Plasma activated water triggers plant defence responses

The study addresses the need for sustainable agriculture that maintains yields while reducing pesticide use. Biotic and abiotic stresses cause significant yield losses globally. Enhancing plant immunity via immunity inducers offers a strategy to mitigate disease impact. Plants naturally generate reactive oxygen and nitrogen species (RONS) as signaling molecules under stress, leading to defense responses such as hypersensitive response, phytoalexin accumulation, and activation of defense genes regulated by key transcription factors. Plasma activated water (PAW), produced by exposing water to cold atmospheric plasma, contains long-lived RONS (e.g., H2O2 and NO3−) that participate in signaling pathways governing plant development and stress responses. The purpose of this study is to investigate whether PAW can induce plant defense responses in micropropagated periwinkle shoots, periwinkle plants, and grapevine plants by assessing transcriptional changes in defense-related genes and post-transcriptional regulation via small RNAs.

Prior work shows that intensive agriculture has environmental costs and that immunity inducers (synthetic elicitors, proteins, chitosan oligosaccharides, microorganisms) can trigger systemic acquired resistance. RONS act as signaling molecules in stress responses, with bursts leading to secondary metabolite and defense gene activation. Hydrogen peroxide and nitric oxide have been implicated in PAL induction and defense signaling leading to hypersensitive responses. PAW has been reported to enhance plant growth, and pioneering studies suggested PAW can induce resistance in plants (e.g., tomato). The phenylpropanoid pathway, including PAL, CHS, STS, and production of phytoalexins like resveratrol, is central to defense in grapevine. In periwinkle, alkaloid biosynthesis enzymes such as strictosidine β-glucosidase (SGD) and deacetylvindoline-O-acetyltransferase (DAT) are involved in defense-related alkaloids (vincristine, vinblastine). Small RNAs, including miRNA families like miR159, miR165/166, miR395, miR396, miR398, regulate stress responses, ROS detoxification, and nutrient homeostasis.

PAW production: 80 mL of sterile distilled water (SDW) was exposed to a nanosecond pulsed dielectric barrier discharge (DBD) operating in ambient air for 10 minutes with a peak voltage of 19 kV and a pulse repetition frequency of 1 kHz. Immediately after treatment, PAW aliquots were frozen. The next day, PAW was thawed, pH measured, and concentrations of H2O2 and nitrates quantified using Amplex Red Hydrogen Peroxide Assay (Thermo Fisher Scientific) and nitrate/nitrite colorimetric assay (ROCHE) per manufacturers’ protocols; absorbance measured with a microplate reader. Short-lived species such as nitrites decayed below detection within minutes after plasma exposure; H2O2 and NO3− remained stable for 24 h, even after freezing/thawing.

Plant materials and growth conditions: Micropropagated periwinkle (Catharanthus roseus) shoots were maintained in vitro (24±2 °C, 16 h light). Greenhouse-grown healthy periwinkle plants (derived from micropropagation) and 2-year-old grapevine plants cv Chardonnay were maintained under insect-proof conditions (16 h light at 30 °C, 8 h dark at 24 °C, RH 70–75%).

Treatments: Periwinkle shoots (5 replicates per treatment) were submerged for 25 min in 20 mL of PAW, SDW (negative control), or a 2.5 g/L fosetyl aluminium solution (FoAl; positive control for PR protein induction). An additional synthetic solution control matched PAW’s H2O2 (13.5 mg/L), NO3− (81.9 mg/L), and pH (adjusted with HCl), which showed no plant effect. Shoots were sampled at 0, 7, 24, 48, 96, and 120 h post-treatment (pt); untreated shoots at each time served as calibrators. Tissues were flash frozen and stored at −80 °C.

Grapevine plants (3 plants per treatment) were acclimatized with irrigation devices; roots were drenched for 25 min in 450 mL PAW, SDW, or FoAl. The 4th–6th leaves were sampled at 16, 26, and 36 h pt; untreated shoots at each time used as calibrators. Tissues were frozen and stored at −80 °C.

For small RNA high-throughput sequencing (HTS), periwinkle plants at the 5th–6th leaf stage (3 replicates per treatment) were immersed upside down for 25 min in 650 mL PAW or SDW and collected at 16 h pt; tissues were frozen at −80 °C.

RNA extraction: Total RNA from micropropagated periwinkle shoots was extracted using Qiagen RNeasy Plant Minikit with on-column DNase. Grapevine RNA was extracted using a CTAB/PVP protocol with LiCl precipitation and organic extractions. For small RNA HTS, total nucleic acid was extracted from periwinkle using a phenol-chloroform protocol. RNA quantity/quality were assessed by Nanodrop and agarose gel; samples were diluted to 250 ng/µL.

RT-qPCR: cDNA was synthesized with M-MLV reverse transcriptase and random hexamers. qPCR used SYBR Green chemistry with three technical replicates and ≥3 biological replicates. Reference genes: ubiquitin (periwinkle); actin, ubiquitin, GAPDH (grapevine). Target genes in periwinkle: CrCalS11 (callose synthase), CrPAL1 (phenylalanine ammonia-lyase), CrSGD (strictosidine-β-glucosidase), CrDAT (deacetylvindoline-O-acetyltransferase), and CrCHS (chalcone synthase). In grapevine: VvPAL1, VvCHS2, VvCHS3, VvSTS (stilbene synthase). Thermal profile: 95 °C 10 min; 40 cycles of 95 °C 15 s, 60 °C 1 min; melt curves performed. Primer efficiency by LinRegPCR; specificity verified by dissociation curves. Expression reported as 2^−ΔCT relative to reference(s). Statistics: ANOVA (P ≤ 0.05) followed by Student’s test.

Small RNA library preparation and sequencing: Six libraries (3 PAW, 3 SDW) were prepared; small RNA fraction purified on polyacrylamide gel, adapters ligated, cDNA synthesized and PCR amplified, gel-purified, and sequenced on Illumina HiSeq2000 (sRNA-seq). Raw reads deposited in NCBI GEO (GSE146177). Adapters trimmed (CLC Genomics Workbench v11); good quality reads (16–28 nt) retained. Reads mapped to C. roseus transcriptome (allowing one mismatch); expression normalized to reads per million (RPM). Differential expression analyzed using Baggerley’s test with FDR correction (P < 0.05). Additional stringency applied via Distributional Fold Change (DFC) and Interquartile Range (IQR) criteria (differences >2 and >5, respectively). Clustering and visualization via Clustvis; heat maps and PCA generated.

miRNA target prediction and GO analysis: Targets predicted using C. roseus transcriptome and confirmed with psRNATarget, CLC Genomics Workbench, and Blast2GO; only shared predictions retained. GO annotation and enrichment performed with Blast2GO; Fisher test with Bonferroni correction; significance P < 0.01 and FDR < 0.01.

• PAW chemistry: Compared to SDW (pH 5.5 ± 0.1; H2O2 0 mg/L; NO3− 0 mg/L), PAW had pH 2.78 ± 0.47, H2O2 13.5 ± 1.3 mg/L, and NO3− 81.9 ± 3.4 mg/L. Nitrites declined below detection shortly after treatment; H2O2 and NO3− remained stable for 24 h, including after freeze–thaw.
• Periwinkle micropropagated shoots (qRT-PCR): PAW induced significant overexpression of defense/phytoalexin pathway genes. • CrCalS11: increased 3.8-fold at 7 h pt (FoAl 7.5-fold at 7 h). Associated with callose deposition as a general stress response. • CrPAL1: increased 2.6-fold at 24 h pt (FoAl showed no increase, likely due to absence of pathogen). Induction consistent with H2O2 and NOx signaling effects. • CrSGD: increased ~5-fold (24 h), 5.4-fold (48 h), 6.8-fold (96 h) pt, indicating activation of alkaloid biosynthesis linked to antimicrobial activity. • CrDAT: slight late overexpression at 120 h pt. • CrCHS: no significant change.
• Grapevine cv Chardonnay (qRT-PCR at 16 h pt): PAW increased expression of: • VvPAL1: 2.9-fold; VvCHS2: 3.9-fold; VvCHS3: 1.9-fold; VvSTS: 4.0-fold. VvCHS1 showed no change. Results support induction of phenylpropanoid/stilbene (resveratrol) pathway.
• Small RNA sequencing (periwinkle plants, 16 h pt): • Libraries yielded ~18.86 million (SDW) and ~21.16 million (PAW) high-quality reads; 24-nt most abundant, then 21-nt. A total of 81 miRNAs (36 families) showed significant differential expression after PAW. • Down-regulated families: miR157, miR172, miR393, miR5368, miR8016. • Up-regulated families: miR159, miR165, miR319, miR395, miR396, miR398, miR399. • Heatmap and PCA showed clear separation between PAW and SDW treatments across biological replicates. • Functional insights: Upregulation of miR159, miR395, miR398 aligns with H2O2 exposure responses. miR165/166 target ABI1/ABI2 PP2C phosphatases linked to H2O2/glutathione signaling. miR398 targets SOD genes (CSD1/2/3), potentially modulating ROS detoxification and sustaining H2O2 signaling. Downregulation of miR157 (negative regulator of GST) supports oxidative response activation. miR395/399 implicated in nutrient deficiency responses, potentially enhancing N, P, and S uptake.
• Control comparisons: FoAl effects were more variable. A synthetic solution matching PAW’s H2O2, NO3−, and pH behaved like SDW (no effect), indicating PAW’s activity arises from a complex mixture of species rather than simple H2O2/NO3−/pH alone.

The findings demonstrate that PAW acts as an elicitor of plant defense pathways through its content of long-lived reactive oxygen and nitrogen species. In periwinkle, PAW triggered early callose synthase (CrCalS11) expression consistent with a general stress and defense response, and activated the phenylpropanoid/alkaloid pathway via PAL and SGD, leading to biosynthesis of defense-related indole alkaloids. In grapevine, PAW induced key nodes of the phenylpropanoid network (PAL, CHS2/3) and the stilbene synthase pathway (STS), which produce resveratrol and related phytoalexins known to combat pathogens such as Erysiphe necator, Plasmopara viticola, and Botrytis cinerea. The lack of effect from a synthetic solution with equivalent H2O2/NO3−/pH suggests synergy among multiple ROS/RNS and possibly other plasma-generated factors drives the biological response. At the post-transcriptional level, miRNA profiling revealed systemic modulation of stress-associated miRNAs. Upregulation of miR398 implies a transient reduction in SOD transcripts, potentially maintaining higher H2O2 levels to amplify defense signaling. Upregulated miR165/166 targeting ABI1/2 aligns with ABA/ROS cross-talk, while miR395 and miR399 implicate adjustments in nutrient signaling pathways that could support stress tolerance and metabolic demands during induced defenses. Downregulation of miR157, coupled with CrCalS11 overexpression, supports cytoskeletal and cell wall-associated changes like callose deposition. Collectively, transcriptional and miRNA data converge to indicate that PAW primes or induces defense responses without pathogen presence, supporting its potential use in integrated plant disease management to reduce pesticide reliance. The variable response to fosetyl aluminum under non-infected conditions underscores that PAW may effectively trigger defenses even in the absence of pathogen cues. These results encourage further development of PAW applications in greenhouse and field settings, focusing on optimizing plasma parameters and understanding the specific bioactive species responsible for elicitation.

PAW, generated via nanosecond pulsed DBD in air, contains stable ROS/RNS that elicit plant defense responses. In periwinkle and grapevine, PAW enhanced expression of key enzymes in phytoalexin biosynthesis (alkaloids in C. roseus; stilbenes in V. vinifera) and modulated stress-responsive miRNAs, indicating activation of oxidative signaling and defense pathways. A synthetic mimic of H2O2/NO3−/pH failed to reproduce PAW’s effects, highlighting the importance of the complex mixture produced by plasma. These results provide a foundation for applying PAW technology in plant disease management to improve yield and quality while reducing chemical inputs. Future work should: (i) scale up PAW generation and delivery systems; (ii) identify and optimize the specific PAW components driving resistance; (iii) evaluate durability and breadth of induced resistance under pathogen challenge; and (iv) assess agronomic performance and environmental impacts under greenhouse and field conditions.

• The study primarily assesses molecular markers (gene expression and miRNA profiles) without direct pathogen challenge assays or disease outcome measurements.
• Sample sizes were modest (e.g., 5 micropropagated shoot replicates per treatment; 3 grapevine plants per treatment), potentially limiting statistical power.
• Time points focused on short-term responses (hours to 5 days); longer-term durability of induced resistance was not assessed.
• The synthetic solution control suggests complexity in PAW’s active components, but specific identities and relative contributions of reactive species were not resolved.
• Fosetyl aluminum effects were variable in the absence of pathogens, complicating comparisons to standard resistance inducers.