Chlorophyll Metabolites Research Articles

Concurrent Photooxidation and Photoreduction of Catechols and Para-Quinones by Chlorophyll Metabolites

Photosynthesis is initiated when the sun’s light induces electron transfer from chlorophyll to plastoquinone, a para-quinone. While photosynthesis occurs in the intact chloroplasts of living plants, similar photochemical reactions between dietary chlorophyll metabolites and quinones are likely and may affect health outcomes. Herein, we continue our studies of the direct photoreduction of para-quinones and ortho-quinones that were generated by the photo-oxidation of catechols. Chlorophyll metabolites, including pheophorbide A, chlorin e6, and pyropheophorbide A, as well as methylene blue were employed as photosensitizers. We detected hydrogen peroxide using horseradish peroxidase following the photo-oxidation of the catechol dopamine, even in the presence of EDTA, a tertiary amine electron donor. Under ambient oxygen, hydrogen peroxide was also detected after the photoreduction of several para-quinones, including 2,3-dimethoxy-5-methyl-p-benzoquinone (CoQ0), methoxy-benzoquinone, and methyl-benzoquinone. The combinations of methylene blue and EDTA or pheophorbide A and triethanolamine as the electron donor in 20% dimethylformamide were optimized for photoreduction of the para-quinones. Chlorin e6 and pyropheophorbide A were less effective for the photoreduction of CoQ0 but were equivalent to pheophorbide A for generating hydrogen peroxide in photo-oxidation reactions with photosensitizers, oxygen, and triethanolamine. We employed dinitrophenylhydrazine to generate intensely colored adducts of methoxy-benzoquinone, methyl-benzoquinone, and 1,4-benzoquinone.

Far-red light mediates light energy capture and distribution in soybeans (Glycine max L.) under the shade

Shade avoidance syndrome (SAS) is an important measure of how plants respond to shade. Shade conditions are characterized by low light density and low red-to-far-red light (R/FR) ratio. However, low light intensity and low R/FR ratio play considerable and diverse roles in SAS. In the present study, soybean plants were grown in the presence and absence of additional FR at two light levels to investigate the different effects on soybean plants. Compared with normal light intensity, net photosynthetic rate significantly decreased in low light, but increased in low R/FR ratio. Chlorophyll a fluorescence transient curves showed that the absorption of light flux increased under low light intensity and low R/FR ratio, whereas the number of photo reaction centers decreased under low light intensity. In addition, the content of porphyrins and chlorophyll metabolites (magnesium protoporphyrin IX, protochlorophyllide, and chlorophyll a and b) was significantly enhanced by low light intensity and the relative gene expression levels of protoporphyrin IX magnesium chelatase, chlorophyll a oxygenase and protochlorophyllide oxidoreductases involved in the porphyrin pathway were significantly increased. The inhibitory effects of low light on the photosynthetic rate and biomass of soybean plants were alleviated by a low R/FR ratio. A low R/FR ratio optimized light capture and achieved efficient light energy utilization under low light intensity by fine-tuning the chlorophyll level and the relative gene expression level of critical chlorophyll biosynthesis-related enzymes.

Overexpression of plastid lipid-associated protein in marine diatom enhances the xanthophyll synthesis and storage.

Plastoglobules, which are lipoprotein structures surrounded by a single hydrophobic phospholipid membrane, are subcellular organelles in plant chromoplasts and chloroplasts. They contain neutral lipids, tocopherols, quinones, chlorophyll metabolites, carotenoids and their derivatives. Proteomic studies indicated that plastoglobules are involved in carotenoid metabolism and storage. In this study, one of the plastid lipid-associated proteins (PAP), the major protein in plastoglobules, was selected and overexpressed in Phaeodactylum tricornutum. The diameter of the plastoglobules in mutants was decreased by a mean of 19.2% versus the wild-type, while the fucoxanthin level was increased by a mean of 51.2%. All mutants exhibited morphological differences from the wild-type, including a prominent increase in the transverse diameter. Moreover, the unsaturated fatty acid levels were increased in different mutants, including an 18.9-59.3% increase in eicosapentaenoic acid content. Transcriptomic analysis revealed that PAP expression and the morphological changes altered xanthophyll synthesis and storage, which affected the assembly of the fucoxanthin chlorophyll a/c-binding protein and expression of antenna proteins as well as reduced the non-photochemical quenching activity of diatom cells. Therefore, metabolic regulation at the suborganelle level can be achieved by modulating PAP expression. These findings provide a subcellular structural site and target for synthetic biology to modify pigment and lipid metabolism in microalgae chassis cells.

Melatonin delays leaf senescence in pak choi (Brassica rapa subsp. chinensis) by regulating biosynthesis of the second messenger cGMP

Melatonin (MT) is a low molecular weight compound with multiple biological functions in plants. It is known to delay leaf senescence in various species. However, no data are available on the MT signaling pathway in postharvest vegetables. This study demonstrates that MT increases cGMP concentration and the expression of the cGMP synthesis gene BcGC1 in pak choi. The cGMP inhibitor LY83583 destroys effect of MT delaying the leaf senescence. LY83583 also prevents MT treatment from reducing the expression of chlorophyll metabolism-related genes (BcNYC1, BcNOL, BcPPH1/2, BcSGR1/2, and BcPAO) and senescence genes (BcSAG12 and BcSAG21). It also inhibits MT from reducing the activity of the key chlorophyll catabolism enzymes Mg-dechelatase, pheophytinase, and pheide a oxygenase. Thus, the ability of MT to maintain high levels of chlorophyll metabolites is also destroyed. The Arabidopsis cGMP synthetic gene mutant atgc1 was used to confirm that delayed leaf senescence caused by MT is mediated, at least in part, by the second messenger cGMP.

HPLC-MS2 Analysis of Chlorophylls in Green Teas Establishes Differences among Varieties.

Green teas are nonfermented teas, the quality of which is measured by the green color. However, this category encompasses a high number of tea varieties that differ in cultivation and processing. For example, leaf or stem/bubble tea, plants cultivated under a light or shadow regime, powdered or unpowdered tea, etc. These variables determine the different qualities among green teas (Matcha, Sencha, Gyokuro, etc.) and consequently their different values on the market. Our purpose is to determine if these variables can exert an influence on the chlorophyll profile and to establish a characteristic profile for specific green teas. With such an aim, we analyzed the chlorophyll profiles of 6 different green tea varieties via HPLC-hr ESI/APCI–MS2 and identified up to 17 different chlorophyll compounds. For the first time, 132-hydroxy-chlorophylls, 132-hydroxy-pheophytins, and 151-hydroxy-lactone-pheophytins have been identified in green teas. Shadow teas (Matcha and Sencha) and light-regimen green teas can be statistically differentiated by the total chlorophyll content and the a/b ratio. However, only Matcha tea contains a higher proportion of chlorophylls a and b among the green tea varieties analyzed, justifying the higher quality and price of this variety. Other chlorophyll metabolites (pheophytins, pyropheophytins, and oxidized chlorophylls) are indicative of the various processing and storage conditions.

Photo-oxidation and Photoreduction of Catechols by Chlorophyll Metabolites and Methylene Blue.

While plant-derived oxidants can protect cells from oxidative damage, limited research has examined the role of dietary chlorophyll. Photoreduction of ubiquinone by chlorophyll metabolites and red light has been reported in vitro and in animal models. Herein we examined photo-oxidation and photoreduction reactions of catechols, dopamine and hydrocaffeic acid. Photo-oxidation of dopamine by methylene blue and the chlorophyll metabolites pheophorbide A, chlorin e6 and sodium copper chlorophyllin was studied by monitoring aminochrome, the cyclized product of the dopamine o-quinone with its amine. Singlet oxygen scavengers including sodium azide, ascorbate and glutathione decreased aminochrome formation by methylene blue and pheophorbide A. Addition of EDTA, a tertiary amine electron donor, to the reaction of dopamine, photosensitizer and red light decreased aminochrome formation. Photoreduction of the dopamine o-quinone produced by mushroom tyrosinase was achieved by both methylene blue and pheophorbide A only when an electron donor was included. Due to limited solubility, photo-oxidation and photoreduction reactions by pheophorbide A required 5-7.5% dimethylformamide for optimal reactivity. Catalytic photoreduction of 2,3-dimethoxy-5-methyl-p-benzoquinone by methylene blue or pheophorbide A and tertiary amine electron donors was observed. Among the chlorophyll metabolites, pheophorbide A was more effective than chlorin e6 or sodium copper chlorophyllin in photo-oxidation of dopamine and photoreduction reactions. Singlet oxygen inhibited lactate dehydrogenase A activity, and higher molecular weight protein cross-links were observed on SDS-PAGE. Hydrocaffeic acid competed with lactate dehydrogenase A for reaction with singlet oxygen produced by methylene blue; however, no protection by hydrocaffeic acid (HCA) was observed when pheophorbide A was used. Cysteine modification of lactate dehydrogenase A by the o-quinone of hydrocaffeic acid was detected using a redox cycling stain. Inclusion of an electron donor decreased protein labeling.

Biosurfactant assisted design treatments for remediation of petroleum contaminated soil and metabolomics based interactive study with Brassica nigra L

Soil contaminated with petroleum hydrocarbons has been considered as a serious problem for more than two decades, as it inhibits microbial population, soil enzymatic activity and exhibits abiotic stress in plants as well as affects their metabolites. In the present study, a novel and effective treatments for managing petroleum contaminated soil has been developed. The design treatments were grouped in five classes (T1, T2, T3, T4 and T5) which include petroleum contaminated soil, cow dung, cow urine and crude biosurfactant in various concentrations. After, 90 days of treatment the soils of each group were screened for microbial population dynamics, enzymatic activity, oil reduction assay, SEM and FTIR analysis. The treated soils were further employed for growing Brassica nigra L and targeted metabolomics based approach study was executed to demonstrate the effect of the treatments on the chlorophyll metabolites. The results revealed that treatments (T1, T2, T3 and T4) enhanced the microbial population dynamics and enzymatic activity of the soil with maximum 67% oil reduction in the treatment T1 (600 g Petroleum Contaminated Soil + 400 g Cow Dung + 200 ml Cow Urine + 50 ml crude Biosurfactant + 250 ml Water). Further, the results of growth parameters and targeted metabolomics based model study complemented the effectiveness of the design treatments as variation of growth parameters and chlorophyll metabolites were observed. Hence, the treatments and metabolomics model developed and can be applied commercially as an effective way for the restoration of petroleum contaminated wastelands .

The degradation of chlorophyll pigments in dairy silage: the timeline of anaerobic fermentation.

Detection of chlorophyll metabolites in milk has recently been suggested to be an indicator of a grass-fed diet fed for cattle. Such a means of detection, however, is complicated when the grazing season is over because cattle can be fed fermented silage ingredients, such as alfalfa and corn silage. During fermentation, chlorophyll compounds and other pigments undergo degradation due to the accumulation of lactic acid and the resultant decline in pH. We monitored degradation of chlorophyll compounds by measuring the fluorescence and absorption spectra of silage extracts. The spectroscopic evidence supports the hypothesis that chlorophylls are converted into fluorescent products, such as pheophytin, and further cleaved into pheophorbide. The degradation starts with dechelation and removal of the magnesium ion to produce pheophytin. Further removal of the phytol chain from pheophytin results in the production of pheophorbide. The fluorescence intensity of these degradation products is reduced compared to that of the parent molecule. These findings are important in understanding the fluorescent signal in milk when cows consume silage rather than fresh pasture grass. © 2020 Society of Chemical Industry.

Interaction Between Induced and Natural Variation at oil yellow1 Delays Reproductive Maturity in Maize

We previously demonstrated that maize (Zea mays) locus very oil yellow1 (vey1) encodes a putative cis-regulatory expression polymorphism at the magnesium chelatase subunit I gene (aka oil yellow1) that strongly modifies the chlorophyll content of the semi-dominant Oy1-N1989 mutants. The vey1 allele of Mo17 inbred line reduces chlorophyll content in the mutants leading to reduced photosynthetic output. Oy1-N1989 mutants in B73 reached reproductive maturity four days later than wild-type siblings. Enhancement of Oy1-N1989 by the Mo17 allele at the vey1 QTL delayed maturity further, resulting in detection of a flowering time QTL in two bi-parental mapping populations crossed to Oy1-N1989. The near isogenic lines of B73 harboring the vey1 allele from Mo17 delayed flowering of Oy1-N1989 mutants by twelve days. Just as previously observed for chlorophyll content, vey1 had no effect on reproductive maturity in the absence of the Oy1-N1989 allele. Loss of chlorophyll biosynthesis in Oy1-N1989 mutants and enhancement by vey1 reduced CO2 assimilation. We attempted to separate the effects of photosynthesis on the induction of flowering from a possible impact of chlorophyll metabolites and retrograde signaling by manually reducing leaf area. Removal of leaves, independent of the Oy1-N1989 mutant, delayed flowering but surprisingly reduced chlorophyll contents of emerging leaves. Thus, defoliation did not completely separate the identity of the signal(s) that regulates flowering time from changes in chlorophyll content in the foliage. These findings illustrate the necessity to explore the linkage between metabolism and the mechanisms that connect it to flowering time regulation.

Distribution Map of Plant Fluorescence Spectrum in Three-Dimensions Created by a Laser-Induced Fluorescence Spectrum (LIFS) Lidar Observations

We have developed a vegetation monitoring lidar called a laser-induced fluorescence spectrum (LIFS) lidar which is able to get the living status of plants by observing their fluorescence remotely. The features of its operation are; daytime observations possible even outdoors, mobility and self-sufficiency, capability of mapping plant living information, and a user-friendly operation by unifying the controls of different equipment of the lidar using software. These features make observations by our LIFS lidar possible at any time and any place. In forest observations, we could depict three-dimensional structures of fluorescence spectrums. We also discuss in this work the possibility of monitoring other plant physiological information such as the concentration of chlorophyll and photosynthesis secondary metabolites in this work.

Chlorophyll Oxidative Metabolism During the Phototrophic and Heterotrophic Growth of Scenedesmus obliquus.

Different cultivation strategies have been developed with the aim of increasing the production rate of microalgal pigments. Specifically, biotechnological approaches are designed to increase antioxidant metabolites as chlorophyll and carotenoids. However, although significant advances have been built up, available information regarding both the chlorophyll metabolism and their oxidative reactions in photobioreactors is scarce. To unravel such processes, the detailed chlorophyll and carotenoid fraction of Scenedesmus obliquus has been studied by HPLC-ESI/APCI-hrTOF-MS from phototrophic and heterotrophic cultures. Scenedesmus is provided with a controlled strategy of interconversion between chlorophyll a and b to avoid the formation of reactive oxygen species (ROS) at high irradiances in addition to the photoacclimation of carotenoids. Indeed, precise kinetics of 132-hydroxy- and 151-hydroxy-lactone chlorophyll metabolites shows the existence of a chlorophyll oxidative metabolism as a tool to manage the excess of energy at high light conditions. Unexpectedly, the oxidation under phototrophy favored chlorophyll b metabolites over the chlorophyll a series, while the heterotrophic conditions exclusively induced the formation of 132-hydroxy-chlorophyll a. In parallel, during the first 48 h of growth in the dark, the chlorophyll fraction maintained a promising steady state. Although future studies are required to resolve the biochemical reactions implied in the chlorophyll oxidative metabolism, the present results agree with phytoplankton metabolism.

An allele of ZmPORB2 encoding a protochlorophyllide oxidoreductase promotes tocopherol accumulation in both leaves and kernels of maize.

Phytol is one of the key precursors for tocopherol synthesis in plants, however, the underlying mechanisms concerning the accumulation of tocopherol remain poorly understood. In this study, qVE5, a major QTL affecting tocopherol accumulation in maize kernels was identified via a positional cloning approach. qVE5 encodes a protochlorophyllide oxidoreductase (ZmPORB2), which localizes to the chloroplast. Overexpression of ZmPORB2 increased tocopherol content in both leaves and kernels. Candidate gene association analysis identified a 5/8-bp insertion/deletion (InDel058) in the 5' untranslated region (UTR) as the causal polymorphism in affecting ZmPORB2 expression and being highly associated with tocopherol content. We showed that higher expression of ZmPORB2 correlated with more chlorophyll metabolites in the leaf following pollination. RNA-sequencing and metabolic analysis in near isogenic lines (NILs) support that ZmPORB2 participates in chlorophyll metabolism enabling the production of phytol, an important precursor of tocopherol. We also found that the tocopherol content in the kernel is mainly determined by the maternal genotype, a fact that was further confirmed by in vitro culture experiments. Finally, a PCR-based marker based on Indel058 was developed in order to facilitate the high tocopherol (vitamin E) maize breeding.

First-Pass Metabolism of Chlorophylls in Mice.

The dietary intake of chlorophylls is estimated to be ≈50 mgd-1 . However, their first pass metabolism and systemic assimilation is not well characterized. A group of 30 mice are fed a diet rich in chlorophylls, while 10 mice received a standard diet without chlorophylls (control group). Liver extracts are analyzed every 15 days by HPLC-ESI(+)/APCI(+)-hrTOF- MS/MS to measure the accretion of specific chlorophyll metabolites. The chlorophyll profile found in the livers of mice fed a chlorophyll-rich diet shows that the formation and/or absorption of pheophorbides, pyro-derivatives, and phytyl-chlorin e6 require the occurrence of a precise first-pass metabolism. In addition, the apical absorption of pheorphorbide a-rich micelles is significantly inhibited in Caucasian colon adenocarcinoma-2 cells pre-incubated with BLT1. Pheophorbide a absorption is, at least partly, protein-mediated through SR-BI. This active absorption process could explain the specific accumulation of pheophorbide a in the livers of animals fed a chlorophyll-rich diet. A complementary mechanism could be the de-esterification of pheophytin a in the liver, yielding pheophorbide a and phytol, which can explain the origin of phytol in the liver. Hence, the results suggest two molecular mechanisms responsible for the accumulation of the health-promoting compounds pheophorbide and phytol.

Non-fluorescent and yellow chlorophyll catabolites in Japanese plum fruits (Prunus salicina, Lindl.)

Although several chlorophyll metabolites have been shown to exert prominent benefits to human health when consumed, the battery of linear chlorophyll derivatives (phyllobilins) presents in fruits is poorly understood. Yellow chlorophyll catabolites (YCCs) are a new kind of phyllobilins recently identified in senescent leaves, probably arising from an oxidative process of the terminal chlorophyll catabolites, NCCs (non-fluorescent chlorophyll catabolites). This work deals with the characterization by first time of this kind of phytochemicals in edible fruits. Two YCCs have been identified in yellow Japanese plums, one (Ps-YCC1) previously described in the senescent leaves of Cercidiphyllum japonicum Siebold & Zucc. and Ps-YCC2, a chlorophyll catabolite structure described by first time in the edible parts of Japanese plum fruits. These YCCs were characterized by high-resolution MS/MS, describing the specific fragmentation (ring A) and the absence of the typical cleavage of phyllobilins (ring D), as a consequence of the unsaturated bond at C15–16 typical of YCCs, allowing the differentiation from NCCs. To the already known array of phenolic acids, anthocyanins and carotenoids, NCCs and YCCs may contribute to the antioxidant potential of these fruits, a potential that deserves attention and future research, considering the photochemical and photophysical behaviour of this group of tetrapyrrolic breakdown products.

Sequestration of ubiquitous dietary derived pigments enables mitochondrial light sensing.

Animals alter their physiological states in response to their environment. We show that the introduction of a chlorophyll metabolite, a light-absorbing pigment widely consumed in human diets, to Caenorhabditis elegans results in animals whose fat mass can be modulated by exposure to light, despite the worm consuming the same amount of food. In the presence of the chlorophyll metabolite, exposing the worms to light increased adenosine triphosphate, reduced oxidative damage, and increased median life spans, without an effect on animal reproduction. Mice fed a dietary metabolite of chlorophyll and exposed to light, over several months, showed reductions in systemic inflammation as measured by plasma α-macroglobulin. We propose that dietary chlorophyll metabolites can enable mitochondria to use light as an environmental cue, by absorbing light and transferring the energy to mitochondrial coenzyme Q.

A Novel Recombinant Chlorophyllase1 from Chlamydomonas reinhardtii for the Production of Chlorophyllide Derivatives.

Natural chlorophyll metabolites have exhibited physiological activity in vitro. In this study, a recombinant chlorophyllase1 gene from Chlamydomonas reinhardtii (CrCLH1) was isolated and characterized. Recombinant CrCLH1 can perform chlorophyll dephytylation and produce chlorophyllide and phytol. In a transient assay, the subcellular localization of CrCLH1-green fluorescent protein was determined to be outside the chloroplast. Biochemical analyses of the activity of recombinant CrCLH1 indicated that its optimal pH value and temperature are 6.0 and 40 °C, respectively. Enzyme kinetic data revealed that the recombinant CrCLH1 had a higher catalytic efficiency for chlorophyll a than for chlorophyll b and bacteriochlorophyll a. According to high-performance liquid chromatography analysis of chlorophyll hydrolysis, recombinant CrCLH1 catalyzed the conversion of chlorophyll a to pheophorbide a at pH 5. Therefore, recombinant CrCLH1 can be used as a biocatalyst to produce chlorophyllide derivatives.

The Green Gut: Chlorophyll Degradation in the Gut of Spodoptera littoralis.

Chlorophylls, the most prominent natural pigments, are part of the daily diet of herbivorous insects. The spectrum of ingested and digested chlorophyll metabolites compares well to the pattern of early chlorophyll-degradation products in senescent plants. Intact chlorophyll is rapidly degraded by proteins in the front- and midgut. Unlike plants, insects convert both chlorophyll a and b into the corresponding catabolites. MALDI-TOF/MS imaging allowed monitoring the distribution of the chlorophyll catabolites along the gut of Spodoptera littoralis larvae. The chlorophyll degradation in the fore- and mid-gut is strongly pH dependent, and requires alkaline conditions. Using LC-MS/MS analysis we identified a lipocalin-type protein in the intestinal fluid of S. littoralis homolog to the chlorophyllide a binding protein from Bombyx mori. Widefield and high-resolution autofluorescence microscopy revealed that the brush border membranes are covered with the chlorophyllide binding protein tightly bound via its GPI-anchor to the gut membrane. A function in defense against gut microbes is discussed.

Turnover rates in microorganisms by laser ablation electrospray ionization mass spectrometry and pulse-chase analysis

Biochemical processes rely on elaborate networks containing thousands of compounds participating in thousands of reaction. Rapid turnover of diverse metabolites and lipids in an organism is an essential part of homeostasis. It affects energy production and storage, two important processes utilized in bioengineering. Conventional approaches to simultaneously quantify a large number of turnover rates in biological systems are currently not feasible. Here we show that pulse-chase analysis followed by laser ablation electrospray ionization mass spectrometry (LAESI-MS) enable the simultaneous and rapid determination of metabolic turnover rates. The incorporation of ion mobility separation (IMS) allowed an additional dimension of analysis, i.e., the detection and identification of isotopologs based on their collision cross sections. We demonstrated these capabilities by determining metabolite, lipid, and peptide turnover in the photosynthetic green algae, Chlamydomonas reinhardtii, in the presence of 15N-labeled ammonium chloride as the main nitrogen source. Following the reversal of isotope patterns in the chase phase by LAESI-IMS-MS revealed the turnover rates and half-lives for biochemical species with a wide range of natural concentrations, e.g., chlorophyll metabolites, lipids, and peptides. For example, the half-lives of lyso-DGTS(16:0) and DGTS(18:3/16:0), t1/2 = 43.6 ± 4.5 h and 47.6 ± 2.2 h, respectively, provided insight into lipid synthesis and degradation in this organism. Within the same experiment, half-lives for chlorophyll a, t1/2 = 24.1 ± 2.2 h, and a 2.8 kDa peptide, t1/2 = 10.4 ± 3.6 h, were also determined.

Recycling and Treatment of Herbal Pharmaceutical Wastewater Using Scenedesmus quadricuada

Globally, herbal pharmaceutical industries are among the leading pharmaceutical industries. They generate large volume of wastewater during processing and production, which is highly biodegradable in nature and cannot be discharged into environment as such. Hence efforts are being made to evaluate the toxicity of herbal pharmaceutical effluents using green algae Scenedesmus quadricauda . Physico-chemically treated effluents (PCTEs) as well as biologically treated effluents (BTEs) were observed after the application of S. quadricauda . Also, S. quadricauda showed higher growth rate after the addition of PCTE and BTE. The highest yield of algae was observed in BTE up to 15 days of incubation by synthesis of chlorophyll and cell metabolites, even with 10-100% dilution of effluents. The present study also discusses the evaluation of biotoxicity and recycling on herbal pharmaceutical wastewater along with heavy metal removal.

In search of photosensitising compounds in the annual forage legume Biserrula pelecinus L.

Biserrula is an annual pasture legume endemic to the Mediterranean basin and has been recently domesticated for use in Mediterranean environments in southern Australia. Over the past 10 years the species has been associated with isolated cases of what appears to be a primary photosensitisation in sheep that graze green pastures in winter and spring. Whole-top samples of biserrula pasture were taken from a range of farmers’ paddocks over 2 years (including paddocks where photosensitisation had been observed) and methanolic extracts were screened by high-performance liquid chromatography and liquid chromatography coupled to mass spectrometry for known primary photosensitising compounds belonging to the classes furanocoumarins and dianthrones. None of these were detected. Pyrrolizidine alkaloids were also not detected and this supports the view that a secondary photosensitisation is not involved. Chlorophyll profiles were relatively unchanged between samples and this suggested that chlorophyll metabolites are unlikely to be responsible for a primary photosensitisation. A series of luteolin and apigenin mono- and diglycosides were identified in the extracts, but these are not regarded as photosensitising compounds. Further work is required to establish the photosensitising agent(s) in biserrula.