**3. Discussion**

This study represents the first published report on quantification and distribution of secondary products, including flavonoids, phenolics, proanthocyanidins, and phytoestrogens, in aerial tissues of several annual pasture species (Tables 1–5, Figure 3). The metabolic profiling and metabolomics analyses employed in this study provided a comprehensive overview of the specific metabolites pertaining to the biosynthesis and regulation of flavonoids and related metabolites, particularly those with adverse impacts on grazing livestock. The accurate identification of secondary metabolites was facilitated by the use of quantitative liquid chromatography quadrupole time-of-flight mass spectrometry (LC-MS-QToF), allowing their detection and quantification at low concentrations (ng kg−1), with compounds identified by comparison with authentic standards or annotated using the METLIN library of secondary metabolites.


**Table 4.** Scientific, common, and cultivar names of annual pasture legumes evaluated in this study.

Along with various isoflavonoids, coumestans are produced in large quantities by members of the *Fabaceae*, commonly known as legumes, and most of these metabolites contribute to plant defense. Plant-produced coumestans are known to be associated with various biological activities, many of which can be attributed to their function as phytoestrogens and polyphenols [42]. 4- methoxycoumestrol has quantified using coumestan as surrogate standard [43,44]. Our findings sugges<sup>t</sup> that the greatest concentration of coumestans occurs in leaf and stem tissues, in contrast to floral tissues which exhibited only trace quantities. This is in agreemen<sup>t</sup> with previous findings in traditional pasture legumes [45]. In addition, our results sugges<sup>t</sup> that consumption of pure stands of novel annual pasture legumes such as biserrula, French serradella, yellow serradella, and arrowleaf clover at physiological maturity would likely pose no threat to herd fertility given the lower levels of coumestans than threshold suggested in the literature. In direct contrast, gland and bladder clover exhibited concentrations of coumestans at levels above the suggested tolerance limit of ≈40 mg kg−<sup>1</sup> in leaf tissues at approximately 48 and 70 mg kg−1, respectively.


**Table 5.** Phytoestrogens from pasture legume species identified in this study by LC-MS-QToF in positive ionization mode.

a Basis for identification codes: AM [43,44]—match to accurate mass/molecular formula and MS/MS spectra; STD—match to accurate mass and retention time of analytical standards.

The most prevalent traditional pasture legumes in Australia, lucerne and subterranean clover, have been reported to consistently accumulate higher concentrations of coumestans at flowering in both leaf and stem tissues, but recurrent selection by plant breeders has recently resulted in reduced levels in many commercial cultivars. Our results indicated that both lucerne and subterranean clover cultivars selected in this study produced substantial concentrations of both metabolites, leading to a total concentration of 52 and 38 mg kg−1, respectively; levels which may exceed livestock tolerance limits (≈40 mg kg−<sup>1</sup> DM). Considerably higher concentrations of coumestans in leaf and stem tissues of gland and bladder clover sugges<sup>t</sup> that these cultivars have experienced relatively limited genetic improvement through breeding at this stage (Angelo Loi, personal communication), and produce significant levels of secondary products that may adversely impact livestock health. This observation further supports the hypothesis that coumestans play a role in plant defense against herbivory when established in natural settings [46]. Our findings also imply that these cultivars should be avoided as sole sources of forage for grazing livestock.

Naturally occurring isoflavones found predominately in the *Fabaceae* have been well described, largely based on their pharmacological activities, including estrogenic effects, in humans and animals. The isoflavonoids profiled in the present study demonstrated clear intra-species variation as well as variation associated with tissue type. Other researchers have also noted some chemical variance within the genus *Trifolium* [47]. The ability of plants to regulate production of secondary metabolites in response to biotic and abiotic stressors, including climate change, has been well documented [48–51] and recent studies have shown that these abilities extend to production of flavonoids [12,13].

Qualitative and quantitative variation in flavonoids and associated phytoestrogens profiled in our study is an indicator of both species and tissue specific adaptations to the environment, resulting in modulation in the expression of associated genes [52]. Although recent pasture studies have suggested that isoflavones are highly abundant in *Medicago* and *Trifolium spp*. and are frequently highest before full-bloom [53], considerable variation in total concentration has been reported in mature *Trifolium pratense* pastures, with those surveyed ranging between 9000 and 27,000 mg kg−<sup>1</sup> DM [48,54,55]. Variations in isoflavone concentration may have been due to environmentally induced responses, time of sampling, extraction efficiency and recovery, and previous analytical workflows employed to profile individual isoflavones [21]. We specifically attempted to address these issues noted in previous published works by conducting experimentation in uniform and replicated field sites, creating composite replicated samples for each pasture plot, and performing sampling at similar stages of physiological maturity in both 2016 and 2017 growing seasons. In addition, we utilized an automated high-pressure extraction device to rapidly and uniformly extract all samples, thereby performing uniform extraction across treatments and replicates, and reducing the possibility of plant to plant variation or inefficiencies associated with manual extraction protocols.

Several phytoestrogenic flavonoids, such as formononetin, the most abundant isoflavone detected followed by and genistein, were prevalent at physiological maturity (50% flowering) in all pasture legumes. These observations are in agreemen<sup>t</sup> with previous reports pertaining to various *Trifolium* species [48,56–58]. Recent findings also suggested a greater abundance of phytoestrogens and isoflavonoids in leaf tissues when compared to stems and inflorescence [53]. Biosynthesis and subsequent distribution of isoflavones among leaf, stem, and floral tissues was impacted by cultivar, physiological growth stage, and climatic conditions under which plants were maintained [59]. In *Trifolium pratense* (perennial red clover), isoflavone concentrations were also found to be impacted by growth stage, with inflorescence tissue containing equivalent concentrations of isoflavones to those in leaf tissue in initial bloom stages, with subsequent declines in floral tissue as the crop entered full bloom stage [48]. The disproportionately high levels of formononetin in bladder clover observed in our study suggests that modulation of the phenylpropanoid pathway may, in fact, be species specific and is important in determination of terminal isoflavone accumulation in the pathway.

Only arrowleaf clover, gland clover, bladder clover, and subterranean clover exhibited total isoflavone concentrations above the livestock-safe threshold level of ≈280 mg kg−<sup>1</sup> DM (Table 4) and therefore, all four of these *Trifolium* spp., could theoretically adversely impact the reproductive performance of livestock. Hashem et al., 2016 similarly reported that cattle fed solely on *Trifolium alexandrinum* (berseem clover) containing isoflavones at high concentrations of ≈280 kg−<sup>1</sup> DM exhibited hormonal disruption and reduced fertility, comparable to similar cattle grazing other species [18]. Interestingly, lambs grazing *Trifolium pratense* with high levels of isoflavones exhibited weight gain compared to cultivars low in isoflavones, but macromolecular interactions impacting plant nutrition and subsequent weight gain were not fully explored in that study [60]. Future studies investigating the production of phytoestrogens, including coumestans and isoflavones at various growth stages will be important to optimize seasonal grazing.

Isoflavones are synthesized as part of the phenylpropanoid pathway (Figure 4), which has multiple branches common to both legumes and non-legumes. The phenylpropanoid pathway leads to the generation of lignins, anthocyanins, phytoalexins and flavonoids, including isoflavones, as a means of plant protection against stress or predation [29]. Encoding enzymes in this pathway are both developmentally and tissue-specifically regulated, and environmental stressors such as exposure to UV light, drought, prolonged cold, pathogen attack, and nutrient deficiency may also influence end products. Isoflavone synthase (IFS) is a key enzyme involved in the production of an array of isoflavones from naringenin, a common phenylpropanoid pathway intermediate [27]. Legumes can produce both daidzein from the intermediate compound liquiritigenin, and genistein from the intermediate naringenin chalcone in alternative branches of the phenylpropanoid pathway (Figure 4). The presence of multiple copies of enzymes such as chalcone synthase and IFS in species within the *Fabaceae* allows for the differential regulation of isoflavone biosynthesis in response to both developmental and environmental stimuli [61,62].

The flavonoid precursors and various isoflavones profiled in this study varied qualitatively and quantitatively among the annual pasture species investigated, suggesting that these species differ with respect to their metabolic dynamics and ability to regulate flavonoid biosynthesis. Daidzein and formononetin are produced from one branch of the phenylpropanoid pathway while genistein is produced from another branch. Both branches originate from *p*-coumaroyl-CoA, but the bias of the pathway towards one branch or the other is typically determined by the equilibrium between the enzymes chalcone synthase (CS) and chalcone reductase (CR) [27]. Greater production of formononetin in the pasture legume extracts profiled in this study, in contrast to the other isoflavones (daidzein and genistein) (Table 2), suggests that flavonoid biosynthesis in some pasture legumes is biased towards the branch terminating in production of formononetin as opposed to the alternate pathway ending in genistein. These findings are also consistent with previous observations [27,63]. Of note, close metabolite clustering and a higher concentration of genistein in both cultivars of biserrula and serradella species (as opposed to the more typical daidzein and formononetin) suggested potential overexpression of the gene encoding CS for the conversion of *p*-coumaroyl-CoA to naringenin chalcone in biserrula and serradella (Figure 4). This observation is consistent with recent results of a study investigating isoflavone profiles in food-related species in the *Fabaceae* family where higher genistein concentrations discriminated the genera *Biserrula* and *Ornithopus* from other members of the family [52].

**Figure 4.** Biosynthetic pathway leading to the formation of various isoflavones including phytoestrogenic isoflavones (boxed) [29,64].

Isoflavones tend to accumulate at highest concentrations at physiological maturity of the plant in *Trifolium* spp. as they commence and complete flowering [65]. To assess the impact of growth stage on the abundance of key phytoestrogens, we further profiled phytoestrogens in two common biserrula cultivars, first commercialized in Australia in 2001, at five different growth stages. Interestingly, we observed no significant differences in coumestan concentrations between the two cultivars, despite the fact that the cultivars were initially isolated from geographically separate locations in the Mediterranean. This suggests that the phenylproponoid pathway was highly conserved in this species, particularly in early growth stages, and was not a ffected by genotypic di fferences. However, the production of phytoestrogenic isoflavones varied significantly at 50% bloom and full bloom stages (Figure 2a,b). A previous study also described a significant di fference in the accumulation of isoflavonoids (formononetin and biochanin A) in di fferent cultivars of red clover, peaking at 50% bloom and full bloom stage [48]. Our results demonstrated that concentrations of all phytoestrogens assessed were lowest at senescence, and are in agreemen<sup>t</sup> with earlier observations in red clover [65]. As noted previously, the genes encoding enzymes for the biosynthesis of isoflavones are developmentally regulated and are frequently influenced by environmental stressors and various biotic factors [29,49,62], some of which are likely experienced in southeastern Australia during a typical growing season. Breeding programs in annual pasture legumes can therefore exploit the bias of the phenylpropanoid pathway by targeted manipulation of genes involved in the biosynthesis of genistein and daidzein through ectopic expression of specific transcription factors over various growth stages [66].

Pasture legume samples collected from multi-year field trials were also subjected to quantification of extractable and bound total polyphenol content (TPC) and total proanthocyanidin content (TPAC) (Table 3). The important role of TPAC in pasture legumes in reducing herbivory by impacting palatability attributes has been elucidated previously [46]. This is the first study to report on both TPC and TPAC in annual self-regenerating pasture legumes at physiological maturity and our findings support previous reports suggesting a similar range for other related pasture legumes [33,67,68]. The range of TPAC in *Ornithopus* reported in a previous study [32] was between 2 g 100 g<sup>−</sup><sup>1</sup> and 2.5 g 100 g<sup>−</sup>1, which is also in agreemen<sup>t</sup> with the current study. However, proanthocyanidins in *Onobrychis viciifolia* (sainfoin) were reported to be up to 10 times higher than measured in this study and this discrepancy could be associated with species, methodology, or growth stage of sampling [69]. Of note is that previous studies using similar assays did not detect measurable TPAC in lucerne [70,71]. This could be due to di fferences in cultivar genetics and growth conditions; however, we also observed that lucerne exhibited the lowest extractable TPC and TPAC levels of all legumes surveyed. A high concentration of extractable TPAC was noted in biserrula cv. Casbah; both cultivars also exhibited high levels of bound TPAC in their cell walls. High TPAC levels sugges<sup>t</sup> the potential for biserrula to limit pathogen and herbivore attack or reduced palatability to grazing livestock [72], but may potentially offer a cost-e ffective opportunity for this species to be integrated into multi-species mixtures to reduce parasite burden, an outcome suggested by recent replicated trials with biserrula on grazing sheep. At this time, specific bioactive proanthocyanidins in biserrula remain unidentified.

This study employed metabolic profiling approaches for identification and quantification of a number of more common secondary plant metabolites, particularly flavonoids. Our results clearly demonstrate that (a) this collection of annual pasture legumes produces a diverse array of flavonoids and other phytochemicals associated with plant defense, and in some cases less desirable phytoestrogens or proanthocyanidins and (b) the regulation of the biosynthesis of flavonoids and related metabolites such as coumestans through the phenylpropanoid pathway occurs at multiple branch sites and as reported could be impacted by elicitation in response to various biotic factors including predation and herbivory. Plants have thus developed various evolutionary adaptations for making regulatory decisions to produce an array of secondary metabolites which can provide them with multiple ecological benefits. In some cases, a single compound or related family of compounds can exhibit multiple biological functions in plants [64,73]. Despite significant di fferences in biological functions of various plant metabolites which are frequently concentration dependent, related compounds often share common biosynthetic pathways while others, including flavonoids, can originate from diverse biosynthetic pathways and precursors [51,74]. Evolutionarily, this chemical diversity or flexibility provides higher plants with a cost-e ffective strategy for further resource allocation or reallocation. As an example, our results sugges<sup>t</sup> that both lucerne and gland clover have the ability to upregulate the synthesis of formononetin from daidzein while bladder clover evolved to upregulate biosynthesis of genistein from 2,4,5,7 tetrahydroxy-isoflavone (Figure 4).

Our study results also support the hypothesis that high concentrations of key phytoestrogens, TPC and TPAC in annual pasture legumes are associated with flavonoid abundance. Interestingly, the relative abundance of total molecular features profiled through non-targeted metabolic profiling was highest in biserrula leaf tissues followed by serradella, while gland clover leaf tissue exhibited the fewest molecular features. In contrast, gland clover exhibited a higher concentration of flavonoids and their glycosides, suggesting that trade-offs in metabolite production and regulation occur in pasture legume species; i.e., if flavonoid synthesis is upregulated, then the expression of TPAC, TPC, and other polyphenols may be downregulated.

#### **4. Materials and Methods**
