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

Stomata, formed by pairs of guard cells, balance CO2 uptake for photosynthesis with water loss by adjusting their aperture in response to environmental cues such as blue light, photosynthetic activity, CO2, and stress signals including abscisic acid (ABA). Blue light strongly promotes stomatal opening via activation of the guard cell PM H+-ATPase through phosphorylation of a penultimate threonine, leading to membrane hyperpolarization, K+ uptake, and guard cell swelling. ABA conversely inhibits PM H+-ATPase phosphorylation to promote closure. While major components such as phototropins, BLUS1, BHP, and PP1 are known, additional regulators likely exist. Small molecules are powerful tools to dissect and manipulate stomatal signaling. Fusicoccin (FC) stabilizes phosphorylated PM H+-ATPase–14-3-3 complexes, microtubule drugs have implicated cytoskeletal roles, and prior chemical screens identified stomatal-closing compounds (SCL, PI, SIM series), with SCL1 conferring drought tolerance via an ABA-independent pathway but lacking more potent analogs. Motivated to find more potent, potentially safer and longer-lasting stomatal opening inhibitors amenable to structure-activity relationship (SAR) optimization, the authors screened a pharmacologically annotated library.

Prior work established blue light signaling via phototropins to PM H+-ATPase activation with 14-3-3 binding upon C-terminal Thr phosphorylation; genetic and biochemical studies confirm PM H+-ATPase’s central role in opening. ABA signaling promotes closure via SLAC1 activation and inhibition of PM H+-ATPase phosphorylation. Chemical biology has elucidated stomatal pathways: fusicoccin stabilizes the phosphorylated PM H+-ATPase–14-3-3 complex; microtubule perturbation suggested cytoskeletal contributions. Previous chemical screens in Commelina benghalensis identified stomatal-closing series (SCL1–9, protease inhibitor PI series, SIM series), with SCL1 (IC50 ~4.62 µM) suppressing wilting via an ABA-independent mechanism. Isothiocyanates (ITCs), electrophiles that covalently modify proteins (notably Cys/Lys), have mammalian targets (Keap1, cytochrome P450s, tubulin) and are Brassicales defensive metabolites in plants, but plant cellular targets are largely unknown. Reports show allyl ITC affects stomatal movements without altering FC-induced PM H+-ATPase phosphorylation, and BITC can promote stomatal closure via redox and ion changes. This study builds on these findings to explore BITC’s role in inhibiting PM H+-ATPase phosphorylation and to improve potency through SAR.

- Chemical screening: 380 compounds from the International Drug Collection were screened at 50 µM using Commelina benghalensis leaf discs incubated in basal buffer under red and blue light; stomatal apertures were quantified after tiered qualitative and quantitative rounds. - Cross-species assays: Stomatal opening assays were conducted in Arabidopsis thaliana and Chrysanthemum leaves; blue light and FC-induced opening were measured. - PM H+-ATPase phosphorylation: Guard-cell immunohistochemistry with anti-phospho-Thr antibody assessed effects on blue light- and FC-induced phosphorylation; mesophyll cell protoplast (MCP) immunoblotting evaluated FC-induced phosphorylation. - Enzyme activity: In vitro vanadate-sensitive ATP hydrolytic activity of PM H+-ATPase was measured in microsomal fractions with/without BITC. - Upstream signaling tests: Phototropin 1 autophosphorylation band-shift assays evaluated photoreceptor activity in MCPs; ABA signaling impact assessed via AKS phosphorylation in Vicia faba guard cell-enriched epidermis, Arabidopsis seed germination, and ABA-responsive gene expression (RAB18, RD29B), including tests in ABA-insensitive mutants (abi1-1, ost1-2). - SAR study: Systematic modifications of BITC’s ITC moiety, aryl core, linker length, and substituents were synthesized and evaluated for IC50 against light-induced opening in C. benghalensis. Multi-ITC derivatives (para/meta bis- and 1,3,5-tris-ITCs; cyclohexyl-cored bis-ITC) were synthesized and tested, including phosphorylation assays and cytotoxicity (FDA) checks. - Transcriptomics: Arabidopsis leaf discs treated with BITC (50 µM) or m-bis-BITC (5 µM) for 3 h underwent RNA-seq (4 biological replicates); DEGs were identified (edgeR) and GO enrichment performed (PANTHER). - Drought-tolerance assays: Chrysanthemum bouquets had leaves dipped in solutions (with spreading agent), irradiated 3 h, then dehydrated to assess wilting and stomatal conductance; longer-term persistence (3 vs 48 h) compared ABA, BITC, m-bis-BITC. Brassica rapa potted plants were foliar-sprayed with m-bis-BITC (50 µM) or control, then subjected to 24 h drought in greenhouse conditions; growth/visible toxicity monitored for up to 10 days. - General plant growth, protoplast preparation, immunoblotting, and detailed protocols are provided in Methods with reagent compositions and conditions.

- Primary hit: BITC strongly inhibited light-induced stomatal opening in C. benghalensis with IC50 = 29.1 µM (no cytotoxicity), and reduced FC-induced opening; also effective in A. thaliana guard cells. - Mechanism: BITC suppressed PM H+-ATPase phosphorylation in guard cells under blue light and in response to FC; similarly inhibited FC-induced phosphorylation in mesophyll protoplasts. In vitro ATP hydrolytic activity of PM H+-ATPase was not significantly affected by 50 µM BITC, suggesting action on phosphorylation regulation rather than catalytic function. - Pathway specificity: BITC did not inhibit phototropin 1 autophosphorylation (contrast with staurosporine) and did not activate canonical ABA signaling (no induction of AKS phosphorylation in Vicia guard cells, no effects on Arabidopsis seed germination or RAB18/RD29B expression); it inhibited opening in ABA-insensitive mutants (abi1-1, ost1-2), indicating an ABA-independent mechanism. - SAR insights: Activity requires the ITC moiety; isomers/analogues (thiocyanate, isocyanate, benzylamine) were weak/inactive. Aromatic core preferred; thiophene tolerated (IC50 20.6 µM), cyclohexylmethyl ITC inactive. Chain length: phenyl ITC (short linker) inactive; longer linkers tolerated but less effective at high concentrations. Substituents on phenyl ring widely tolerated; bulky iodo and phenyl substitutions improved potency. Biphenyl-ITCs showed enhanced activity regardless of substitution position (e.g., meta-biphenyl-ITC IC50 5.3–7.0 µM). - Multi-ITCs: Adding multiple ITC groups greatly increased potency: p-bis-BITC IC50 3.5 µM (8.5-fold), m-bis-BITC IC50 1.7 µM (17-fold), 1,3,5-tris-BITC IC50 0.44 µM (66-fold). A cyclohexyl-cored bis-ITC retained activity (IC50 11.4 µM) but was less potent than aryl bis-ITCs. Multi-ITCs also inhibited PM H+-ATPase phosphorylation in guard cells without cytotoxicity. - Transcriptomics: m-bis-BITC (5 µM) upregulated 334 and downregulated 155 genes; 76% of up- and 81.3% of down-regulated genes overlapped with BITC (50 µM). Overlapping DEGs enriched for responses to abiotic stress (up) and biotic/defense responses (down). m-bis-BITC-specific DEGs showed limited additional categories (notably response to heat), suggesting similar or weaker transcriptional impact relative to BITC. - Drought tolerance and persistence: In intact Chrysanthemum leaves, multi-ITCs achieved inhibition at ~50-fold lower concentrations than BITC; tris-BITC was less effective than m-bis-BITC in fully suppressing opening in leaves. BITC and m-bis-BITC reduced wilting after 1.5 h dehydration. Over longer periods, inhibitory effects of ABA (100 µM) and BITC (2,500 µM) were alleviated within 48 h, whereas m-bis-BITC (50 µM) maintained suppression of stomatal opening and lowered conductance; in Arabidopsis leaves, m-bis-BITC suppressed PM H+-ATPase phosphorylation for at least 2 days (alleviated by day 5). - Safety: BITC at 2,500 µM caused withering within 3 days despite suppressing wilting; m-bis-BITC was effective at 50 µM without visible damage over 10 days. In Brassica rapa, foliar 50 µM m-bis-BITC improved drought tolerance (reduced wilting at 24 h) without growth inhibition or visible damage over 10 days. - Comparative potency: The most potent derivative (tris-BITC, IC50 0.44 µM) exhibited greater activity than ABA (IC50 ≈ 2.9 µM in comparable assay conditions).

The study addressed the need for potent, ABA-independent chemical tools to suppress stomatal opening and enhance drought tolerance. Identification of BITC as an inhibitor of PM H+-ATPase phosphorylation reveals a previously unrecognized plant role for this Brassicales metabolite and indicates that its action is conserved across tissues (guard and mesophyll) and species. SAR optimization demonstrated that aryl substitution and especially multiplication of ITC groups dramatically enhance potency, yielding multi-ITCs with activities comparable to or exceeding ABA and with longer functional persistence on leaves. The action is independent of phototropin activation and canonical ABA signaling, reducing potential ABA-associated side effects. Transcriptomic profiling suggests that the improved derivative, m-bis-BITC, exerts similar regulatory impacts as BITC but with a generally limited and partly weaker transcriptional footprint, supporting specificity for the stomatal opening pathway. These findings provide promising agrochemical candidates for conferring drought tolerance and mechanistic probes to uncover unknown components that regulate PM H+-ATPase phosphorylation. The unknown molecular target may involve kinases, regulators, or the ATPase itself; the enhanced potency with multiple electrophiles suggests multivalent covalent interactions or improved noncovalent affinity. While effective and seemingly safe in short-term surface applications to mature plants, broader physiological impacts require evaluation given PM H+-ATPase roles in growth, nutrient uptake, and hydraulics.

BITC was identified as a potent, ABA-independent inhibitor of stomatal opening by suppressing PM H+-ATPase phosphorylation. Structure-guided optimization produced multi-ITC derivatives (notably m-bis-BITC and tris-BITC) with up to 66-fold higher potency, longer-lasting effects on leaves, and negligible toxicity at effective concentrations. These compounds reduced wilting in cut Chrysanthemum leaves and conferred drought tolerance to Brassica rapa without visible growth penalties, establishing multi-ITCs as promising drought tolerance-conferring agrochemicals. Future work should identify the direct molecular target(s) and binding mode of BITC/multi-ITCs, refine scaffold design (e.g., optimal positioning and spacing of ITC groups), and assess species breadth, application methods, environmental stability, and potential off-target impacts on other PM H+-ATPase-dependent processes.

- The direct molecular target(s) of BITC and multi-ITCs remain unidentified; conclusions on mechanism are based on phosphorylation assays and pathway exclusion (phototropin, ABA) rather than target engagement. - BITC required high concentrations for intact leaf efficacy and caused withering at effective doses (2,500 µM) over several days, although m-bis-BITC mitigated this. - Tris-BITC, while most potent in disc assays, was less effective in completely suppressing opening in intact Chrysanthemum leaves compared to m-bis-BITC, indicating context-dependent performance. - Long-term and developmental stage-specific effects, as well as impacts under nutrient limitations, were not comprehensively assessed; PM H+-ATPase functions in growth, nutrient uptake, and hydraulics could be affected. - Persistence and reversibility were evaluated over a limited timeframe (days); environmental fate and field performance remain to be tested.