Identification and improvement of isothiocyanate-based inhibitors on stomatal opening to act as drought tolerance-conferring agrochemicals

Stomatal pores, controlled by guard cells, regulate gas exchange in plants, balancing CO2 uptake for photosynthesis and water loss. The primary driving force for stomatal opening is the plasma membrane (PM) H+-ATPase, activated by phosphorylation via a signal transduction pathway initiated by blue light perception. Understanding this pathway is crucial for improving plant productivity and drought resistance. Previous studies identified components of this pathway, including phototropins, BLUS1, BHP, and PPI, leading to PM H+-ATPase phosphorylation and subsequent K+ uptake, causing guard cell swelling and stomatal opening. Conversely, abscisic acid (ABA) inhibits PM H+-ATPase phosphorylation, promoting stomatal closure. However, some components remain unidentified. Chemical screening offers a valuable tool to investigate and manipulate stomatal movement. Previous work using this approach identified various stomatal-regulating compounds, including SCL1, which showed potential as a drought-tolerance agrochemical. This study aims to identify and improve more potent stomatal opening inhibitors for further research and potential agrochemical development.

Studies have used compounds like fusicoccin (FC) to elucidate the PM H+-ATPase activation mechanism. FC stabilizes the binding of phosphorylated PM H+-ATPase and 14-3-3 protein, inhibiting dephosphorylation and promoting stomatal opening. Microtubule-targeting drugs have also highlighted the role of microtubule rearrangement in stomatal movement. Chemical screening previously identified BHP, a Raf-like kinase, as a signaling component in blue light-induced stomatal opening, along with several stomatal closing compounds (SCLI-SCL9, protease inhibitors, and aminated oxazoles). Among these, SCL1 showed the highest activity and effectively suppressed wilting, suggesting its potential as a drought-tolerance agrochemical. However, efforts to improve SCL1's bioactivity through structure-activity relationship (SAR) studies were unsuccessful. This prompted a search for more potent inhibitors for further SAR research and molecular improvement.

A chemical library of 380 compounds was screened using *Commelina benghalensis* leaf discs to identify inhibitors of light-induced stomatal opening. Benzyl isothiocyanate (BITC) emerged as a potent inhibitor. Its effects were further investigated using *Arabidopsis thaliana*. The mode of action of BITC was studied by examining its impact on PM H+-ATPase phosphorylation using immunohistochemistry and immunoblotting. In vitro ATPase activity was assessed to determine if BITC directly affected the enzyme's catalytic activity. The effects of BITC on other signaling components, such as phototropin and ABA signaling, were also investigated. A structure-activity relationship (SAR) study was conducted to generate BITC derivatives with enhanced bioactivity. Various BITC derivatives were synthesized and tested for their ability to inhibit stomatal opening in *C. benghalensis*. The most active derivatives, termed multi-ITCs, were further analyzed for their effects on PM H+-ATPase phosphorylation and their transcriptomic impact using RNA-seq. Finally, the drought-tolerance effects of BITC and multi-ITCs were evaluated using Chrysanthemum bouquets and *Brassica rapa* plants under water-deficit conditions. Leaf wilting was assessed, and stomatal conductance was measured to gauge the effects on plant water status. Fluorescein diacetate (FDA) assay was used to assess guard cell viability after BITC treatment. Seed germination and reverse transcription-PCR analyses were employed to determine any effect on ABA related responses.

BITC, a Brassicales-specific metabolite, was identified as a potent inhibitor of light-induced stomatal opening. BITC suppressed PM H+-ATPase phosphorylation, but did not directly inhibit its in vitro ATPase activity. The inhibitory effect was observed in both *C. benghalensis* and *A. thaliana*, suggesting a conserved mechanism. BITC's action was independent of ABA signaling. A SAR study led to the development of multi-ITCs, demonstrating up to a 66-fold increase in inhibitory activity compared to BITC. Multi-ITCs also suppressed PM H+-ATPase phosphorylation and showed minimal toxicity. RNA-seq analysis revealed that m-bis-BITC triggered a qualitatively similar, but less extensive, transcriptomic response compared to BITC. Importantly, both BITC and, more effectively, multi-ITCs suppressed leaf wilting in short- and long-term drought experiments in Chrysanthemum bouquets and *B. rapa*. m-bis-BITC exhibited prolonged effectiveness compared to BITC and ABA. In *B. rapa*, 50 µM m-bis-BITC showed significant drought tolerance without visible toxicity.

This study reveals a novel biological function of BITC in plants – the inhibition of PM H+-ATPase phosphorylation, independent of the ABA pathway. The significantly improved activity of multi-ITCs, particularly m-bis-BITC, makes them promising candidates for drought-tolerance agrochemicals. Their prolonged effectiveness and low toxicity, compared to ABA, are significant advantages. The lack of significant effect on ABA related responses further supports the conclusion that the mechanism is ABA independent. While m-bis-BITC showed improved activity, its transcriptomic impact was less extensive than BITC, suggesting greater specificity. The identification of BITC's molecular target is crucial for a more in depth understanding of PM H+-ATPase regulation and for developing further optimized agrochemicals. The observed effects in various plant species suggests a conserved mechanism of action.

This research uncovered a novel role for BITC in regulating stomatal opening by inhibiting PM H+-ATPase phosphorylation and developed highly effective multi-ITC derivatives. m-bis-BITC, in particular, demonstrated superior activity and duration of action compared to ABA, making it a promising lead compound for drought-tolerant agrochemicals. Future research should focus on identifying the molecular target of BITC to further refine the design of optimized agrochemicals and to gain a complete understanding of the signaling pathway involved.

While the study demonstrated the effectiveness of m-bis-BITC in suppressing wilting and enhancing drought tolerance, further research is needed to fully assess potential long-term effects on plant growth and development, especially in different growth stages or under nutrient limitation. Although the FDA assay showed no significant cytotoxicity on guard cells, effects on other plant tissues need investigation. The effects of prolonged exposure to multi-ITCs in different environmental conditions requires further study. The specific molecular target of BITC remains to be identified.