Distribution of photosensitive fagopyrin in buckwheat flowers and its potential biological relevance

Abstract

Fagopyrum esculentum (Moench) is a valuable pseudo-cereal valued for its highly nutritious, gluten-free seeds. Despite being recognized as a 21st -century superfood, buckwheat remains non-competitive in seed yield compared to common cereals. Low productivity is mainly caused by abnormalities in female gametophyte development and frequent flower and embryo abortion. Buckwheat flowers accumulate high levels of phototoxic fagopyrin (FAG), whose physiological role remains unclear. FAG and its precursor (PFAG) are light-sensitive compounds with absorbance spectra in the green-yellow range (549–593 nm, peak at 590 nm), which makes their accumulation potentially responsive to light conditions. To address this, plants were cultivated under different light spectra, and the content of FAG and PFAG was analyzed in distinct floral organs (stamen, pistil, petal, and receptacle) using LC-MS, with their spatial distribution assessed by the MALDI-MS imaging. Pistil showed statistically the highest FAG and PFAG contents, while petals contained the lowest levels. A high density of FAG surrounding the ovary indicates a potential role in the reproductive part. Moreover, negative correlations were detected between flower production and FAG levels in the receptacles and pistils under specific light treatments. These results suggest that FAG may influence flower production and female gametophyte development, linking light environment to reproductive success in buckwheat.

Introduction

Common buckwheat (Fagopyrum esculentum Moench) is economically the most valued species within the genus Fagopyrum^1,2. Its wild ancestor, F. esculentum subsp. ancestrale Ohnishi originates from the mountain area of Yunnan province in China³. Currently, common buckwheat is a well-known species cultivated worldwide^4,5. Buckwheat botanically belongs to the Polygonaceae family, but it is categorized as a pseudo-cereal crop^6,7. It is an annual plant with a short vegetation period, prized for its highly nutritious seeds and multipurpose use of different parts of the plant⁴. The health-promoting properties of buckwheat result from the content of many bioactive components such as dietary fiber, phenolic acids, flavonoids, and vitamins⁸. Buckwheat seeds are gluten-free and contain a well-balanced amino acid composition⁹. They have higher nutritional value than many common cereals. Therefore, buckwheat is considered one of the super-foods of the 21 st century^10,11. Despite many advantages, buckwheat cultivation progressively decreased in the last few decades due to low and unstable seed yield compared to the other cereals². The causes of the low seed yield of buckwheat are associated with specific flowering biology and aspects related to reproduction¹⁰. Buckwheat is characterized by non-self-terminating flowering and self-incompatibility due to the heterostyly of flowers. Its single flower has a one-day lifespan; therefore, insects have limited time for pollination^10,12. Among the most important factors limiting the seed yield are abnormalities within the development of female gametophytes^13–15, and frequent embryo and flower abortion^16–20. The establishment of a seed set appears during or right after fertilization, affecting the future yielding potential. These processes are sensitive to biotic and abiotic stresses, frequently resulting in flower and seed abortion²¹.

Buckwheat flowers are rich in phenolic compounds such as flavonoids²² or photosensitive fagopyrin (FAG)²³. FAG was isolated for the first time in 1941 from the blossom of the red-flowering genotype of common buckwheat^24,25, and the chemical structure of this compound was described first in 1979²⁶. FAG is an anthraquinone derivative, characterized by a polycyclic skeleton with two piperidine molecules²⁷. There are six possible forms of FAGs (marked as A-F)²⁸ among which three types (A, E, and F) have been identified using liquid chromatography with tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR)^23,29,30. FAG has similar photosensitive properties as well-known hypericin (HYP) isolated from Hypericum perforatum L. (St. John’s wort)³¹. FAG and HYP are structurally similar and belong to the naphthodianthrones that appear as red pigment³². In buckwheat plants, the photosensitizer is present in the basic form of protofagopyrin (PFAG), which upon exposure to sunlight converts to phototoxic FAG²³. Ingesting parts of buckwheat containing FAG can lead to sunlight-induced skin irritation, resulting in fagopyrism—a phototoxic reaction observed in livestock that consumes buckwheat³³. These photosensitizer molecules, by absorbing light in specific wavelengths, start the production of reactive oxygen species (ROS), which leads to selective cellular destruction³⁴. FAGs in common buckwheat occur in smaller quantities in stems (0.04 ± 0.03 mg g⁻¹) and in leaves (0.08 ± 0.01 mg g⁻¹), but they are notably most abundant in flowers (0.31 ± 0.03 mg g⁻¹). However, this metabolite was not detected in buckwheat seeds²⁹. HYP exhibits many therapeutic features and was studied as a photosensitizer in photodynamic therapy³⁵. There is a lack of information about the photodynamic properties of FAG²⁸. The biological function of FAG in buckwheat is not clear. Recent studies report the antifungal³¹ and antibacterial properties of FAG²⁸. Possible ways of FAG biosynthesis and production in buckwheat plants have been proposed³⁶. Kreft et al.³⁷. consider its possible involvement in protecting the plant from UV-B radiation as do other metabolites with aromatic rings of six carbon atoms, mainly phenols. However, FAGs are not activated by UV light but respond to a different part of the sunlight spectrum³⁸. FAG and PFAG have absorbance spectra within the green and yellow wavelengths range, specifically from 549 to 593 nm³⁹, with a peak absorbance at 590 nm²³. Light, in terms of intensity, duration, and quality, plays a vital role in regulating plant metabolism. Plants detect specific wavelengths of light through specialized photoreceptors, activating a variety of physiological processes⁴⁰. Among the wavelengths, blue and red light are the most effective for photosynthesis, and they are the most efficiently absorbed close to the leaf surface. In contrast, green light penetrates deeper into the leaf tissue and lower on the canopy layers, suggesting its potential role in supporting photosynthesis in areas where other wavelengths may be limited⁴¹. The function of yellow light in plant physiology is diverse, influencing plant growth and accumulation of carotenoids and flavonoids in some species⁴². Light-emitting diodes (LEDs) have emerged as a promising light source for plant cultivation in controlled environments due to their long lifespan, energy efficiency, and ability to customize spectral composition⁴³.

Our study aimed to identify the precise location of PFAG and FAG in the flowers of common buckwheat and to investigate their potential relevance using liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI). FAG production can be influenced by light, while temperature may affect PFAG formation under in vitro conditions³⁹. Considering the high abundance of FAG in the flower, compared to other plant parts, the research seeks to elucidate potential relations between light-induced changes and the distribution of this photosensitizer. We investigated if the light of different wavelengths affects the production of PFAG and FAG, and how it correlates with flower production. We hypothesize that FAGs accumulate in critical reproductive structures, such as pistils and the floral receptacle, and may contribute to flower and seed development via their photosensitive and cytotoxic properties. Additionally, we propose that FAG content varies in different flower parts depending on light spectrum conditions, potentially influencing overall reproductive success by affecting the number of flowers produced or their abortion.

Materials and methods

Plant material and growth conditions

The seeds of Fagopyrum esculentum (Moench) cv. Panda were purchased from the Malopolska Plant Breeding in Polanowice, Poland. The experiment was performed in a cultivation room with isolated growth chambers with different LED (Light Emitting Diodes) lamp combinations, encompassing blue to red wavelengths including the green (550 nm) and the yellow (580 nm) absorbance range of the studied photosensitizer³⁹. The breeding room has access to sunlight. The plants were cultivated in plots (20 × 20 × 22 cm; three plants per plot; ten plots per treatment) using a commercial soil substrate (pH = 5.8) mixed in a 1:1 (v/v) ratio with perlite. In the growth chambers with LED light, plants were cultivated under a 16-h photoperiod with fixed light intensity from 6:00 a.m. to 10:00 p.m. Buckwheat plants were cultivated for eight weeks from May until August 2023 at 20 ± 2 °C day/night and 50–60% RH. Buckwheat plants in the growth chambers were exposed to solo LED light (combined in various arrangements of blue, red, green, and yellow radiation and Full Spectrum LED Flood Light 4000 K, 45 W, Color Temp.: 380–840 nm) and average 100 µmol (photons) m^–2 s^–1 of PPFD (photosynthetic photon flux density) at the plant germination and growth stages. The growth chambers contain combinations of different LEDs: RB (red and blue), RBGY (red, blue, green, and yellow), RBY (red, blue, and yellow), and RBG (red, blue, and green) (Fig. 1B-E). The plants grown under sunlight were exposed to north-west solar radiation (Fig. 1A), and the natural daylight duration lasted from 5:25 a.m. (sunrise) to 7:55 p.m. (sunset) at the beginning of the experiment and from 5:56 a.m. to 7:30 p.m. at the end. The maximum sunlight intensity ranged between 1300 and 1600 µmol (photons) m^–2 s^–1 of PPFD during the cloudless day and on average around 600 µmol. The breeding room was situated at latitude 50° 00′ 26.2″ N and longitude 20° 15′ 32.0″ E. The DLI (daily light integral) value for the sunlight was 37 mol m⁻² day⁻¹ for an average day, while for the growth chamber, it was 5 mol m⁻² day⁻¹. The light spectrum was measured using the Lighting Passport Pro spectrometer (Asensetek, Taiwan) and the Spectrum Genius Cloud software (Taiwan).

Fig. 1.

Characteristics of the lighting spectrum during the cultivation of buckwheat plants: sunlight spectrum represents figure A; light spectrum emitted by LED lamps used in growth chambers (B-E) where figure B represents red and blue diodes (RB); C – red, blue, and green diodes (RBG); D – red, blue, and yellow diodes (RBY); E - red, blue, green, and yellow diodes (RBGY).

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI)

The method described by Slazak et al.⁴⁴, with modifications, was used. The fully-open flowers were collected from eight-week-old plants into Eppendorf tubes, embedded in gelatin (100 mg/mL in sterile water), and snap-frozen in liquid nitrogen. The longitudinal sections of the flower tissue, with a thickness of 15–20 μm, were prepared using a cryostat microtome (Leica CM3050S UV, Leica Microsystems, Welzlar, Germany) and thaw-mounted onto conductive indium tin oxide (ITO) glass slides for MALDI imaging (Bruker Daltonics, Bremen, Germany). The sections were dried under a nitrogen stream and placed for 15 min in RT in a desiccator. Three different matrix solutions for MALDI were used in separate experiments on fresh slides: (1) 2,5-dihydroxybenzoic acid (DHB) at a concentration of 35 mg/mL in 50% acetonitrile (ACN) and 0.2% trifluoroacetic acid (TFA); (2) α-cyano-4-hydroxycinnamic acid (CHCA) at a concentration of 5 mg/mL in 50% ACN and 0.2% TFA; (3) CHCA (5 mg/mL) in 70% methanol and 0.2% TFA. Matrices were applied to the samples using an automated matrix sprayer (TM-Sprayer; HTX Technologies, Carrboro, NC, United States). The sprayer operated at a flow rate of 70 µl/min, a stage speed of 1100 mm/min, track spacing of 2 mm in a crisscross pattern, 6 psi of nitrogen pressure, and a spray nozzle temp. at 95 °C for 6 passes. The sections on slides were scanned using a flatbed scanner (Epson Perfection V500) at a resolution of 4800 dpi to generate optical images. In parallel, methanol extracts from different flower parts were spotted on a metal plate target, mixed with DHB or CHCA matrix solutions, and dried before MALDI-MS. Data from the extracts were collected manually and saved as a sum spectrum for each matrix preparation.

MALDI-MSI analyses were conducted utilizing a MALDI tims TOF flex instrument (Bruker Daltonics, Bremen, Germany) operating in positive ion mode with a smartbeam 3D laser or a MALDI Fourier-transform ion cyclotron resonance (FTICR) (solariX 7T-2ω, Bruker Daltonics, Bremen, Germany) mass spectrometer equipped with a Smartbeam II 2 kHz laser. The MS acquisition parameters were set as per the manufacturer’s recommendations and adjusted for optimal performance. Data collection was carried out at a lateral resolution of 10 μm on the MALDI tims TOF fleX instrument and 20 μm on the MALDI FTICR. The images were produced and analyzed using SCiLS or flexImaging software (Bruker Daltonics, Bremen, Germany).

Flower dissection

Flower dissection was made by separating parts (Fig. 2) from five fully open flowers as one biological replicate sample. The analyses were made in four biological replicates per treatment. Flowers were collected from top inflorescence exposed to the light source.

Fig. 2.

Flower parts used for LC-MS analyses of PFAGs and FAGs: stamen (st), pistil (pi), petal (p), and receptacle (rc), bar = 0.5 cm.

Extraction of PFAGs and FAGs from dissected tissues

One biological sample was defined as specific parts of the flower (crone petals, pistils, stamens, and receptacles) collected from five flowers of buckwheat plants grown at the same experimental condition. Four replicates per experimental condition were obtained. Each sample was weighed immediately after collection, frozen in liquid nitrogen, and stored at −80 °C for further steps in separate 2 ml Eppendorf tubes. Before extraction, samples were freeze-dried (Freeze Dry System, Labconco) and then finely-powdered using a TissueLyser (Qiagen, Germantown, MD), for 1 min at 25 Hz. PFAGs and FAGs were extracted in methanol. One sample contained a maximum of up to 9 mg of FW, therefore the samples were normalized to the FW by adding the solvent in the proportional amount of 200 µl per each 1 mg of tissue. The samples were extracted in darkness at RT for 24 h, and sonicated for 15 min. In the end, extracts were centrifuged and the supernatant was collected to the new Eppendorf tubes and stored at −20 °C for further LC-MS analyses.

Liquid chromatography-mass spectrometry (LC-MS) analysis

The analyses were carried out using Xevo G2-XS quadrupole time-of-flight (QToF) mass spectrometer coupled with a Premiere Acquity UPLC (Waters Corp. Milford, MA, USA), equipped in C18 column (Kinetex 1.7 μm C18 100 Å, LC Column 100 × 2.1 mm). The extracts were separated in a 10-minute gradient of 5%−95% acetonitrile (ACN), 0.1% Formic acid (FA) in Mili-Q water, and 0.65 ml min-1 flow rate. Data was collected in the positive ion mode. Peaks for particular compounds were generated using accurate molecular weights (MW) and a mass filtering tool (Table 1) and integrated. The area under the curve (AUC) for each compound conveyed the relative abundance. All analyses were made in MassLynx software (Waters Corp.).

Table 1.

The theoretical and experimental monoisotopic m/z used for mass filtering.

Compound	Elemental composition	Calculated monoisotopic m/z	Experimental monoisotopic m/z LC-MS (m/z error [ppm])	Experimental monoisotopic m/z MALDI-MSI (m/z error [ppm])
Protofagopyrin E	C39H35N2O8	659.2393	659.2360 (5.0)	659.2401 (1.2)
Protofagopyrin F	C40H37N2O8	673.2550	673.2496 (8.0)	673.2557 (1.0)
Fagopyrin E	C39H33N2O8	657.2237	657.2250 (2.0)	657.2246 (1.4)
Fagopyrin F	C40H35N2O8	671.2393	671.2386 (1.0)	671.2396 (0.4)

The average number of flowers

The number of flowers was counted in eight-week-old buckwheat plants that grew under different light quality (S, RB, RBG, RBY, RBGY), with four replicates per treatment.

Statistical analysis

Due to the non-normal distributions (Shapiro-Wilk test), and lack of homogeneity of variance (Levene’s test), the statistical significance was calculated on log10-transformed relative abundance data obtained from LC-MS analyses. The data were analyzed using one-way ANOVA and a post hoc Tukay’s test (P < 0.05) using the STATISTICA 13 package (StatSoft, Tulsa, OK, USA). Values show the means ± SE (Standard Error) of row data.

Results

PFAGs and FAGs content in different parts of buckwheat flower

The PFAG and FAG (E and F) were analyzed in different parts of buckwheat flowers: stamen, pistil, petal, and receptacle. The content of PFAG E was statistically the lowest in the petal, while significantly higher levels were observed in the pistil, stamen, and receptacle. In the case of FAG E, the content was statistically the highest in the pistil and statistically the lowest in the petal. In the case of PFAG F and FAG F, the pistil showed statistically the highest content, whereas the petal contained statistically the lowest levels. The percentage content of PFAG E in pistils to its total content in the whole flower is 45%, PFAG F − 76%, FAG E − 42%, and FAG F − 76% (Fig. 3A-D).

Fig. 3.

Comparison of PFAGs and FAGs (forms E and F) relative content in different parts of buckwheat flowers (stamen, pistil, petal, and receptacle) and their percentage contribution to total content in buckwheat flowers. Values represent means (n = 20) ± SE, different lowercase letters indicate significant differences between parameters evaluated separately for each compound on log-transformed data analyzed with one-way ANOVA followed by Tukey’s post-hoc test (P < 0.05).

Spatial distribution of PFAGs and FAGs in buckwheat flower

Each adjustment of the methodology was done on a fresh separate glass slide with sections of the flower. Primarily, a MALDI-MSI method, for imaging the distribution of PFAGs and FAGs on the flower sections, was developed. The usage of our standard procedures with 2,5-dihydroxybenzoic acid (DHB) or α-cyano-4-hydroxycinnamic acid (CHCA) matrix sprayed in 50% acetonitrile (ACN), 0.2% trifluoroacetic acid (TFA) in water, and analyzed by two different MALDI MSI instruments (timsTOF fleX MALDI-2 or solariX Fourier-transform ion cyclotron resonance, FTICR, Bruker Daltonics, Bremen, Germany), didn’t provide any successful results. The peaks with m/z corresponding to PFAGs and FAGs were not detected. To rule out the possibility of photo-degradation during laser desorption and the influence of different types of matrix, flower extracts were spotted, mixed with DHB or CHCA, and analyzed by MALDI mass spectrometry. The FAG peaks were visible in the spectra in the case of both matrices used. Finally, to test the impact of solvent, the CHCA matrix was sprayed onto the flower slides in 70% methanol, and 0.2% TFA in water and analyzed by MALDI FTICR MSI. This approach enabled ion distribution images of PFAGs and FAGs.

All analyzed forms of PFAGs and FAGs were detected in cross-sections of buckwheat flowers. The PFAG E (m/z 659.2401) and FAG E (m/z 657.2246) are broadly dispersed across the flower cross-section, with their highest concentrations localized around the ovary (ov) and receptacle (rc) regions, and the lowest concentrations in the corolla petals (p). The spatial distribution of PFAG F (m/z 673.2557) and FAG F (m/z 671.2395) is notably concentrated around and above the ovary, with a reduced presence near the receptacle, and minimal amounts in the petals. These distribution patterns are visible in the two provided cross-sections of buckwheat flowers (Fig. 4).

Fig. 4.

Distribution of PFAGs and FAGs (forms E and F) in the buckwheat flower as depicted by MALDI-MS imaging. The spatial distribution of the studied compounds is shown in the images from the second to the fifth row: PFAG E with m/z 659.2401 (second row), PFAG F with m/z 673.2557 (third row), FAG E with m/z 657.2246 (fourth row), and FAG F with m/z 671.2395 (fifth row). The heat color scale indicates intensity levels: from black-blue (0%) to red-white (100%). Specific parts of the flower are labeled with lowercase letters: ov – ovary, p – petal, rc – receptacle. Optical micrographs of the flower specimens were taken after matrix application (first row). Scale bar = 200 μm.

PFAGs and FAGs content under different light quality

LC-MS analyses were done on extracts from different parts of flowers (stamen, pistil, petal, and receptacle) obtained from buckwheat plants that grew under different light spectrums (Table 2).

Table 2.

The relative content of PFAGs and FAGs (forms E and F) in different parts of flowers (stamen, pistil, petal, and receptacle) collected from buckwheat plants grown under different light conditions. S – sunlight; RB – LED lamps made by red and blue diodes; RBG – LED lamps made by red, blue, and green diodes; RBY – LED lamps made by red, blue, and yellow diodes; RBGY – LED lamps made by red, blue, green and yellow diodes.

Flower part	Light treatment	PFAG E	PFAG F	FAG E	FAG F
Stamen	Sunlight	1000 ± 242.4 a	3150.5 ± 1327 a	6830 ± 1310.9 b	15668.3 ± 4099.6 c
	RB	2705.7 ± 981.6 a	7648.3 ± 3101.9 a	18257.3 ± 2386.2 a	38071.5 ± 4951.2 a
	RBG	3056 ± 484.2 a	4926.5 ± 820 a	11,720 ± 735.2 ab	18774.3 ± 2282.9 b
	RBY	2669 ± 965.1 a	3988.5 ± 1322.5 a	12851.8 ± 2847.8 ab	18173.5 ± 1922.9 b
	RBGY	1433.3 ± 393.7 a	1290.7 ± 206.8 a	13357.8 ± 2472.6 ab	22465.3 ± 210.8 ab
Pistil	Sunlight	2830.3 ± 699.8 a	28,480 ± 4529 a	18046.3 ± 6271.5 abc	165,225 ± 26585.9 b
	RB	8621.7 ± 560.1 a	52246.3 ± 1747.6 a	49749.3 ± 4178.1 a	419085.5 ± 114759.2 a
	RBG	3838.3 ± 1716.4 a	21153.7 ± 1313.7 a	17269.3 ± 2858.7 bc	114,711 ± 7744.1 b
	RBY	4562.3 ± 2262.9 a	35,665 ± 15587.4 a	13414.7 ± 5096.8 c	150,849 ± 60215.5 b
	RBGY	3586.3 ± 1907.6 a	20411.7 ± 5412.8 a	35596.7 ± 8863.7 ab	208849.3 ± 6134.3 ab
Petal	Sunlight	562.3 ± 121.8 ab	858 ± 100.6 ab	3298 ± 828.4 a	6508 ± 769.9 a
	RB	904 ± 236.1 a	1655 ± 165.8 ab	6366.8 ± 966.9 a	11,252 ± 1644.5 a
	RBG	955.8 ± 219.1 a	2900.3 ± 753.9 a	3213 ± 637.2 a	9721.3 ± 3045.3 a
	RBY	191.7 ± 32.9 b	816.8 ± 297.1 b	2665 ± 697 a	5105.3 ± 821.8 a
	RBGY	177 ± 39.5 b	559.8 ± 7.6 b	4253 ± 1101.5 a	8216.7 ± 238.6 a
Receptacle	Sunlight	4568 ± 854.5 a	8584.3 ± 1936 a	33338.3 ± 9838.2 ab	60059.5 ± 18697.8 ab
	RB	4556.8 ± 1676.1 a	6214 ± 626.2 a	37375.7 ± 1048.2 a	68880.3 ± 19576.5 a
	RBG	2479 ± 461.1 ab	2595 ± 714.9 ab	7493.3 ± 1000.3 b	17217.3 ± 5014.8 c
	RBY	1154 ± 226.5 b	2933.3 ± 1079.9 ab	17,160 ± 5043.8 ab	20676.3 ± 3729.2 bc
	RBGY	1187.5 ± 240.1 b	1659.5 ± 458.1 b	10971.3 ± 3015.5 ab	22,000 ± 4356.2 bc

In stamens, no statistically significant differences were observed in the content of the studied PFAGs across all tested light conditions. However, for FAG E in the stamens, significant differences were found between plants grown under RB light, where the highest content was detected, compared to those grown under sunlight, where the content of this metabolite was the lowest. The content of FAG F in stamens varied significantly among plants grown under different lighting conditions. The highest FAG F content was recorded in plants grown under RB and RBGY light, while the lowest was recorded under sunlight (Table 2).

In pistils, no statistically significant differences were found in the content of the studied PFAGs across all tested light conditions. In the case of FAG E content, significant differences were observed only between plants grown under RB light, where the highest content was detected, and under RBG and RBY light, where the content of this metabolite was the lowest. The content of FAG F in pistils significantly differed among plants grown under RB light, where the highest content was detected, and under RBG, RBY, and sunlight, where the content of this metabolite was the lowest (Table 2).

In petals, the content of PFAG E showed statistically significant differences between plants grown under RB and RBG light, with the highest amount detected, and under RBY and RBGY light, with the lowest content of this metabolite. Similarly, for PFAG F in petals, significant differences were found between plants grown under RBG light, where the highest content was detected, and under RBY and RBGY light, with equally low content. No statistically significant differences were detected in the content of studied FAGs from plants grown across all tested light conditions in petals (Table 2).

The contents of PFAGs and FAGs varied in receptacles collected from plants grown under different light qualities. For PFAG E, significant differences were observed among plants grown under sunlight and RB light, where the content was highest, and under RBY and RBGY light, where it was lowest. Regarding PFAG F in receptacles, significant differences were found between plants grown under sunlight and RB light, exhibiting the highest detected content of this compound, and plants grown under RBGY light, displaying the lowest content. The content of FAG E showed significant differences between plants grown under RB light, with the highest content of the studied metabolite, and plants under RBG light, with the lowest content. Similarly, for FAG F in receptacles, significant differences were detected in plants grown under sunlight and RB light, showing the highest content, and under RBG light, exhibiting the lowest content of this metabolite (Table 2).

Values represent means (n = 4) ± SE, different lowercase letters within columns separately for each flower part indicate significant differences between parameters evaluated apart for each compound on log-transformed data analyzed with one-way ANOVA followed by Tukey’s post-hoc test (P < 0.05).

The average number of flowers in plants cultivated under different light-quality

The average number of flowers was calculated in eight-week-old buckwheat plants that grew under different light spectra (S, RB, RBG, RBY, RBGY). The average number of flowers per plant was statistically significantly higher under RBG, RBY, and RBGY light, whereas it was significantly lower under sunlight and RB light (Fig. 5).

Fig. 5.

The average number of buckwheat flowers collected from eight-week-old plants cultivated under different light treatments. S – sunlight; RB – LED lamps used red and blue diodes; RBG – LED lamps used red, blue, and green diodes; RBY – LED lamps used red, blue, and yellow diodes; RBGY – LED lamps used red, blue, green and yellow diodes. Values represent means (n = 4) ± SE, different lowercase letters indicate significant differences between parameters evaluated with one-way ANOVA followed by Tukey’s post-hoc test (P < 0.05).

Correlation analyses

A weak negative correlation was found between the number of produced flowers and the content of produced PFAG F in the stamens (Table 3). Negative correlations ranging from moderate to strong between the number of produced flowers and the content of all studied metabolites were detected in pistils. A weak positive correlation was found between the number of flowers and the content of PFAG F in petals. A statistically significant negative correlation was observed between the number of produced flowers and the content of the studied metabolites in the receptacle.

Table 3.

Pearson coefficients of linear correlation (P < 0.05) between the number of flowers and studied metabolites, calculated separately for each flower structure: stamen, pistil, petal, and receptacle.

	Flower part	PFAG E	PFAG F	FAG E	FAG F
No. of flowers	Stamen	−0.067	−0.114	0.027	0.075
	Pistil	−0.528	−0.449	−0.434	−0.388
	Petal	−0.055	0.145	−0.026	0.020
	Receptacle	−0.564	−0.617	−0.596	−0.493

Statistically significant correlations were indicated by the color red. The classification scheme for describing the results was adopted based on the absolute value of r as follows: no correlation (0–0.1), weak (0.1–0.3), moderate (0.3–0.5), strong (0.5–0.7), and very strong (> 0.7).

Discussion

Little is known about the role of FAG in buckwheat flowers, where it was previously detected in significant amounts^23,45. In the current study, we focused on the spatial distribution of photosensitive FAG in buckwheat flowers. We analyzed PFAG (precursor) and FAG in two forms, E and F, in the following floral parts: stamens, pistils, corolla petals, and receptacles. The main findings of this study show that, within the entire flower parts, the highest contents of PFAGs and FAGs were found in pistils, while the lowest were detected in petals. Current biological research on plant-derived photosensitizers includes investigating the mechanisms of photo-degradation of biomolecules, exploring their role in plant defense, examining plant-herbivore coevolution involving sensitizers, and applying photosensitized reactions for disease treatment⁴⁶. HYP, a popular photosensitizer structurally similar to FAG, also appears in the highest concentrations in the flowers among all parts of H. perforatum^47,48. It was shown that HYP content strongly correlates with dark gland occurrence and the highest density of dark glands was reported in stamens⁴⁹. The positioning of photosensitizers like HYP within glandular structures is thought to have evolved as a strategy to prevent the potential self-toxicity of these substances⁵⁰. However, buckwheat does not produce dark glands. The potential role of HYP in plant physiology was studied by Knox & Dodge⁵¹. Their research showed that the photodynamic effect of HYP leads to the induction of photo-oxidative damage, as evidenced by pigment depletion and ethane generation. Until now, FAGs have not been explored in terms of their function in plants.

Our study confirmed the previous report of Kim & Hwang²³ that the F form is the most abundant FAG in buckwheat flowers (Fig. 3D). Our results verified that in the pistil, the content of PFAG F and FAG F makes 76% of their total amount in the whole flower, separately for each compound. This fact may indicate a special role of the F form, both as a precursor and as a photosensitizer itself, in buckwheat pistils. Photosensitizers are characterized by their ability to absorb light at specific wavelengths, initiating activation processes that result in the selective destruction of target cells³⁴. Kim et al.²⁸. showed that extracts containing a high concentration of FAG F, when exposed to blue light, generated ROS. Hence, the high content of PFAG F and FAG F in buckwheat pistils should be further investigated.

To our best knowledge, MALDI-MS imaging of PFAGs and FAGs or similar compounds’ distribution in plant tissues has never been performed before and required some development. The compounds could not be observed on slides sprayed with DHB matrix, even though they are visible in LC-MS analyses of extracts from different parts of the flower. Because MALDI-MSI typically employs a UV laser, it may be challenging to detect photosensitizers like FAGs. For example, Low et al.⁵² demonstrated that photolabile azidobenzoyl peptides undergo varying degrees of photoactivation and degradation in MALDI-MS and to avoid that, the CHCA matrix should preferably be used. However, the application of CHCA gave a similar result with a lack of detection of m/z corresponding to FAGs. The MALDI-MS of spotted extracts showed the presence of FAGs in the case of both matrices, which proved that the issue is not the photolability. Finally, a solvent system with a higher percentage of organic solvent (70% methanol) was used for matrix spraying , enabling detection of the FAGs. Apparently, FAGs require a higher percentage of organic solvents to be extracted from the tissues and co-crystalized with the matrix, allowing laser desorption and detection.

PFAGs and FAGs in both forms were detected in the mass spectra generated by MALDI-MSI scans of longitudinal sections of flower tissue. The spatial distribution of the analyzed PFAG and FAG forms, as revealed by MALDI-MSI, was consistent with the results obtained from LC-MS analyses in each of the corresponding optical micrographs. The spatial distribution of the E-form PFAGs and FAGs was broader across the flower tissue compared to the F-form, although both forms were predominantly localized around the ovary. Specifically, PFAG F and FAG F were highly concentrated in the region surrounding the ovary in buckwheat flowers. Notably, all detected compounds were nearly absent in the petals. Previous studies on common buckwheat have indicated that elevated levels of abscisic acid, a stress hormone, in open flowers may signal imminent abscission²⁰. In research on maize, reduced antioxidant capacity has been identified as a factor contributing to increased ovary abortion, as it leads to the over-accumulation of H_₂O_₂⁵³. Thermal stress (30 °C) significantly reduces the percentage of properly developed ovules in open flowers of common buckwheat²⁰. In vitro studies have demonstrated that high temperatures significantly stimulate FAG accumulation³⁹. Given our discovery of the primary localization of cytotoxic FAG F around the ovary, this relationship should be further explored through exogenous FAG treatment on ovule sac development, followed by histological analyses. Our findings indicate a potential role of photoactive FAG F at the ovary site, highlighting the need for further detailed investigation into its function in this region.

Abiotic stressors, including light, lead to the overproduction of ROS in plant cells. ROS include both free radicals such as superoxide (O₂^•–) and hydroxyl radicals (^•OH), as well as non-radical forms like hydrogen peroxide (H₂O₂) and singlet oxygen (¹O₂). ROS plays a regulatory role in gene expression, influencing a variety of processes such as growth, cell cycle, signaling, and programmed cell death (PCD). ROS are highly reactive and can damage cellular components leading to oxidative stress, which can trigger PCD in plants⁵⁴. PCD in plants is categorized into developmental PCD (dPCD), induced by internal factors, and environmental PCD (ePCD), induced by external factors⁵⁵. The dPCD is involved in processes such as growth, reproduction, senescence and specialization of plant tissues and organs e.g. embryogenesis^56,57, while ePCD is a response to external stimuli such as light or temperature⁵⁸.

FAG, when exposed to light, absorbs energy and transitions to an excited state. The absorbed energy can be released through two mechanisms: (1) via photochemical reactions where FAG emits orange to red fluorescence, or (2) by transferring the energy to an acceptor molecule. In the second case, the photodynamic molecule shifts from a singlet to a triplet state. Upon returning to the singlet state in the presence of triplet oxygen as the acceptor, singlet oxygen is produced. These radicals oxidize cellular components such as lipids, proteins, and nucleic acids. However, the formation of superoxide radicals is also possible as a result of this energy transfer³². For example, in a study on HYP, this photosensitizer underwent a one-electron transfer to singlet oxygen at pH 7.0, but at a higher pH of 9.7, superoxide radical production primarily occurred through electron transfer between excited HYP and its ground state, with minimal involvement of singlet oxygen⁵⁹. Thus, the energy transfer pathways are highly dependent on the environment. Effective cell death has been demonstrated when sufficient light, oxygen, and the evaluated photosensitizer are present. The excitation process is also undoubtedly influenced by the light³². The singlet state has a relatively short duration compared to the triplet state, where electrons take longer to return to the ground state. And excited light sensitive molecule has a very brief lifespan and must dissipate excess energy to revert to its ground state. Because of the brief digestion time, any effects resulting from excitation (such as oxidative stress) occur at or near the site of the photosensitive molecule³⁴. Therefore, the results of MALDI-MSI analyses indicating the precise localization of the photosensitive FAG F around the ovary should be thoroughly investigated.

Light quantity (photoperiod and intensity) and quality (wavelengths) serve as fundamental factors that govern plant physiology. Plants utilize light for both its role as an energy source in photosynthesis (assimilative function) and as a signaling mechanism to initiate and regulate various pivotal processes (control function)⁶⁰. The results showed a varied response of PFAGs and FAGs to the spectrum of the tested light. However, the most stimulating effect on the production of both forms of FAGs in stamens, pistils, and receptacles was exhibited by the RB light (Table 2). These results correspond with the findings reported by Kim et al.²⁸. demonstrating that the content of FAGs E and F increased after exposing PFAG E and F to sole blue, infrared, or red-light spectra, with an increase observed as early as 1 h post-irradiation. The photochemical reactions of photosensitizers like FAGs are initiated by the absorption of photons of light³⁴. In the current study, RB light appears to demonstrate the most efficient impact on FAGs production.

Green light added to the primary RB colors increased flower production per plant (Fig. 5). This may refer to the properties of the green light to penetrate plant tissues more efficiently and support plant growth by increasing carbon fixation^61,62. However, the increased production of both forms of FAGs was observed in pistils and receptacles in plants cultivated under RB light, while the addition of G and Y wavelengths to the RB light decreased FAGs content. RB light, which stimulates the production of FAGs, significantly reduces the number of flowers produced in buckwheat. A significant negative correlation was found between the number of produced flowers and each of the studied metabolites, both PFAGs and FAGs, in the floral receptacle (Table 3). Flower and fruit abortion can be triggered by various environmental factors, including high temperatures and far-red radiation (FR; 700–800 nm)^63,64. In sweet pepper, increased auxin levels in shoot apices under supplemental FR light are thought to promote fruit abortion by increasing competition for assimilates, thereby limiting their transport to flowers⁶⁵. This process is regulated by the auxin-ethylene balance, with ethylene promoting abscission and auxin hampering it by reducing sensitivity to the abscission zone⁶⁶. Unlike sweet pepper, buckwheat does not form an abscission layer in the pedicel, which may contribute to higher flower and seed abortion rates^67,68. In our study, plants grown under S and RB light treatments produced significantly fewer flowers, with increased FR detected only under the S light condition. However, a significantly higher level of FAGs was observed in the pistils and flower receptacles of plants grown under RB treatment. Therefore, the mechanism reducing the number of flowers in these two light treatments may differ. The confirmed correlations may suggest that FAGs contribute to flower and seed abscission processes. However, determining FAGs presence in the pedicel and conducting experimental investigations are important next steps. A strong negative correlation was detected between the number of produced flowers and the studied photosensitizers in pistils. FAG exhibits phototoxic and anti-proliferative properties. For example, the purified extracts containing FAG under irradiation showed strong inhibition of the protein kinases⁶⁹. Protein kinases play a crucial role in the signal transduction pathway which regulates cell growth and differentiation⁷⁰. Since the 1950 s the topic of defective embryo sac formation and flower abortion that causes poor seed yield of buckwheat plants has been widely studied^{10,16–20,71–73}. Due to the activity of indeterminate meristems, the production of reproductive structures in buckwheat is potentially endless. Therefore, the abortion processes influencing meristems and flower activity, are the mechanisms contributing to the arrest of morphogenesis⁷⁴. The signal responsible for inducing the arrest of meristem activity, observed in various species, is the subject of many investigations. There are two frequently proposed not mutually exclusive mechanisms to elucidate this process: (1) exhaustion of a critical nutrient, and (2) generation of an inhibitory signal^74,75. Flowers within a buckwheat inflorescence do not share an equal likelihood of maturing into ripe seeds. Their development is notably influenced by their position within the raceme: flowers located at the base of the inflorescence are more prone to undergo normal development and yield mature seeds compared to those flowers positioned at the top of the inflorescence⁷⁶ which are directly exposed to light. Perhaps the localization of PFAGs and their subsequent conversion into FAGs in the receptacle serves to trigger flower abortion under the light of a specific wavelength. However, if this process is not completed, photosensitizers may reach the pistil and by surrounding the ovary disrupt the development of the female gametophyte.

Conclusions

The highest concentrations of PFAG F and FAG F were observed in the pistil of buckwheat flowers, particularly around the ovary. Light conditions during buckwheat growth significantly impacted the levels of PFAGs and FAGs, suggesting that RB light could enhance the production of these compounds. Additionally, the same light treatment was shown to decrease the number of flowers produced per plant. The detected correlations indicate that PFAGs and FAGs may play a role in flower production and could affect the development of the female gametophyte.

Future research on the biological function of FAGs and their precursors in common buckwheat is crucial, particularly regarding their cytotoxic properties and their strategic localization around the ovary and in the receptacle.

Abbreviations

ACN

acetonitrile

CHCA

α–cyano–4–hydroxycinnamic acid

DHB

2,5–dihydroxybenzoic acid

DLI

Daily Light Integral

FAG

fagopyrin

HYP

hypericin

LC

MS–Liquid Chromatography–Mass Spectrometry

LC

MS/MS–Liquid Chromatography with Tandem Mass Spectrometry

LED

Light Emitting Diodes

m/z

mass–to–charge ratio

MALDI MSI

Matrix–Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

NMR

Nuclear Magnetic Resonance

PFAG

protofagopyrin

PPFD

Photosynthetic Photon Flux Density

RB

red and blue

RBG

red, blue, and green

RBGY

red, blue, green, and yellow

RBY

red, blue, and yellow

ROS

Reactive Oxygen Species

S

Sunlight spectrum

TFA

trifluoroacetic acid

Author contributions

MH: planned and designed the research, participated in all the experiments, and drafted the manuscript. MK-M: aided in experiment preparation. AN, and PA: assisted with the MALDI-MSI experiments, data analysis, and interpretation. UG: contributed to the LC-MS experiments and data analyses. ZM: reviewed the manuscript and supervised the study. BS: participated in all experiments and supervised the study. All authors contributed to revising and finalizing the manuscript.

Data availability

The datasets used in this study are available on Zenodo at https://zenodo.org/records/14000367.

Declarations

Competing interests

The authors declare no competing interests.

Conflict of interest

The authors declare no conflict of interest.