The shikimate pathway and its products play a role in host-parasite relationships. Phenolic acids and AAAs are the first products of the shikimic acid pathway and are considered the source of building blocks for a very wide number of primary and secondary products in the plant kingdom (Vogt, 2010). In the current study, we defined the profiling changes in phenolic acid and AAAs of the two faba bean varieties and associated O. crenata parasites during four stages of infestation. Healthy and infected faba bean plants of the two faba bean varieties were collected 16 weeks after planting. Then, infected plants were classified into four groups depending on the developmental stages of the attached O. crenata parasite.
Differences in phenolics and AAAs profiling between infected and non-infected faba bean plants at four stages of Orobanche infestation.
Phenolic acids are naturally occurring antioxidants that are considered one of the main classes of plant phenolic compounds and serve as a main precursor for the synthesis of polyphenol compounds including flavonoids, tannins, lignin, lignans, coumarins, and other phenolic compounds (Kumar and Goel, 2019). Firstly, we evaluate the changes in phenolic acid compositions, total phenolics, total flavonoids, and their antioxidant activities in the infected and non-infected plants of two faba bean varieties throughout four Orobanche infestation stages. In this study, three benzoic-derived phenolic acids, protocatechuic, p-hydroxybenzoic, and vanillic, as well as three cinnamic-derived phenolic acids, caffeic, coumaric, and ferulic acids, were detected in the roots and leaves of the healthy and infected faba bean plants with different concentrations. In line of these results, six phenolic acids, namely, p-hydroxybenzoic acid, vanillic acid, salicylic acid, ferulic acid, benzoic acid, and cinnamic acid, were previously identified in the roots and leaves of faba bean plants (Cen et al., 2023).
The tendency for phenolic acids, total phenolics, total flavonoids, and antioxidant activity to increase in parasitized roots and, to a lesser extent, in parasitized leaves as compared to those of healthy plants was noticed at the most infestation stages. These results are in accordance with those of Zakaria et al. (2015), Briache et al. (2020), and El-Mergawi et al. (2022). In this regard, Karou et al. (2005) reported that the plants responded to pathogen attack by accumulating phenolic phytoalexins, such as hydroxycoumarins and hydroxycinnamates. The enhancement effect of infection on phenolic acids may be connected with their antioxidant defense function against parasites or with their involvement in preventing attached parasite from developing into the host vascular system (Rubiales and Fernández-Aparicio 2012). Our findings showed that the phenolic acids in roots varied in their enhancement effects with parasite infestation. The levels of both vanillic acid and caffeic acid in roots showed a significant increase as affected by Orobanche at the most infestation stages. Variations in the response of phenolic acid in Carthamus glaucus plants to the Cuscuta babylonica parasite were observed by Asan and Ozen (2016). They found that among the identified phenolic acids, the greatest increase possessed gallic acid (2.4 fold), caffeic acid (1.7 fold), and protocatechuic acid (PCA) (2.1 fold). The results declared that the enhancement effect of Orobanche infestation on the different phenolic constituents varied between the tested varieties. Such differences may be related to the differences between the two varieties in their resistance to the parasite (Zakaria et al., 2015). In this concern, Serghini et al. (2001) found that resistance to the Orobanche parasite is related to increasing the levels of phenolics in host roots, suggesting that the increase of phenolics is a tolerance mechanism to maintain redox homeostasis and improve plant health. Moreover, the identified phenolic constituents in leaves showed variations in their responses to the different stages of parasite infection. The difference between caffeic, ferulic, and coumaric acids that possessed faba bean shoots in their response to Orobanche infestation was previously observed by El-Akkad et al. (2002). Caffeic acid in the infected leaves tended to exhibited a continuous increase at all infestation stages when compared with the leaves of healthy plants. In accordance with these results, the greatest increase in the levels of caffeic (1.7-fold) was observed when parasitizing Carthamus glaucus plants with Cuscuta babylonica (Asan and Ozen, 2016). Meanwhile, the maximum increase in the level of all identified phenolics in the roots of both tested varieties due to Orobanche infection was observed at the first infestation stage, and caffeic acid achieved the greatest increase (six fold). The accumulation of phenolic compounds in the infected roots might induce a toxic release near the parasite (Pérez-de-Luque et al., 2009), which may play a defense role against the penetration of the parasite haustorium into the host (Asan and Ozen, 2016).
The results revealed that the levels of free AAAs profiled in the roots and leaves of two faba bean varieties were changed consequently by Orobanche parasitism. Levels of the free phenylalanine, tyrosine, and tryptophan in the roots of the two faba bean varieties showed significant increases as affected by the most stages of Orobanche infestation. In line with these results, a significant increase in the levels of the two amino acids serine and methionine was observed in Broomrape-infected carrot roots when compared with the healthy roots (Nandula et al., 2000). Such enhancement effects of parasite infection on free AAAs in host roots may be due to the higher demand for these amino acids in the infected roots as a result of parasite attachments (Nandula et al. 2000). On the contrary, we observed that the Orobanche parasitism tended to reduce the levels of AAAs in faba bean leaves of both faba bean varieties during different infestation stages. Leaves served as a sink for the metabolites; decreasing levels of free AAAs in infected leaves could reflect the additional susceptible sink that parasite plants represent (Clermont et al. 2019). Meanwhile, we observed differences between the two faba bean varieties in terms of changes in the accumulation of AAAs in roots or leaves upon infestation by O. crenata. Variations in the response of O. crenata infestation to fatty acid composition between the tested faba bean varieties were reported by Abbes et al. (2020). Such variations in response between the two varieties may be related to the differences between them in their resistance to the parasite (Zakaria et al., 2015).
Differences in metabolic profiling of phenolics and AAAs between host and parasite during four infestation stages.
Orobanche, similar to all other holoparasite plants, are chlorophyll-lacking parasites, unable to undergo photosynthesis, and totally dependent on their hosts for carbohydrates, minerals, and water (Abbes et al., 2020). In the current study, the phenolic components and AAAs metabolic profiling of the parasite was compared across its developmental stages and directly against the respective roots and leaves of the two host varieties. The results of the current study provide many evidences that indicate O. crenata is able to self-regulate its phenolic and AAA metabolites during its developmental stages.
The first evidence was concerned with the quantitative differences in the phenolics and AAAs in the parasitic tissues that occurred at four growth stages of the parasite as it developed on two host varieties. We observed that the level of detected phenolic acids, total phenolics, total flavonoids, and antioxidant activities in the parasite's tissues showed a noticeable change during its developmental stages, and the changes in these compounds did not have the same pattern. We found that the levels of caffeic acid, the major cinnamic-derived phenolic acid, showed to be in the range of 343 µg g− 1 and 5445 µg g− 1 during different developmental stages of the parasite. A qualitative change in the phenolic acids, P-coumaric, ferulic, and caffeic compositions during various developmental stages of the O. crenata parasite was previously observed by El-Akkad et al. (2002). Many investigators showed significant variations in the metabolic composition during different developmental stages of Phelipanche aegyptiaca and Phelipanche ramosa (Nativ et al., 2017; Clermont et al., 2019). These results indicated that the holoparasite can self–regulate its own metabolites during their developmental stages (Kumar et al., 2022). Another good example of the ability of the O. crenata parasite to regulate its own metabolites is that this parasite showed a great variation in its content from phenylalanine (372–663 µg g− 1), tyrosine (138–603 µg g− 1), and tryptophan (31–170 µg g− 1), during the tested developmental stages. In accordance with these findings, a great difference in the levels of AAAs in Phelipanche aegyptiaca holoparasite was observed during its developmental stages (Nativ et al., 2017; Kumar et al., 2022). Moreover, a great change in the levels of phenolic compounds and AAAs was observed between the developmental stages of the parasite, which developed on the two different host varieties. For example, caffeic acid in parasite grown on Nubaria 4 showed great variations in their level to be constituted between 343µg g− 1 at the second stage, and 5445 µg g− 1 at the fourth stage. Whereas, the level of this compound in parasite grown on Nubaria 5 showed its minimum value at the fourth stage (428 µg g− 1) and its maximum value at the third stage (4067 µg g− 1). Significant differences were observed in the phenolics identified in Cuscuta reflexa that developed on the different hosts, suggesting that the holoparasite can self-regulate its metabolites (Kumar and Amir 2021).
There is much other evidence concerned with the qualitative and quantitative differences in metabolic profiling of phenolics and AAAs between host and parasite during different infestation stages. In addition to the six phenolic compounds detected in host tissues, HPLC analysis showed that syringic acid is considered to be characteristic of the parasite at all developmental stages. Many secondary metabolites were detected only in the parasite tissues; for example, phenylethanoid glycosides are considered to be characteristic of parasites, and they usually do not occur in the host (Scharenberg and Zidorn, 2018). In this regard, Emran et al. (2020) reported that among the detected carotenoids, zeaxanthin only occurred in the holoparasite Phelipanche aegyptiaca, which is grown on carrots. Moreover, we observed that the levels of detected phenolic acids in the roots and leaves of the two host varieties did not show permanent relationships with the levels of these compounds that were present in the respective parasite tissue at all infestation stages. The parasite tended to produce a significant increase in vanillic and caffeic acid content as compared with those of the host roots and host leaves of the two host varieties at all infestation stages. The parasite showed a great increase in caffeic acid content at the fourth infestation stage of the Nubaria 4 variety and at the third for the Nubaria 5 variety, making the concentration of this compound in the parasite reach more than 100 times and 20 times its concentration in the respective host roots, respectively. On the contrary, the parasite developed on both host varieties continued to accumulate less coumaric and ferulic acids than those accumulated by the roots and leaves of the respective hosts at all infestation stages. Additionally, the levels of p-hydroxybenzoic acid in parasitic tissues tended to decrease compared with those of host roots at all infestation stages. A difference in the detected phenolic acids between the O. crenata parasite and its faba bean host was observed by El-Akkad et al. (2002). The polyphenol profile of Phelypaea tournefortii (parasite) was different from that of Tanacetum polycephalum (host), indicating the presence of an independent biosynthetic pathway in the holoparasite (Piwowarczyk et al., 2020). Moreover, there was an increase in total phenolic and total flavonoid contents in parasite tissues when compared with those of the respective host roots of the two examined varieties. The parasite developed on Nubaria 5 roots possessed the maximum increase in total flavonoid contents; their levels reached 4- to 6-fold those of host roots at all infestation stages. In line with these results, Hacham et al. (2016) found that total phenols in the Orobanche parasite increased by 3.3-fold compared to the roots of infected tomatoes.
Great variations between the host and parasite in their contents from the free AAAs were observed during different infestation stages. The concentration of free phenylalanine in the parasite ranged between 2.2 and 5.5 times that of the host roots at all stages of infestation. But the parasite tended to accumulate lower amounts of free tyrosine and tryptophan compared to those found in the respective roots and leaves of two host varieties. These findings suggest that the parasite can self-regulate its own AAAs. The difference in AAAs between tomato roots and the Phelipanche aegyptiaca parasite attached to them was also observed by Hacham et al. (2016). Who found that the Phelipanche aegyptiaca contents from Phe, Trp, and Tyr are higher by 37, 8, and 40-fold, respectively, compared with the host tomato roots. The differences in the results of this study and our findings may be due to the differences between the host and parasite examined in the two studies.
Among the 17 amino acids identified in the two host parts and associated parasite at the third infestation stage, a remarkable variation in their levels was observed. The difference in phenylalanine and tyrosine contents was observed between the parasite and the respective host roots. Also, we found a remarkable variation in the levels of the other amino acids between two host parts and attached parasites. The parasite that grew on the two host varieties accumulated higher amounts of alanine, valine, methionine, and isoleucine, along with lower amounts of serine, glycine, and threonine than those accumulated by the roots and leaves of the two host varieties. In accordance with these results, a significant difference in amino acid contents was shown between the tomato roots and the Phelipanche aegyptiaca tubercles attached to them, suggesting that, in addition to amino acids that are transported from the host, the parasite can synthesize its own amino acids (Hacham et al., 2016). The dependence of P. aegyptiaca holoparasite completely on its own production of AAAs and the other amino acids was previously implied by Shilo et al. (2016).
Based on these results, we proposed that O. crenata has its own metabolic mechanism that enables the parasite to modify synthesize phenolic acids and AAAs according to its own needs, which differ from those of its faba bean host. These findings suggest the presence of an independent biosynthetic pathway for the synthesis of aromatic compounds in O. crenata holoparasite (Piwowarczyk et al., 2020). Theoretically, phenolic acids and AAAs are synthesized mainly throughout the shikimate pathway (Kumar and Goel 2019); hence, our findings imply the ability of this parasite to synthesize its own aromatic compounds through the activity of its shikimate pathway enzymes. Indications of the presence of functional pathways in O. crenata were recently noted after studying the effect of glyphosate herbicide targeting the EPSPS enzyme in the shikimate pathway (El-Mergawi and El-Dabaa, 2021). The results of Kumar et al. (2022) indicated that the Phelipanche aegyptiaca parasite has its own EPSPS enzyme that is significantly more sensitive to glyphosate herbicide than their host. The presence of activity for shikimate pathway enzymes in the holoparasite tissues can serve as direct evidence that the parasite has its own machinery for AAA and phenolic acid biosynthesis.