# Insights on the Adaptation of _Foericulum vulgar_ Mill to Iron Deficiency
## 3 Results and Discussion
### Effect of HCO\({}_{3}\)-Induced Fe Deficiency on Phenolic Compounds  
The antioxidant and chelating properties of phenolic compounds are crucial in the adaptive response to Fe deficiency [16]. For this reason, monitoring of total phenolic content (TPC) and total flavonoid content (TFC) was conducted in hydromethanolic extracts obtained from control and treated fennel plants. As shown in Figure 4, the levels of phenolic compounds were higher in leaves than in roots. Curiously, when submitted to Fe deficiency conditions, values of TPC and TFC decreased in leaves, compared to the control plants (from 3.93 \(\pm\) 0.27 to 2.92 \(\pm\) 0.21 mg GAE/g DWplant and from 2.14 \(\pm\) 0.31 to 1.82 \(\pm\) 0.04 mg CE/g DWplant, respectively), while the opposite trend was observed in roots (from 0.49 \(\pm\) 0.02 to 1.07 \(\pm\) 0.14 mg GAE g/DWplant and from 0.37 \(\pm\) 0.08 to 0.88 \(\pm\) 0.02 mg CE/g DWplant, respectively). Figure 4: Levels of total phenolic compounds (**A**) and of flavonoids (**B**) in leaves and roots of fennel plants grown under Fe sufficiency (+Fe) or under Fe deficiency conditions (+Fe +Bic). Data represent the mean \(\pm\) standard deviation of three independent assays. Values with different superscripts are significantly different at \(p<\) 0.05. RP-HPLC analysis was herein used to further understand the impact of iron deficiency on the levels of individual phenolic components for both organs. Eight phenolic compounds, already reported for this plant species [12], were identified by RP-HPLC analysis of the hydromethanolic extracts from leaves, namely, the gallic acid, chlorogenic acid, ferulic acid and _trans_-cinnamic acid, and the flavonoids isorhamnetin, rutin, kaempfer and quercetin. Fe starvation did not cause an appreciable qualitative modification on the chromatograms, but it changed the levels of the individual phenolic compounds (Supplementary Materials Figure S1). In particular, the amounts of gallic acid and kaempfer (i.e., the main identified hydroxybenzoic acid and flavonoid, respectively) were decreased by about 26% and 35%, respectively, whereas major hydroxycinnamic acids showed an opposite trend (increasing from 0.075 to 0.160 mg/g DW for chlorogenic acid and from 0.070 to 0.135 mg/g DW for _trans_-cinnamic acid) (Figure 5). Overall, it is of note that the phenolic acids pool (hydroxybenzoic plus hydroxycinnamic acids) was maintained, unlike that of flavonoids, that was significantly decreased, causing a total decrement in phenolic compounds (in agreement with the TPC and TFC results, in Figure 4). Figure 5: Contents of individual phenolic acids (**A**) and flavonoids (**B**) (mg/g DW) in leaves (L) and roots (R) of fernnel plants grown in the presence of Fe (+Fe) or in the presence of Fe plus bicarbonate (+Fe +Bic). GA, gallic acid; Chl A, chlorogenic acid; CA, caffe acid; SA, syringic acid; FA, ferulic acid; t-Cin A, _trans_-hydroxycinnamic acid; Iso, isorhamnetin; R: rutin, IsoQ: isoquercetin; K, kaempfer; Q, quercetin; ND, non-detected. Data represent the mean \(\pm\) standard deviation of three independent assays. Values with different superscripts are significantly different at \(p<0.05\). In turn, in comparison to the control, the hydromethanolic extracts obtained from bicarbonate-treated root organs, showed a remarkable increase in the levels of phenolic compounds, including flavonoids (isoquercetin and isorhamnetin) and phenolic acids (gallic acid, chlorogenic acid, syringe acid, ferulic acid, caffeic acid and _trans_-cinnamic acid), compared to control plants. Notably, some of these compounds (e.g., isoquercetin and ferulic acid) were not detected in the extracts from the control plants, suggesting that, under Fe stress, roots may be able to synthesize new phenolic compounds (Figure S1 and Figure 5). Phenolics represent the main classes of organic ligands exuded by strategy I plants under Fe-deficiency [38], due to their ability in the mobilizing of Fe in the rhizosphere by interfering two based mechanisms, including reduction and complexation [38; 39].