Towards Increased Crop Yields with Molecular Heater Technology

doi:10.21203/rs.3.rs-3466431/v1

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Towards Increased Crop Yields with Molecular Heater Technology

https://doi.org/10.21203/rs.3.rs-3466431/v1

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Food security is fast becoming one of the most pressing issues of the 21^st century. With increasing climate instability, crop growth is now routinely affected by adverse weather patterns, which can result in reduced crop yields and even crop death. We are developing an environmentally responsible molecular heater to help boost plant growth, to expand the geographic regions suitable for such growth and to protect plants against cold weather. Our molecular heater, sinapic diacid (SDA, 2-(4-hydroxy-3,5-dimethoxybenzylidene)malonic acid), acts as a light-to-heat agent, converting solar energy into heat energy delivered to the plant. We have designed and optimised a sustainable chemical synthesis of SDA, characterised its photophysical properties using a combination of steady-state and ultrafast laser spectroscopy techniques and complementary computational studies, assessed its light-to-heat energy transfer performance to plant leaves in vivo, and explored key environmental and toxicological endpoints through in silico studies. Our spot-test results demonstrate that adsorbed SDA efficiently converts solar energy to heat energy, increasing the local plant leaf temperature by ~ 0.2 °C after sustained irradiation, with no alerts to environmental fate and human toxicity, and with impressive resilience to prolonged solar irradiation. Our studies reveal the hitherto untapped potential of molecular heaters such as SDA for helping to boost plant growth and protect plants against climate instability, with transformative potential towards food security.

Physical sciences/Chemistry/Physical chemistry

Biological sciences/Biotechnology/Environmental biotechnology

Health sciences/Risk factors

Extreme weather events are known to cause damage in plants, including crops of significant importance for food supply.¹ This damage constitutes a severe threat to food supplies and food security.^2–5 In fact, the likelihood of frost damage to crops may increase due to climate change, since starker temperature variations, with periods of relatively warm weather followed by extreme cold, may prove more damaging to crops than long periods of cold weather.⁵ Such extreme temperature variations are increasingly being observed around the world, causing an estimated economic loss of € 3.3 billion across Europe in 2017.⁶ Mitigating the adverse effects of these weather events is an essential step towards improved food security, in line with Goal 2 of the United Nations’ Sustainable Development Goals for 2030 and the Zero Hunger initiative by the United Nations’ Department of Economic and Social Affairs.⁷

To address the adverse physiological effects of weather damage, we aim to develop molecularheater technology by creating a foliar spray containing molecular heaters to be applied directly on to the plant surface. The application of a molecular heater has the potential to raise the leaf temperature – thereby boosting plant growth and likely expanding the geographic region capable of supporting such plant growth – and to mitigate against daytime chilling damage and potentially prevent frost damage. The new class of molecules developed in this work converts photon energy afforded by absorption of ultraviolet radiation (UVR, 280‑400 nm) to thermal energy (heat) through an efficient, energy-conserving relaxation mechanism. Other materials that have been shown to offer such a light-to-heat conversion capability are typically homogeneous structures like graphene and carbon nanotubes.^8,9 However, it is well known that photoexcitation of small organic molecules can result in vibrational temperatures of thousands of Kelvin.^10–12 The high vibrational temperatures achieved by these systems present a hugely promising source for energy transfer from the heater to the surrounding environment.

A molecular heater for crop protection must meet several key criteria: first, it should absorb solar radiation efficiently in a region of the electromagnetic spectrum that does not overlap with that required for photosynthesis.¹³ Second, it should be able to dissipate the energy gained by solar radiation absorption into heat with close-to-unity quantum yields across the range of environments to which it is exposed. Finally, and crucially, its application onto the plant should not adversely affect plant physiology or the surrounding environment, which includes being non-toxic to the soil, water, humans, and other animals.

Here we report the results of studies employing a broad suite of techniques utilized to design and test the effectiveness of a proposed molecular heater, sinapic diacid (SDA, 2-(4-hydroxy-3,5-dimethoxybenzylidene)malonic acid), shown in the inset of Figure 1.¹⁴ We have used ultrafast transient absorption spectroscopy alongside time-dependent density functional theory (TD-DFT) and in vivo plant testing to track the energy transfer following photoexcitation of the molecular heater to the plant leaf. These studies have been complemented with in silico approaches to predict environmental fate and human toxicity of SDA, and comparative photostability studies of SDA and a collection of structurally similar molecules. The present studies of SDA are performed as proof of concept, highlighting the vast potential for expanding molecular heater technology using structurally similar systems.¹⁴

Photodynamical studies

Figure 1 compares the steady-state UV/Vis absorption spectra of SDA solutions prepared in water at various pH values ranging from pH 2.5, where SDA is fully protonated (pKa = 2.77), to pH 7, where SDA is dianionic (pKa = 3.96), demonstrating the sensitivity in absorption maximum (l_max) to pH. Physiological pH of the plant leaf ranges from pH 5-7. Consequently, most of the photodynamical transient absorption (TA) studies presented below employed SDA-buffered solutions. For completeness, additional TA data were acquired for solutions at pH values outside this region, shown in the Supporting Information (Figure S1 and Table S1).

Transient absorption spectroscopy (TAS) studies have been performed for SDA in three different environments: (i) SDA dissolved in a solvent, i.e. bulk solution; (ii) SDA in bulk solution against a substitute cuticle surface; and (iii) as a thin film, where SDA is deposited on the substitute cuticle. The substitute cuticle surface serves as a better mimic of the real-world application of SDA applied as foliar spray on plant leaves. The TAS data obtained for SDA under environments (i) and (iii) are presented in Figure 2. The excitation wavelength for each measurement corresponds to the l_max of SDA under the given conditions, evidenced in Figure 1.

Bulk solution

The TAS measurements for environments (i), shown in Figure 2a, and (ii), shown in Figure S1a, reveal a similar overall picture following photoexcitation at 305 nm (i.e. at l_max), indicating that the photodynamics is driven by bulk effects. Both TAS results, shown in the form of false colour heat maps, display the same features: three excited state absorption (ESA) bands centred at ~ 375 nm, ~ 425 nm, and ~ 650 nm, along with a stimulated emission (SE) feature centred at ~ 475 nm. All four features decay to reveal a residual ESA centred at ~ 390 nm, not immediately apparent in the false colour heat maps but observable in the evolutionary associated decay spectra (EADS) in Figure 2c.

Global sequential fitting of the TAS data also yields similar lifetimes for environments (i) and (ii), presented in Table 1. Our interpretation of these data and the associated lifetimes is based on previous studies on sinapoyl malate (SM) and its derivatives, which show efficient relaxation through trans/cis photo‑isomerisation leading to efficient repopulation of the electronic ground state.¹⁵ In addition, the symmetric substitution at the terminal carbon in the allylic double bond eliminates the possibility of forming an isomer photoproduct, thereby preventing any possible post‑isomerisation reduction in absorption cross-section and any (potential) isomer toxicity.^16–18

Based also on the present TD-DFT calculations, these lifetimes are assigned to the following mechanism: photoexcitation at 305 nm populates the optically bright 1¹ππ* excited state from which the molecule undergoes a geometry relaxation, which takes place within τ₁, reflected by the decay of the ESAs centred at 425 nm and 650 nm and the loss of the oscillator strength on the S₁ surface (see Figure 3). It is likely that τ₁also includes the time for any solvent reorganisation related to the geometry change as the molecule moves out of the Franck-Condon region.^15,19

Following geometry relaxation, the excited-state population evolves towards the energetically accessible intersection between the excited (S₁) and ground (S₀) states within the lifetime of τ₂. The S₁ population undergoes internal conversion through the S₁/S₀ intersection, leading to highly vibrationally excited S₀ state molecules. τ₂ is assumed to encompass the time the population evolves on the S₁ state and the subsequent internal conversion to the S₀ state, as well as its relaxation on the hot ground state. Both the ESA (at ~375 nm) and the SE (~475 nm) features decay within τ₂; consistent with the excited-state lifetimes of similar symmetrically substituted systems.^16,17 Despite the present analysis offering no separate lifetime attributable to vibrational cooling in the S₀ state, the TAS data do show some evidence for this process, with a modest blue shifting of the absorption around 375 nm appearing after the intense ESA (and SE) attributed to internal conversion (Figure S2a).

The final lifetime returned by the fitting, τ_3, persists for the maximum pump-probe time delay in these experiments (Δt = 2 ns) and is assigned to the residual population trapped in an excited state. We note here that the long-lived component could be assigned to photoproduct formation. Still, we discard this hypothesis since (i) the symmetrical substitution within SDA (and SDA^2-) leads to only one ground‑state conformer in solution, and (ii) our steady-state bulk solution photostability measurements (see Section Photostability) show no visible absorbance change in this spectral region (≈ 400 nm) following continuous irradiation with a simulated solar spectrum for 2 hours.

To further explore the excited-state landscape of SDA (specifically SDA^2- at a pH of 5.6 in aqueous solution), we have performed TD-DFT calculations on bare SDA^2- and on SDA^2- dianions microsolvated with up to 15 water molecules.²⁰ These will be identified as SMD/water calculations (where SMD is the acronym for Solvation Model based on Density), more details of which can be found in the SI, Figures S3-S5). The vertical excitation energies varied little as the number of water molecules was augmented (cf. the implicit solvation case with no explicit water molecules). Good agreement between the experimental absorption spectra and the computed S₁ vertical excitation energies was achieved at the ωB97XD/6-31+G(d,p) level of theory. The present calculations show that the phenyl ring and one of the carboxylate groups are coplanar at the minimum energy geometry of the ground state parent molecule. The second carboxylate group is near perpendicular to this plane. After excitation into S₁, the allylic bond twists to nearly 90°, and the two carboxylate groups are almost perpendicular to the plane of the phenol ring at the S₁/S₀ intersection geometry. Similar results were reported previously for other symmetrically substituted sinapates.^17,18,21

Figure 3 (see also Figure S6 in the SI) reveals a barrierless pathway from the Franck-Condon region of the S₁ state towards the S₁/S₀ intersection. This excited state is initially bright but becomes optically dark i.e. the oscillator strength is nearly zero when approaching the S₁/S₀ crossing geometry. These calculations support the assigned relaxation mechanism of SDA^2- via a rapid barrierless internal conversion to the S₀ state.

Thin Film Samples

Thin films of SDA were created by applying and spin-drying a solution of SDA dissolved in ethanol onto a wax substrate, which resulted in ≈ 3 mm thick films (see more details in the Methods section). These films on the wax substrate should be better mimics of the environment in which the molecular heater is expected to perform. Following photoexcitation at 330 nm (i.e. l_max), the photodynamics of thin films of SDA on wax (Figure 2b) consists of a very broad ESA with a prominent maximum centred on 400 nm, similar to that seen in bulk solution, but which now blue shifts over the first 10 ps as it decays. We also observe an apparent red shift in the GSB feature after 10 ps, but this can be justified by the overlap of the ESA and GSB signals at early time delays. There is now a clear ground state bleach (GSB) feature at 350 nm, which persists up to Δt = 2 ns. While such long-lived GSB features are often observed due to molecular degradation,²² these thin film samples of SDA are found to retain 70% of their UV/Vis absorbance for 2 hours following continuous irradiation with a solar simulator (see Section Photostability). These observations contrast with those for sinapoyl malate, a natural photoprotection molecule with a similar structure to SDA, which retains only 9% of its UV absorbance under the same conditions.

These results are now discussed in the context of the proposed relaxation mechanism for SDA, noting the obvious differences between these results and those obtained in bulk solution (see Figure 2). In the case of the thin film samples of SDA, photoexcitation at 330 nm gives rise to a broad ESA spanning almost the full range of the probe spectrum (350-740 nm) with a maximum at ~400 nm, along with a GSB centred at 340 nm. These features decay with a lifetime of τ₁, assigned to the excited-state population undergoing geometry relaxation as it moves towards and through the energetically accessible S₁/S₀ intersection, as discussed supra for the bulk solution. At this juncture, we draw attention to the notably longer τ₂ value (~12 ps) than the bulk solution equivalent (~2.5 ps). This difference could be due to (i) the excited-state population taking much longer to traverse the S₁/S₀ intersection, (ii) the formation of vibrationally hot S₀ molecules which subsequently cool, or some combination of both factors. However, the lack of SE signal in the wax substitute points to a rapid internal conversion, and τ₂is thusassigned to electronic ground state dynamics. Additionally, in solution at pH 5.6, the anionic nature of SDA^2- may increase the effectiveness of the energy dissipation (cf. the neutral species in thin film) through the formation of a hydrogen bonding network with water molecules, thereby increasing the disparity in the lifetimes between the two environments.²³ Finally, τ₃ is assigned to the long-lived GSB centered on 340 nm, which persists beyond the maximum Δt of 2 ns. While a long-lived GSB is (usually) evidence of the excited-state population not recovering to the initial ground-state energy minimum, there are no additional features, such as ESA to suggest photoproduct formation.

Again, we turn to theory to guide our interpretation of the observed TAS data and provide additional insight into the origin of τ₃ in the thin film measurements. Geometry optimisations of neutral SDA were first computed in a vacuum at the ωB97XD/6-31+G(d,p) level of theory. For neutral SDA, the intramolecular hydrogen bond between the two carboxylic groups restricts them to the same plane, but they can assume different orientations, giving rise to different conformers. The two more stable are separated by only 2 kcal/mol: SDA_A (the more stable conformer) has the carbonyl group oriented towards the phenyl ring, keeping the overall planarity of the molecules; in turn, SDA_B has the hydroxyl group oriented towards the phenyl ring, losing planarity, which results in a slight shift in the predicted absorption spectrum and slightly different extinction coefficients for the two conformers (Figure S7 and Table S2).

Modelling a leaf surface is challenging since it is non-crystalline and varies morphologically and compositionally among plant species.²⁴ Usually, the main components are primary and secondary alcohols, ketones, fatty acids, and aldehydes with twenty to forty carbon atoms.²⁵ To simulate the impact of a wax surface on the SDA dynamics, we start with a simple model based on a class of epicuticular wax with platelet morphology, which exhibits single-crystal patterns largely dominated by primary alcohols with carbon chains larger than twenty.^25,26 The chosen model consists of SDA on top of the crystalline structure of 1,21‑henicosanediol (extracted from Cambridge Crystallographic Database, deposition number 153917).²⁷ The SDA_A and SDA_B conformers were optimised in different orientations atop the model surface using TD-DFT, keeping the surface frozen. The two most stable S₀ geometries obtained using this model (SDA_A1 and SDA_A2) are shown in Figure 4. The most stable, SDA_A1, is bonded to the surface dialcohols by the carboxylic acid group of SDA, while SDA_A2 is bonded to the surface through the phenolic hydrogen (see Figures S8 and Table S3).

With these calculations, we now suggest the origin of τ₃ in the thin film measurements. The most stable conformer, SDA_A1, has one of the largest oscillator strengths (Table 2). Following photoexcitation and relaxation to the ground state, some population may relax into other local minima on the S₀ state through a rotation of the allylic group, generating, for example, SDA_B1, which has a lower oscillator strength and manifest as a persistent GSB signal in TAS measurements (viz. Figure 2b). No absorption features attributable to such higher energy conformers are identified, but this is unsurprising given the similarities in their vertical excitation energies (see Table 2).

In summary, our TAS data and the TD-DFT calculations indicate that the absorbed photon energy is rapidly converted to vibrational energy (heat) across all three environments that the SDA molecular heater is likely to encounter on the surface of a leaf.

Photostability studies

Another key criterion for commercial use of any molecular heater is its long-term photostability, which is here assessed in solution and on the mimetic cuticle surface. This part of the study was expanded to include several other structurally similar sinapate-based molecules to place the results from SDA against other possible molecular heaters: these included diethyl sinapate (DES), methyl sinapate (MS) and sinapoyl malate (SM). Results for all four samples (SDA, DES, MS, and SM) following 2 hr irradiation with a solar simulator (intensity I = 1 sun) are shown in Figure 5. Clearly, SDA presents exceptional photostability in solution (Figure 5a insert) with only 9% degradation over 2 hr irradiation at the absorption maximum. Notably, compared with all other samples, SDA also presents the best photostability (23% degradation) as a thin film on the wax substitute cuticle.

Again, we note that symmetric substitution at the terminal carbon atom in the allylic double bond (in DES and in SDA) results in improved relative photostability on the wax substitute cuticle cf. the non-symmetrically substituted molecules MS and SM, Figure 5c and d. The increased photodegradation in comparison with SDA is also clearly evident when inspecting the respective UV absorption spectra at wavelengths below 250 nm, where DES, MS, and SM each exhibit a substantially larger increase in UV absorption attributable to photoproduct formation following the 2 hours of irradiation. This provides further evidence for the favourable photostability (resilience to UVR damage) of SDA. We also note that the present predictions of multiple conformers of SDA on the wax surface are consistent with the isosbestic point observed at ~400 nm (see Figure 5a), where the small increase in absorbance at longer wavelengths is attributed to the formation of the higher energy conformers discussed above; DES, MS and SM do not show this behaviour. These studies demonstrate the critical role of symmetric substitution at the terminal carbon atom in the allylic double bond in boosting photostability and the impact of the surface.

In vivo heat-transfer studies

To explore energy transfer from the proposed molecular heaters to the plant leaf on a macroscopic scale, in vivo spot tests were carried out on the leaves of Nicotiana benthamiana plants under controlled conditions, both in a greenhouse and in a climate chamber containing white light and UV radiation (see Methods). The results of these trials, shown in Figure 6a-d, clearly demonstrate that an increase in SDA concentration results in a progressive increase in the amount of heat transferred to the leaf. In those areas of the plant leaves where a 50 mM solution of SDA had been deposited and allowed to dry, the average temperature increased by 0.18 ^°C (climate cabinet measurements) and 0.12 ^°C (greenhouse measurements), as compared to an area where a formulation blank had been deposited as a control. These macroscopic test results confirm the efficient transfer of the photon energy to the heating of the leaf, as required for a molecular heater. It is assumed a formulation containing the molecular heater would result in a fuller coverage of the leaf which would harvest more incident solar photons and transfer more energy to the leaf and thereby ensure a greater temperature increase.

In silico and in vitro toxicity studies

While the increase in leaf temperature is the key aim of the present work, it is also important to consider the possible effects of applying the proposed molecular heaters into the food chain, especially to human and environmental toxicity.^28,29 To account for these effects, we conducted an in silico analysis of the toxicological effects of the molecular heaters applied to the plant surface. In silico testing allows the examination of key factors such as bioaccumulation, persistence, mutagenicity, carcinogenicity, and endocrine disruption.^30,31 As presented in Table S5 and the accompanying text in the SI, all predictions showed a log bioconcentration factor (BCF) < 3.3 (with good reliability for ISIDA), indicating that SDA is predicted not to be bioaccumulative. Moreover, SDA was predicted to be readily biodegradable according to both VEGA (low reliability) and ISIDA (good reliability) models. Regarding soil and water persistence, all the different predictions point towards non-persistence of SDA in these matrices with good reliability in the case of ISIDA (Table S6). Conflicting results were observed for sediment persistence, with SDA being predicted persistent (156 days, beyond the threshold of 140 days) by the IRFMN model. This prediction, however, was linked to low reliability. On the contrary, ISIDA predicted SDA to be not persistent with optimal reliability. In total, it can be concluded that SDA does not raise concern with regard to environmental fate.

Figure 6e summarises the predicted effects of SDA on different regulatory-relevant toxicological endpoints based on multiple models, including the toxicity estimation software tool (TEST), the VEGA platform, and the LAZAR software (see Methods). For SDA, these studies predicted no mutagenicity or genotoxic potential (SI, Table S7). The predicted values for acute and short-term toxicity (LD₅₀ and NOAEL, respectively) indicate overall low toxicity for SDA, which was confirmed by the read-across analysis (SI, Table S8). In silico modelling also showed no indication of estrogenic or thyroid activity; SDA was predicted to have binding affinity for the estrogen receptor and be active at the androgen receptor. However, the reliability of both predictions was low. The carcinogenicity prediction was equivocal due to conflicting outcomes of the models.

In addition to the computational approaches, in vitro studies were conducted to assess the toxicity of SDA in human cells. Following 24 h of incubation, SDA did not induce any cytotoxicity in human intestinal Caco-2 or liver HepaRG cells in the NRU assay (Figure S13). Induction of DNA damage in Caco-2 or HepaRG cells was further investigated in the γH2AX assay. SDA showed no effect, while the positive control doxorubicin led to a statistically significant increase (Figure S14). Taken together, these findings add confidence for the low toxic potential of SDA. Of note, SDA has been shown to exhibit antioxidant and anti-inflammatory properties.^32,33

We have demonstrated, for the first time, how the application of a candidate molecular heater, sinapic diacid (SDA, 2-(4-hydroxy-3,5-dimethoxybenzylidene)malonic acid), can increase the local temperature of a plant leaf by ~ 0.2°C during solar irradiation. After suitable scale-up, foliar sprays containing such materials could provide a thermal blanket, promoting plant growth and helping protect plants from cold stress. The present study describes a multidisciplinary approach that has led to the shortlisting of SDA as a target molecular heater. We have developed a sustainable chemical synthesis of SDA; characterized its photophysical properties and mechanisms with a combination of steady-state and ultrafast laser spectroscopy methods and computational calculations; assessed its light-to-heat energy transfer performance to plant leaves in vivo; and explored key environmental and toxicological endpoints through in silico studies. SDA clearly demonstrates solar energy to heat energy conversion, with no alerts to environmental fate and human toxicity and impressive resilience to prolonged irradiation. From the present studies, molecular heaters offer a tantalizing new approach towards promoting plant growth and protecting plants against climate instability, thereby contributing to the Zero Hunger initiative by the United Nations.

Photochemistry: Sample Preparation

SDA was synthesised through a sustainable green chemistry method previously reported by Rioux et al.¹⁴ Solutions of SDA were prepared in 10 mM concentrations using 0.4 M aqueous sodium phosphate buffer to maintain a physiological pH value of 5.6.³⁴ Samples of diethyl sinapate, methyl sinapate and sinapoyl malate were made in the previously reported fashion.^16,35

Wax-coated calcium fluoride (CaF₂) circular windows of 25 mm diameter and 2 mm thickness (UQG optics) were prepared by adapting the protocol previously reported by Pambou et al.³⁶ Specifically, Carnauba wax, No. 1 (CAS: 8015-86-9, Sigma Aldrich) was dissolved in chloroform to a concentration of 0.05% by weight. 1 mL of this solution was then pipetted onto a clean UV grade CaF₂ window and spin-coated using a spin coater (Laurell Technologies, WS‑650MZ-23NPP/LITE) programmed to run at 3000 rpm for 20 seconds. These parameters have been shown in reference 36 to give a film thickness between 50 and 100 Å; hence we assume this to be the approximate thickness of the wax coatings on the samples studied in the present work.

Thin films of the chosen sample were then added on top of the wax coatings mentioned above; to achieve this, 1 mL of a 100 mM solution dissolved in ethanol was pipetted on top of a wax-coated CaF₂ window prepared as described above and spun again for 20 seconds at 3000 rpm. The presence of the sample on the surface of the windows was confirmed through UV/Vis absorption spectroscopy, which revealed a peak in the absorption spectrum centred at 330 nm (Figure 1), approximately 15 nm red-shifted from the absorbance of SDA in aqueous solution at pH 5.6. The thickness of the spin-coated films was calculated through the experimentally determined molar absorption coefficient of SDA (15,000 ± 2,000 M^-1cm^‑1_,Figure S15), giving a thickness of 3 µm.

All steady-state UV/Vis absorption spectra were collected on a Cary 60 Spectrometer (Agilent Technologies) using a cuvette of 10 mm path length for solution samples. Along with these studies, solar irradiation to assess the photostability of the samples was provided by Oriel instruments, 91191-1000 solar simulator and was tuned using a neutral density filter to give an irradiation power equivalent to 1 sun (1000 W m^-2).

Transient Absorption Spectroscopy

The TAS set-up used to collect the presented ultrafast spectroscopy data has been described previously;^37,38 hence only specific details about the reported experiments are given here. Photoexcitation (or pump) wavelengths were chosen to correspond to the peak absorption wavelength of the samples of interest (305 and 330 nm), with the white light continuum detection (or probe) beam (330–740 nm) generated by focussing fundamental (800 nm) laser radiation into a vertically translated 2 mm CaF₂ window. Liquid samples flowed through the sample interaction region using a diaphragm pump (SIMDOS 02) and a demountable liquid flow cell (Harrick Scientific Products Inc.); PTFE spacers of 100 µm and 6 µm thickness were used to define the thickness of the liquid samples. Thin film samples in liquid form and spin-coated onto the wax surface were translated in the focal plane of the probe beam. Sample degradation in all three environments was monitored by comparing the ΔOD between each scan cycle; if drops in the signal were observed after a scan cycle, the samples could be replenished before continuing.

To gain quantitative insight into the ultrafast dynamics of SDA (and SDA^2-, vide supra), a global sequential fitting method across all probe wavelengths (330–740 nm) was employed using the TIMP package in R interfaced through Glotaran.^39,40 Each set of TA spectra collected at a series of pump-probe time-delays (Δt) were sequentially fit with a 3-step kinetic model (i.e., , giving rise to 3 Evolutionary Associated Difference Spectra (EADS), modelling the spectral components. Each EADS exponentially decays with an associated lifetime, τ_n. This decay incorporates a convolution with a Gaussian to model the instrument response function (IRF, ≈ 80 fs full-width half maximum (FWHM)) and a third-order polynomial correction for the chirp of the experimental data. The fit returned lifetimes for each dynamical process, along with associated standard errors reported as twice their value. The fit quality was evaluated through the associated residuals (difference between the fit and the data), displayed in the SI Figure S16.

Computational Methods

To account for the different environments, DFT and TD-DFT were used to compute sinapic diacid in water (using implicit and explicit solvent models), in the gas phase, and on a mimic of a wax surface (here referred to as model surface). The deprotonated form (SDA^2‑) was used in aqueous environments, while the neutral (SDA) form was used in the gas phase and model surface calculations, all of which were performed using Gaussian 16 rev a03.⁴¹

Optimizations and energy calculations of SDA^2-and SDA were performed at the ωB97XD/6-31+G(d,p) level. In the calculations including the model surface, the optimizations were done using ωB97XD/6-31G(d) and subsequent energy calculations using 6-31+G(d,p) basis set. The minimum energy S₁/S₀ state intersections were optimised using the penalty function method proposed by Martínez and co-workers,^42,43 implemented in the CIOpt program with an in-house adaptation to work with Gaussian software. The topographies of the potential energy curves were characterised by linear interpolations in internal coordinates connecting the vertically excited S₁ state (at the ground state equilibrium geometry) to the S₁ minimum and the S₁/S₀ intersection. Charge transfer analysis of the ground and low-lying singlet excited states of neutral and dianionic SDA were computed using the TheoDore program⁴⁴ (see Table S4 and Supplementary Information for more details).

To mimic the water environment, we use SMD (Solvation Model Based on Density)²⁰ as an implicit solvation model (here referred to as SDA^2-.(H₂O)₀) and systematically added up to 15 water molecules (SDA^2-.(H₂O)_n, where n indicates the number of water molecules). The ground (S₀) and the first three singlet excited electronic states (S₁, S₂, S₃) for SDA^2- were optimised with implicit solvation, while the S₀and S₁ states were optimised with micro-solvation. These SMD/water results can be viewed in Figure S4 and the reaction coordinates are shown in Figure S6.

To simulate SDA in a wax environment, the fully protonated form was considered since this species is expected to be deposited on the surface. First, optimisations of the S₀and S₁ states of the isolated neutral SDA molecule in the gas phase were undertaken. Thus, the two most stable conformers of SDA in its ground state (see Figure S7 and Table S2) were selected for the simulations with the wax surface model The topography of the S₁ state for these isolated conformers is shown in Figure S10. In the S₀ optimizations of SDA_A and SDA_B in different orientations on the top of the model surface, only the SDA atoms were allowed to move (Figure S8).

Plant Heating

Experiments to measure the local heating of plant leaves were performed in the following manner: 25 μl spots of sinapic diacid, 50 mM dissolved in 10% ethanol, adjusted to pH 4.5-5.6 using 1M KOH, were pipetted onto 39-day old Nicotiana benthamiana leaves. 10% ethanol was used as a control. After evaporation, the plants were moved to a climate cabinet (18°C, 70% RH) or a greenhouse. Images were taken after 4 hr UV-A+B and white light treatment (5 µmol m^-2s^-1 total UV and 190 µmol m^-2s^-1 Photosynthetic Active Radiation, PAR, for the climate cabinet, 18 µmol m^-2s^-1 total UV and 850 µmol m^-2s^-1 PAR for the greenhouse studies, irradiation spectrum is shown in Figure S12).

In Silico investigation

i. Bioaccumulation

Bioaccumulation was evaluated using the bioconcentration factor (BCF), defined as the ratio of the concentration of the test chemical in aquatic organisms to its concentration in the ambient environment. Two different platforms were employed to predict BCF. The VEGA platform provides 4 models (CAESAR, Meylan, KNN/Read-across, and Arnot-Gobas) for which a log value is given, as well as information about the reliability of the prediction (low, moderate, or high). The online tool ISIDA/Predictor is a consensus regression model based on 17 individual models. The outcome is given as a log value with a prediction confidence estimate (outside applicability domain (AD), average, good, and optimal, based on the model predictions). According to the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) regulation, a substance with a BCF higher than 2000 (or log BCF > 3.3) is considered bioaccumulative, while a substance with a BCF higher than 5000 (or log BCF > 3.7) is considered very bioaccumulative.

ii. Biodegradability

Biodegradability was evaluated using the VEGA platform (IRFMN model) as well as the online tool ISIDA/Predictor (consensus regression model based on 15 individual models). For both tools, the outcome is given as qualitative prediction (readily biodegradable/not readily biodegradable), plus information about the reliability of the prediction (low, moderate, or high reliability for VEGA or outside AD, average, good, and optimal for ISIDA/Predictor).

iii. Persistence

Persistence in sediment, soil, and water were evaluated using the VEGA platform. The qualitative IRFMN model gives a statement (not persistent or nP, close to persistence threshold or nP/P, close to very persistent threshold or P/vP, very persistent or vP) while the quantitative IRFMN model gives an estimate on the number of days of persistence in the so-called matrix. Both models also provide information on the reliability of the prediction (low, moderate, or high reliability). In addition, we used the online tool ISIDA/Predictor that provides a consensus model based on 19, 15, and 20 individual models, respectively. The outcome is given as a statement (persistent P or not persistent nP) with a prediction confidence estimate (outside AD, average, good, and optimal).

iv. Mutagenicity and carcinogenicity

The potential mutagenicity and carcinogenicity of SDA was assessed using the different software tools VEGA, TEST, and LAZAR. Previous work has shown the relevance of applying multiple models to increase the predictive power of such in silico investigations.^45,46 Detailed information on the use of these models can be found in previous publications.^18,47 In short, the different predictions generated by the individual models were converted to numeric values ranging between 0 and 1, in which presumed non-mutagenicity/non-carcinogenicity spans the range 0-0.50, while mutagenicity/carcinogenicity ranges from 0.51 to 1 (see Table S7).

The arithmetic mean of the different prediction scores was calculated and plotted in a diagram, divided into three zones: a score > 0.66 means a positive prediction (mutagenic/carcinogenic) with good reliability, while a score <0.33 is a negative prediction (non-mutagenic/non-carcinogenic) with good reliability. A score between 0.33 and 0.66 is considered equivocal: scores between 0.33 and 0.5 are regarded as negative predictions (non-mutagenic/non-carcinogenic) with insufficient reliability, while scores between 0.5 and 0.66 are regarded as positive predictions (mutagenic/carcinogenic), again with insufficient reliability.

v. Endocrine Toxicity

Endocrine toxicity was investigated using the VEGA platform. The term “endocrine toxicity” here encompasses 5 models which give predictions on receptor-mediated effects (4 models: Estrogen Receptor-mediated effect IRFMN/CERAPP, Androgen Receptor-mediated effect IRFMN/COMPARA, Thyroid Receptor Alpha effect NRMEA, and Thyroid Receptor Beta effect NRMEA), as well as on receptor binding affinity (Estrogen Receptor Relative Binding Affinity model IRFMN). A qualitative prediction (yes/no), plus information about the reliability of the prediction (low, moderate, or high reliability), is provided for each model.

vi. Acute and short-term toxicity

Acute and short-term toxicity was investigated using in silico models for oral LD₅₀ and the no-observed-adverse-effect level (NOAEL) from 90-day toxicity studies. The oral LD₅₀ in rats was estimated using T.E.S.T., based on a dataset comprising values from 7413 substances. The NOAEL was estimated using the module NOAEL - IRFMN/CORAL provided within the VEGA platform, based on a dataset comprising 140 different NOAELs from repeated-dose 90-day oral toxicity studies in rodents.

vii. Read-across approach

The read-across approach relies on the structural similarity of SDA and closely-related compounds to predict, by extrapolation, unknown aspects of SDA’s toxicity. Using the OECD QSAR Toolbox (https://www.oecd.org/chemicalsafety/risk-assessment/oecd-qsar-toolbox.htm), we sought to identify a list of structurally similar compounds for which available experimental data for the endpoints of mutagenicity, carcinogenicity and acute toxicity were extracted. Predictions for the test compound (SDA) were then generated with the help of the module « Read-across ». Compounds used in the read-across approach were isoferulic and ferulic acids (CAS 537-73-5 and 1135-24-6, respectively). Sinapic acid is probably the most structurally similar analogue, but it was not considered for the read-across due to a lack of available data in the OCED and QSAR toolbox.

In vitro investigation

i. Chemicals

Dimethyl sulfoxide (DMSO) was purchased from AppliChem (Darmstadt, Germany). Doxorubicin was purchased from Cayman (Ann Arbor, MI, USA) and dissolved in DMSO. All other chemicals were purchased from Merck (Darmstadt, Germany) or Sigma, at the highest purity available, if not stated otherwise below.

ii. Cell culture

Human intestinal Caco-2 cells were cultured as previously described in Voss et al.⁴⁸ Briefly, cells (passages 26–36) were seeded at 10,000 cell/well in 96-well plates in culture medium (DMEM with 4.5 g/L glucose, L-Glutamine, sodium pyruvate, 3.7 g/L NaHCO₃, supplemented with 10% heat-inactivated fetal bovine serum, 100 IU/mL penicillin,100 µg/mL streptomycin). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂. For differentiation into an intestinal epithelial-like monolayer, cells were cultured for 3 weeks with renewal of medium every 2-3 days.⁴⁹ All the assays were performed on differentiated Caco-2 cells.

Human liver HepaRG cells were cultured as previously described in Alarcan et al.⁵⁰ In short, cells (passages 15–20) were seeded at 9,000 cell/well in 96-well plates in culture medium (Williams’ E Medium with stable Glutamine, 2.24 g/L NaHCO₃ and Phenol Red, supplemented with 10% heat-inactivated fetal bovine serum, 100 IU/mL penicillin,100 µg/mL streptomycin, 5 µg/mL insulin, and 50 µM hydrocortisone hemisuccinate). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂. Following 2 weeks maintenance, the cells were cultured in the same medium supplemented with 1.7% DMSO (differentiation medium) for an additional 2 weeks with medium renewal every 2 to 3 days.

iii. Cell viability assay

Neutral Red uptake (NRU) assay was performed to assess the cytotoxicity of SDA in Caco-2 and HepaRG cells. Following 24 h incubation, cell medium was removed, then cells were incubated for 2 h with 100 µL solution of neutral red (4 µg/mL). After washing with PBS, cells were lysed and put under shaking for 10 min before fluorescence measurement at 645 nm (excitation at 530 nm).

iv. γH2AX assay

The γH2AX assay was performed according to Sprenger et al.⁵¹ Cells were treated for 24 h with different concentrations of SDA. Doxorubicin (1 μM) was used as positive control. Following treatment, the cells were washed once with cold PBS and fixed with 50 μl ice-cold methanol for 30 min. Cells were then washed with PBS-T (0.1% Tween-20 in PBS) and blocked with 50 μl of 1% bovine serum albumin (BSA) in PBS-T for 1 h at room temperature. After removal of blocking solution, cells were stained with 40 μl (1:500 dilution in blocking solution) of the primary antibody anti-phospho histone H2A.X (S139) (EMD Millipore, Burlington, MA, USA). After 1 h, the antibody was removed and cells were washed three times with PBS-T before staining with 40 μl (1:400 dilution in blocking solution) of the secondary antibody AlexaFluor 647 (Life Technologies, Carlsbad, CA, USA) for 1 h. The cells were again washed three times with PBS-T, then stained for 30 min with 50 μl DAPI (3 μM; TCI Deutschland, Eschborn, Germany). The fluorescence intensity was measured with the Celldiscoverer 7 microscope using the software ZEN 3.1 for image acquisition and analysis.

Acknowledgements

The authors acknowledge the FET-Open grant BoostCrop (grant agreement 828753) and the entire BoostCrop (https://boostcrop.eu ) team for their interest in the work. The authors also acknowledge the Warwick Centre for Ultrafast Spectroscopy (go.warwick.ac.uk/WCUS) for the use of the Oriel instruments, 91191-1000 solar simulator and the Cary 60 Spectrometer. N.d.N.R. acknowledges funding from the Fundação para a Ciência e Tecnologia (FCT) under the 5th edition of Stimulus for Individual Scientific Employment Call (Concurso para o Estímulo do Emprego Científico, CEEC). T.T.A. thanks the University of Warwick for a PhD studentship through the Chancellor Scholarship scheme. M.T. do C., J.M.T. and M.B. thank the Centre de Calcul Intensif d’Aix-Marseille for accessing its high-performance computing resources and the HPC resources from GENCI-TGCC (Grant 2022- A0110813035 and 2023-AD010813035R1). B.R., M.M. and F.A. thank the Agence Nationale de la Recherche (grant number ANR-17-CE07-0046), as well as the Grand Reims, Conseil Départemental de la Marne, and the Grand Est region for financial support. V.G.S. thanks the Royal Society for an Industry Fellowship.

Author Contributions

J.M.W., N.d.N.R. and V.G.S. acquired and analyzed the time resolved spectroscopic data and prepared the first draft of the manuscript. J.M.T., M.Tdo C. and M.B. performed and analyzed the (TD)DFT calculations. B.R., M.M.M. and F.A. designed and performed the synthesis of the compounds. B.R., M.M.M. and F.A. characterized the compounds. C.G., X.S., T.M. and K.F. acquired and analyzed the in vivo plant data. J.A. and A.B. acquired and analysed the in sillico studies. T.T.A. performed and analysed the steady-state spectroscopic data. W.J.B. and M.N.R.A. critically evaluated the entirety of the analysis. All authors contributed to the writing of the manuscript.

Thakur, P., Kumar, S., Malik, J. A., Berger, J. D. & Nayyar, H. Cold stress effects on reproductive development in grain crops: An overview. Environ. Exp. Bot. 67, 429–443 (2010).
Letcher, T. M. Managing global warming: an interface of technology and human issues. (Academic Press, 2018).
Al-Ghussain, L. Global warming: review on driving forces and mitigation. Environ. Prog. Sustain. Energy 38, 13–21 (2019).
Ma, Q., Huang, J. G., Hänninen, H. & Berninger, F. Divergent trends in the risk of spring frost damage to trees in Europe with recent warming. Glob. Chang. Biol. 25, 351–360 (2019).
Unterberger, C. et al. Spring frost risk for regional apple production under a warmer climate. PLoS One 13, 1–19 (2018).
Vitasse, Y. & Rebetez, M. Unprecedented risk of spring frost damage in Switzerland and Germany in 2017. Clim. Change 149, 233–246 (2018).
United Nations, The 17 Goals. https://sdgs.un.org/goals/goal2 (2021).
Wang, Y., Zhang, L. & Wang, P. Self-Floating Carbon Nanotube Membrane on Macroporous Silica Substrate for Highly Efficient Solar-Driven Interfacial Water Evaporation. ACS Sustain. Chem. Eng. 4, 1223–1230 (2016).
Li, X. et al. Graphene oxide-based efficient and scalable solar desalination under one sun with a confined 2D water path. Proc. Natl. Acad. Sci. U. S. A. 113, 13953–13958 (2016).
Schwarzer, D., Hanisch, C., Kutne, P. & Troe, J. Vibrational energy transfer in highly excited bridged azulene-aryl compounds: Direct observation of energy flow through aliphatic chains and into the solvent. J. Phys. Chem. A 106, 8019–8028 (2002).
Pigliucci, A. & Vauthey, E. Vibrational relaxation dynamics of polyatomic molecules in solution. Chimia (Aarau). 57, 200–203 (2003).
Barbatti, M. Defining the temperature of an isolated molecule. J. Chem. Phys. 156, 204304 (2022).
Moore, R., Clark, D., Kingsley, R. & Stern, R. Botany. (Brown Publishers, 1994).
Rioux, B., Peyrot, C., Mention, M. M., Brunissen, F. & Allais, F. Sustainable synthesis of p-hydroxycinnamic diacids through proline-mediated knoevenagel condensation in Ethanol: An access to potent phenolic UV filters and radical scavengers. Antioxidants 9, 331 (2020).
Baker, L. A. et al. Ultrafast Photoprotecting Sunscreens in Natural Plants. J. Phys. Chem. Lett. 7, 56–61 (2016).
Horbury, M. D. et al. Towards symmetry driven and nature inspired UV filter design. Nat. Commun. 10, 1–8 (2019).
Abiola, T. T. et al. New Generation UV-A Filters: Understanding Their Photodynamics on a Human Skin Mimic. J. Phys. Chem. Lett. 12, 337–344 (2020).
Abiola, T. T. et al. Towards developing novel and sustainable molecular light-to-heat converters. Chem. Sci. 12, 15239–15252 (2021).
Woolley, J. M. et al. The role of symmetric functionalisation on photoisomerisation of a UV commercial chemical filter. Phys. Chem. Chem. Phys. 21, 14350–14356 (2019).
Marenich, A. V., Cramer, C. J. & Truhlar, D. G. Universal solvation model based on solute electron density and on a continuum model of the solvent defined by the bulk dielectric constant and atomic surface tensions. J. Phys. Chem. B 113, 6378–6396 (2009).
M. Toldo, J. et al. Mechanistic Aspects of the Photophysics of UVA Filters Based on Meldrum Derivatives. 125, 5499–5508 (2021).
Woolley, J. M., Losantos, R., Sampedro, D. & Stavros, V. Computational and Experimental Characterization of Novel Ultraviolet Filters. Phys. Chem. Chem. Phys. 2, 25390–25395 (2020).
Woolley, J. M. et al. Unravelling the Photoprotection Properties of Mycosporine Amino Acid Motifs. J. Phys. Chem. Lett. 9, 3043–3048 (2018).
Kolattukudy, P. E. Polyesters in higher plants. Biopolyesters (Springer, 2001).
Ensikat, H. J., Boese, M., Mader, W., Barthlott, W. & Koch, K. Crystallinity of plant epicuticular waxes: electron and X-ray diffraction studies. Chem. Phys. Lipids 144, 45–59 (2006).
Koch, K., Bhushan, B. & Barthlott, W. Multifunctional surface structures of plants: An inspiration for biomimetics. Prog. Mater. Sci. 54, 137–178 (2009).
Nakamura, N., Uno, K. & Ogawa, Y. 1,21-Henicosanediol. Acta Crystallogr. Sect. C 56, 1389–1390 (2000).
Dudka, S. & Miller, W. P. Accumulation of potentially toxic elements in plants and their transfer to human food chain. J. Environ. Sci. Heal. - Part B Pestic. Food Contam. Agric. Wastes 34, 681–708 (1999).
Datta, S. P. & Young, S. D. Predicting metal uptake and risk to the human food chain from leaf vegetables grown on soils amended by long-term application of sewage sludge. Water. Air. Soil Pollut. 163, 119–136 (2005).
Myatt, G. J. et al. In silico toxicology protocols. Regul. Toxicol. Pharmacol. 96, 1–17 (2018).
Valerio, L. G. In silico toxicology for the pharmaceutical sciences. Toxicol. Appl. Pharmacol. 241, 356–370 (2009).
Chen, C. Sinapic acid and its derivatives as medicine in oxidative stress-induced diseases and aging. Oxid. Med. Cell. Longev. 2016, 1–10 (2016).
Hameed, H., Aydin, S. & Başaran, N. Sinapic acid: Is it safe for humans? Fabad J. Pharm. Sci. 41, 39–49 (2016).
Hass, A. R. C. pH determination in Plant Tissue. Plant Physiol. 16, 405–409 (1941).
Dean, J. C., Kusaka, R., Walsh, P. S., Allais, F. & Zwier, T. S. Plant sunscreens in the UV-B: Ultraviolet spectroscopy of jet-cooled sinapoyl malate, sinapic acid, and sinapate ester derivatives. J. Am. Chem. Soc. 136, 14780–14795 (2014).
Pambou, E. et al. Structural features of reconstituted wheat wax films. J. R. Soc. Interface 13, 20160396 (2016).
Greenough, S. E. et al. Solvent induced conformer specific photochemistry of guaiacol. Phys. Chem. Chem. Phys. 16, 16187–95 (2014).
Greenough, S. E. et al. Ultrafast photo-induced ligand solvolysis of cis-[Ru(bipyridine)₂(nicotinamide)₂]²⁺: experimental and theoretical insight into its photoactivation mechanism. Phys. Chem. Chem. Phys 16, 19141–19155 (2014).
Snellenburg, J. J., Laptenok, S. P., Seger, R., Mullen, K. M. & van Stokkum, I. H. M. Glotaran : A Java -Based Graphical User Interface for the R Package TIMP. J. Stat. Softw. 49, 1–22 (2012).
Mullen, K. M. & van Stokkum, I. H. M. TIMP: An R Package for Modeling Multi-way Spectroscopic Measurements. J. Stat. Softw. 18, 1–46 (2007).
Frisch, M. J. et al. Gaussian 16, Revision A.03,. Gaussian, Inc., Wallingford CT, 2016.
Levine, B. G., Ko, C., Quenneville, J. & Martínez, T. J. Conical intersections and double excitations in time-dependent density functional theory. Mol. Phys. 104, 1039–1051 (2006).
Levine, B. G., Coe, J. D. & Martínez, T. J. Optimizing conical intersections without derivative coupling vectors: Application to multistate multireference second-order perturbation theory (MS-CASPT2). J. Phys. Chem. B 112, 405–413 (2008).
Plasser, F. & Lischka, H. Analysis of excitonic and charge transfer interactions from quantum chemical calculations. J. Chem. Theory Comput. 8, 2777–2789 (2012).
Frenzel, F. et al. Use of in silico models for prioritization of heat-induced food contaminants in mutagenicity and carcinogenicity testing. Arch. Toxicol. 91, 3157–3174 (2017).
Glück, J., Buhrke, T., Frenzel, F., Braeuning, A. & Lampen, A. In silico genotoxicity and carcinogenicity prediction for food-relevant secondary plant metabolites. Food Chem. Toxicol. 116, 298–306 (2018).
Rioux, B. et al. Sustainable synthesis, in silico evaluation of potential toxicity and environmental fate, antioxidant and UV-filtering/photostability activity of phenolic-based thiobarbituric derivatives. Green Chem. Lett. Rev. 15, 114–125 (2022).
Voss, L. et al. Intestinal and hepatic effects of iron oxide nanoparticles. Arch. Toxicol. 95, 895–905 (2021).
Sambuy, Y. et al. The Caco-2 cell line as a model of the intestinal barrier: infuence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biol. Toxicol. 21, 1–26 (2005).
Alarcan, J. et al. Metabolism of the marine phycotoxin PTX-2 and its effects on hepatic xenobiotic metabolism: Activation of nuclear receptors and modulation of the phase i cytochrome P450. Toxins (Basel). 9, (2017).
Sprenger, H. et al. Use of transcriptomics in hazard identification and next generation risk assessment: A case study with clothianidin. Food Chem. Toxicol. 166, 113212 (2022).

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Towards Increased Crop Yields with Molecular Heater Technology

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Abstract

Figures

Introduction

Results and Discussion

Photodynamical studies

Bulk solution

Thin Film Samples

Photostability studies

In vivo heat-transfer studies

In silico and in vitro toxicity studies

Conclusions

Methods

Photochemistry: Sample Preparation

Transient Absorption Spectroscopy

Computational Methods

Plant Heating

In Silico investigation

i. Bioaccumulation

ii. Biodegradability

iii. Persistence

iv. Mutagenicity and carcinogenicity

v. Endocrine Toxicity

vi. Acute and short-term toxicity

vii. Read-across approach

In vitro investigation

i. Chemicals

ii. Cell culture

iii. Cell viability assay

iv. γH2AX assay

Declarations

References

Additional Declarations

Supplementary Files

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