the air plasma over the glass lamella.

Fig 2. Direct and indirect plasma effects on germination rate of Arabidopsis thaliana seeds.

The boxplots show the effects of different applied plasmas on the testa rupture. Observations are made on four independent assays at t0+24h. Six hundred seeds are counted for each batch. Wilcoxon test has been used for the significance between the means, the p-value is: * < 0.05; ***<0.002 (For data see S1 Fig).

Fig 3. Air plasma stimulated germination rate of A. thaliana.

Boxplots show control (cyan) and treated (magenta). A. Testa rupture as a function of time between treatment and imbibition (times t₀, t₂, t₇ and t₉ are given in days), observed 24 h after imbibition. B. As panel A but observations made at 40 h after imbibition. C. Endosperm rupture (observations at 40 h). Six hundred seeds are counted for each of the four considered batches. Wilcoxon test has been used to test the significance between the means, the p-value is: * < 0.05; *** < 0.002 (For data see S1 Fig).

Fig 4. Class III peroxidases activity and reactive oxygen species detection.

Class III peroxidases activity was detected seeds by using guaiacol as substrate (A and B). Superoxide ion O₂^.- was observed y with NBT (C and D). Control seeds (A and C) and seeds treated with air plasma (B and D). The intensity of the coloration appeared to be identical at the same level of germination for both cases (control or plasma treated seeds). Each scale bar corresponds to 100 μm.

Fig 5. A. thaliana seed permeability assay.

Seeds were incubated for 24 h in tetrazolium red at 28°C and formazan production assayed by measuring absorbance of the incubation solution at 492 nm. For plasma treated (purple bar), the air plasma was used and seeds were incubated with tetrazolium immediately following treatment. Control (blue bar) were treated similarly but without activating the plasma. Water (white bar) treatment omitted the tetrazolium. Bars show mean of 4 assays and the error bars are 0.05 (For data see S1 Fig).

Fig 6. Scanning electron microscopy (SEM) images of A. thaliana seeds.

(A, C, E) are the control while (B, D, F) are the air plasma treated. Scale bars: 100 μm A, B; 50 μm C, D; 10 μm E, F.

Fig 7. Effects of plasma-activated tap water (PAW_tap) on A. thaliana growth.

Photos after 28 days of development watered by tap water (A) and PAW_tap (B). Diameter of the rosette (C); A. thaliana seedling leaf number (D); area of the leaves (E), and total number of flowering plants (F). Wilcoxon test was used to test the significance between the means with p-value indicated as: · < 0.1; *<0.05; **<0.005; ***<0.002 (For data see S1 Fig).