The biologically active molecules karrikinolide (KAR₁) and trimethylbutenolide (TMB) present in wildfire smoke play a key role in regulating seed germination of many plant species. To elucidate the physiological mechanism by which smoke-water (SW), KAR₁, and TMB regulate seed germination in photosensitive ‘Grand Rapids’ lettuce (Lactuca sativa), we investigated levels of the dormancy-inducing hormone abscisic acid (ABA), three auxin catabolites, and cytokinins (26 isoprenoid and four aromatic) in response to these compounds. Activity of the hydrolytic enzymes α-amylase and lipase along with stored food reserves (lipids, carbohydrate, starch, and protein) were also assessed. The smoke compounds precisely regulated ABA and hydrolytic enzymes under all light conditions. ABA levels under red (R) light were not significantly different in seeds treated with TMB or water. However, TMB-treated seeds showed significantly inhibited germination (33%) compared with water controls (100%). KAR₁ significantly enhanced total isoprenoid cytokinins under dark conditions in comparison with other treatments; however, there was no significant effect under R light. Enhanced levels of indole-3-aspartic acid (an indicator of high indole-3-acetic acid accumulation, which inhibits lettuce seed germination) and absence of trans-zeatin and trans-zeatin riboside (the most active cytokinins) in TMB-treated seeds might be responsible for reduced germination under R light. Our results demonstrate that SW and KAR₁ significantly promote lettuce seed germination by reducing levels of ABA and enhancing the activity of hydrolytic enzymes, which aids in mobilizing stored reserves. However, TMB inhibits germination by enhancing ABA levels and reducing the activity of hydrolytic enzymes.

Natural biostimulants such as plant growth-promoting rhizobacteria (PGPRs) and seaweed extracts are used in agriculture to improve crop yield in an environmentally friendly approach. However, interaction effects between different biostimulants are seldom investigated. This study aimed to determine the combined effect of PGPRs and Kelpak^® (KEL), a seaweed-derived extract, on the growth and biochemical composition of Amaranthus hybridus, a traditional leafy vegetable of South Africa. A pot trial was conducted with 6 treatments—control (distilled water), Bacillus licheniformis (BL) inoculum, Pseudomonas fluorescens (PF) inoculum, 1% KEL, and combination treatments of KEL + BL and KEL + PF. These were applied 3 times to A. hybridus seedlings at 2 weekly intervals prior to harvesting after 6 weeks. Growth parameters and biochemical content were quantified in the harvested material. Kelpak^® significantly improved growth (leaf number and area, shoot and root length, and fresh weight) and chlorophyll and carotenoid content. Treatments with 2 microorganisms had a significant negative effect on growth and pigment content. However, the combination treatments significantly improved growth and photosynthetic pigment content to similar levels as the KEL treatment. The mineral content was significantly increased for most minerals when treated with PF and decreased when treated with KEL and KEL + BL. Treatment with 2 bacterial strains and KEL caused a significant decrease in superoxidase dismutase activity with an additive effect when applied in combination. This suggests stress reduction in A. hybridus. Combined use of PGPR and seaweed extracts promises to be an eco-friendly strategy to improve the production and mineral content of traditional leafy vegetables such as A. hybridus.

Karrikins (KARs) are plant growth regulators that promote seed germination and the subsequent growth and development of seedlings of many plant species. In nature they are generated and released by combustion of plant material and promote the restoration of burned ecosystems. Smoke water can be artificially prepared as a saturated extract of all substances in smoke produced by burning plants, and it has various horticultural and agricultural applications. We have developed, validated and applied the first fast, specific and sensitive method, based on ultra-high performance liquid chromatography–tandem mass spectrometry, for quantifying KARs in smoke water. To assist these efforts and further analyses, standards of the main KARs (which are not commercially available) were synthesized. Due to the complex matrix of smoke waters, two quantification approaches (standard dilution with a structural KAR analogue and standard addition) were compared. The standard addition method allowed absolute quantification of KARs in six of eight smoke water samples of diverse origins and ages. Our findings reveal differences in both total and relative levels of KARs in smoke water, and indicate that differences in its KAR composition may be linked to variations in its bioactivity.