Karrikins: A novel plant growth regulator with emerging agronomic applications

Introduction

Plant growth regulators (PGRs), commonly referred to as plant hormones or phytohormones, are organic molecules that govern fundamental aspects of plant growth and development. These include the regulation of branching, shoot elongation, return bloom, fruit thinning, and maturity modification. Phytohormones are typically produced in small amounts at a specific site within the plant (the source) and then transported to a other site (the target) where they exert their effects. All plant groups, including algae, produce phytohormones, and these molecules serve roles analogous to those found in higher plants (Tarakhovskaya et al., 2007) [25] . Some phytohormones are also found in microorganisms such as unicellular fungi and bacteria, though they don't have a hormonal activity so it is better to classified as secondary metabolites (Rademacher, 1994) [22] . The classical group of phytohormones including auxins, gibberellins, cytokinins, abscisic acid, and ethylene. In recent decades, novel hormones including brassinosteroids, salicylic acid, strigolactones, and polyamines have also been added to this repertoire. Among the most recently discovered is karrikinsbutenolide compounds that are not endogenously produced within plants but are generated externally during the combustion of plant material (Flematti et al., 2013) [6] . Unlike conventional PGRs, karrikins are fire-derived exogenous signals that exhibit remarkable biological activity across a wide range of plant species, including important agricultural crops. The present review comprehensively summarizes the discovery, chemistry, mechanism of action, comparison with strigolactones, smoke water preparation, interaction with other hormones, role under abiotic stress, and effects of karrikins on crop growth and development, with perspectives on future research. [19] . They are notable for their ability to stimulate seed germination across a wide spectrum of plant species. Wildfires, oftenly known as bushfires, are a natural occurrence globallyfrom Australia and California to South Africa. During these events, the combustion of vegetation generates smoke and ash laden with karrikins (KARs). After the fire subsides, KARs accumulate on the soil surface in a dormant state until rainfall occurs. Upon the arrival of rain, karrikins are leached into the soil and stimulate the germination of seeds that had remained dormant in the seed bank (Fig. 1). Certain plant species have evolved to exploit this phenomenon, growing rapidly, flowering, producing seed, and completing their lifecycle within one to two years after a fire. These are referred to as 'fire-followers' or 'fire-ephemerals.' This ecological strategy is highly adaptive: wildfires liberate nutrients that were bound in plant biomass, reduce competition from established vegetation, and create an open environment where seedlings can successfully establish. Importantly, rainfall without a preceding fire does not induce germination in fire-ephemeral species, indicating that these seeds can undergo repeated cycles of imbibition (Wet) and dehydration (dry) while remaining dormant and karrikin-sensitive. Such seeds may persist viably in the soil seed bank for decades between fire events. KARs thus play a significant role in the ecological regeneration of firedevastated landscapes.

Fig 1:

The role of karrikins in revegetation after a fire (Flematti et al., 2015) [4] Discovery of Karrikins:

The first instance of seed germination induced by the combustion of plant material was made by Wicklow in 1997, who demonstrated that charred stem segments placed on damp filter paper could stimulate the germination of dormant seeds.

However, the identification of the active chemical compound responsible for this effect did not occur until 2004. In that year, two scientific groups simultaneously identified the first active chemical in smoke -KAR1 (3-methyl-2H-furo[2,3-c]pyran-2one)using bioassay-guided fractionation of smoke water, gas chromatography-mass spectrometry (GC-MS), and nuclear magnetic resonance (NMR) spectroscopy. Flematti et al. (2004) [5] used burnt cellulose as the smoke source, while Van Staden et al. (2004) [27] employed smoke-saturated water generated from burned plant material.

The butenolide compounds identified from plant-derived smoke were subsequently named 'Karrikins' (KARs), derived from the Noongar Aboriginal word 'karrik' meaning smoke. KAR1 was recognized as the most potent germination stimulator among the karrikins. Structurally, karrikin is a bicyclic molecule with a molecular mass of 150 Da produced during partial combustion of cellulose. It promotes cotyledon expansion and chlorophyll accumulation while inhibiting hypocotyl elongation. KARs improve seedling vigor and photomorphogenesis and play a crucial role in plant abiotic stress tolerance. Biological activity has been observed not only in fire-follower species but also in non-fire followers, including Arabidopsis thaliana, tomato, maize, rice, and lettuce. To date, six karrikin molecules (KAR1 to KAR6) have been identified from plant-derived smoke, each differing in the presence and position of methyl group(s) (Fig. 2).

Preparation of Smoke Water

The water solubility of active smoke compounds was first demonstrated by De Lange and Boucher (1990) [3] , which led to the development of smoke water (SW) as a practical formulation for horticultural and agricultural use. Smoke water is prepared by bubbling smoke generated from the controlled burning of plant material through water (Fig. 3), a process in which the biologically active components rapidly dissolve. This technique, described by Brown and Van Staden (1997) [1] and refined by subsequent researchers (Van Staden et al., 2004; Kulkarni et al., 2011) [27,12] , is valued for being low-cost, simple to execute, and requiring minimal material.

KARs sustain seed germination-promoting activity at concentrations as low as 10 -9 mol/L (Light et al., 2009). Smoke water exhibits a 'dual regulatory' effect on germination: stimulating germination at lower concentrations while inhibiting it at higher concentrations (Light et al., 2002) [15] . The germination-inhibiting compound has been identified as 3,4,5trimethylfuran-2(5H)-one (Light et al., 2010) [13] . Depending on the plant species, smoke water should be diluted at ratios of 1:250, 1:500, 1:1000, 1:1500, or 1:2000 (v/v) before application.

Once autoclaved, smoke water can be stored for extended periods (Van Staden et al., 2004; Light et al., 2004) [27,16] .

Fig 3:

Apparatus designed for the preparation of smoke water (Gupta et al., 2020) [9] .

Comparison of Karrikins and Strigolactones

Karrikins are structurally and functionally analogous to strigolactones (SLs). The first strigolactone, strigol, was isolated from cotton root exudates and named for its ability to stimulate germination in the parasitic plant Striga lutea. Strigolactones promote the growth and branching of symbiotic arbuscular mycorrhizal fungi and regulate shoot branching in plants. Both KARs and SLs are butenolide molecules, yet differ significantly in their origin, biological context, and molecular targets. Table 2 presents a comparative summary of these two classes of plant regulators. [2,18,29] . Two key genes govern the karrikin response: KAI2 (KARRIKIN INSENSITIVE 2) and MAX2 (MORE AXILLARY GROWTH 2). Figure 4 presents a model of KAR and SL signaling pathways. KAI2 belongs to the α/β-hydrolase superfamily and serves as the karrikin receptor. Upon karrikin binding, KAI2 forms a receptor-ligand complex, analogous to the role of DWARF14 (D14) in strigolactone signaling (Guo et al., 2013) [8] . The F-box protein MAX2, a member of the leucine-rich-repeat (LRR) family, forms part of an SKP1-Cullin-F-box (SCF) E3 ubiquitin ligase complex. This complex facilitates substrate-specific polyubiquitination via the 26S proteasome pathway, leading to the targeted degradation of repressor proteins (Waters, 2017) [29] . The downstream repressors in karrikin signaling are SMAX1-LIKE (SMXL) proteins, which function as transcriptional corepressors related to ATP-dependent heat shock proteins (Yao and Waters, 2019) [32] . The SMXL family comprises eight members: SMAX1 and SMXL2 are engaged in KAR signaling; SMXL3-SMXL5 govern phloem development independently of both KAR and SL; and SMXL6-SMXL8 are involved in SL signaling (Stanga et [24,23,28] .

Interaction of Karrikins with other Plant Hormones

Plant growth and development involve complex hormonal crosstalk. Understanding how karrikins interact with other plant hormones is essential for elucidating their role in seed germination and plant development.

1. ABA inhibits the action of KARs on seed germination. 2. KARs promote seed germination by stimulating GA biosynthesis, whereas ABA counteracts this effect by promoting seed dormancy (Nelson et al., 2009) [20] . 3. KARs suppress the expression of IAA (auxin) response genes (Nelson et al., 2011; Gilkerson et al., 2015) [21,7] , potentially accelerating germination by attenuating auxin signaling. 4. KARs inhibit hypocotyl elongation and lateral root development through mechanisms yet to be fully characterized, while simultaneously promoting root hair elongation, nodule development, and arbuscular mycorrhizal colonization (Yang et al., 2019) [31] . 5. The interactions between KARs and gibberellins, abscisic acid, and auxins collectively orchestrate transitions between dormancy and active germination under varying environmental conditions.

A. Role of Karrikins under Abiotic Stress 1. In Arabidopsis, KARs protect seeds from abiotic stress during germination and promote drought resistance by enhancing antioxidant defense, cuticle formation, stomatal closure, and anthocyanin biosynthesis (Yang et al., 2019) [31] . 2. KAR-KAI2 signaling maintains seed dormancy under unfavorable abiotic conditions while promoting germination under favorable conditions (Fig. 5) (Yang et al., 2019) [31] . 3. KARs improve seedling sensitivity to light and enhance seedling establishment under low-light conditions (Waters and Smith, 2013) [30] . 4. Meng et al. (2016) [17] demonstrated that KARs delay soybean seed germination specifically under shaded conditions by promoting ABA biosynthesis and inhibiting GA production. This effect was not observed under white light or darkness, indicating a light-dependent modulation of karrikin activity distinct from its role in Arabidopsis. [11,26] . 2. In maize, soaking kernels in 100 nM KAR1 for 1 hour enhanced post-germination seedling growth compared to untreated controls (Van Staden et al., 2006) [26] . 3. KAR1 has been demonstrated to promote tomato seed germination and subsequent seedling vigor even when grown at sub-optimal (10°C) or supra-optimal (40°C) temperatures. KAR1 also increased plant height, fresh and dry weight, stem thickness, and leaf number (Jain et al., 2006) [10] . 4. KAR2 promotes Arabidopsis seed germination under favourable environmental conditions.

Conclusion and Future Perspectives

Karrikins are a new and intriguing class of plant growth regulators that have important ramifications for both applied agronomy and basic plant biology. The KAI2 receptor, the MAX2 F-box protein, and the SMXL repressor proteins are among the fundamental elements of KAR signaling that have seen significant advancements. Nevertheless, there is still much to learn about the exact molecular role of this route under both normal and stress conditions. The next logical stages in determining the whole mode of action of karrikins are genomic and transcriptome techniques. Furthermore, comprehensive research utilizing integrated physiological, biochemical, and molecular methods is necessary to understand how KARs interact with other plant hormones under various abiotic stress conditions. The practical use of smoke water and pure karrikin formulations in crop production-especially for enhancing seedling establishment, stress tolerance, and germination under severe temperatures-offers significant agricultural potential. A fascinating new area in the fields of molecular plant physiology, plant evolutionary biology, and fire ecology has been made possible by the discovery of karrikins. As our understanding grows, karrikins have the potential to be valuable tools in the establishment of sustainable, stress-resilient cropping systems.

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