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. 2023 Aug 25;64(1):24.
doi: 10.1186/s40529-023-00396-7.

L-DOPA induces iron accumulation in roots of Ipomoea aquatica and Arabidopsis thaliana in a pH-dependent manner

En-Jung Hsieh  1 , Siao-Wei Liao  1 , Ching-Yuan Chang  1 , Chu-Han Tseng  1 , Shan-Li Wang  1 , Louis Grillet  2
Affiliations

L-DOPA induces iron accumulation in roots of Ipomoea aquatica and Arabidopsis thaliana in a pH-dependent manner

En-Jung Hsieh et al. Bot Stud. .

Abstract

Background: Iron deficiency is the leading cause of anemia worldwide, particularly in countries with predominant plant-based diets. Plants constitute the main source of dietary iron. Increasing their iron concentration could reduce the occurrence of anemia. The water spinach Ipomoea aquatica is consumed as a vegetable throughout Asia and tolerates high iron concentrations making it an attractive candidate for iron biofortification. L-DOPA is an allelopathic molecule secreted by some legumes. L-DOPA can trigger the expression of Fe deficiency-inducible genes, and could potentially be used as a biostimulant to increase Fe concentration.

Results: L-DOPA significantly affected root growth of water spinach, and triggered a massive accumulation of Fe in roots. Both effects were exacerbated when L-DOPA was dissolved in KOH, which is surprising given that L-DOPA is less stable at high pH. To check whether a higher pH could indeed increase the bioactivity of L-DOPA, we used Arabidopsis thaliana, which grows at lower pH than water spinach, and subjected the plants to L-DOPA treatments at pH 5.5 and pH 6.0, which are both within the optimal range for Arabidopsis nutrition. At pH 6.0, the root growth of Arabidopsis was more strongly inhibited than at pH 5.5. We found that at higher pH, L-DOPA oxidizes to form a melanin precipitate.

Conclusions: We concluded that the oxidation of L-DOPA that we observed upon solubilization in KOH, or in nutrient solutions at slightly higher pH produces melanin-related molecules that are more potent than L-DOPA itself to trigger the primary root growth inhibition, Fe uptake and root Fe accumulation in water spinach and Arabidopsis.

Keywords: Allelopathy; Iron; L-DOPA; Water spinach; pH.

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Conflict of interest statement

The authors declare having no competing interests.

Figures

Fig.1
Fig.1
Growth of water spinach (Ipomoea aquatica Forssk.) plants subjected to exogenous LDOPA. A Seedlings were germinated on sponges and transferred to black boxes containing ES medium. B 17 day-old water spinach plants, after 3 days of transfer to L-DOPA containingmedium. C Tubes containing the nutrient solution and L-DOPA solubilized either in HCl or in KOH. D Overview of the oxidation pathway of L-DOPA
Fig.2
Fig.2
Phenotypes of 28-day old water spinach. A Root fresh weight was significantly lower and B the length of primary root was significantly shorter in L-DOPA-treated plants. C The number of lateral roots, D shoot fresh weight and E number of leaves were not significantly different between control plants and treated plants. F Three tubes containing ES growth medium without treatment (left), and after 4 days of treatment with either L-DOPA dissolved in 0.1 N HCl (middle) or in 1 M KOH (right). The medium containing KOH-dissolved L-DOPA was noticeably darker and contained a black precipitate. Error bars represent the standard deviation (Student’s t-test. * P < 0.05, n = 8)
Fig.3
Fig.3
Iron and chlorophyll status in water spinach plants after L-DOPA treatment. A Iron concentration in roots. Fe accumulated in roots following L-DOPA treatments, and this effect was stronger with L-DOPA solubilized in KOH. B The root Fe content was also affected, showing that the increased Fe concentration resulted from an increased Fe quantity taken up by plants rather than roots being shorter. C Chlorophyll concentration in shoots was not affected by L-DOPA. D Chlorophyll content was also not affected. E Iron concentration in shoots and F Iron content of shoots per plant were not affected following L-DOPA treatment. Error bars represent the standard deviation (Student’s t-test. ** P < 0.01 and *** P < 0.001; n = 8)
Fig.4
Fig.4
Arabidopsis thaliana (Col-0) were grown hydroponically for 14 days and transferred to nutrient solutions with or without L-DOPA, at pH 5.5 and 6.0. A Pictures of the plants three days after transfer to the different treatments in a representative experiment. DOPA-treated plants appear smaller and darker than control plants that grew without L-DOPA at both pH. The roots and rosettes of plants treated with L-DOPA at pH 6 are smaller than their counterparts from plants treated at pH 5.5. B Fresh weight of rosettes showing the inhibitory effect of L-DOPA on plant growth. The inhibition of rosette size by L-DOPA was exacerbated at pH 6.0. C Ferric chelate reductase (FCR) activities of Arabidopsis roots, which constitutes an estimate of the Fe uptake activity. L-DOPA increased FCR activity at the root surface, regardless of the pH. At pH 6.0, the effect of L-DOPA on Fe reduction by roots was more pronounced. The symbols *, ** and *** indicate statistical significance of P < 0.05, P < 0.01 and P < 0.001 respectively. Results are means of the results of 3 experiments, and error bars correspond to standard deviation (n = 3)
Fig.5
Fig.5
Analysis of L-DOPA and its oxidation products and the influence of pH. A HPLC analysis of L-DOPA 10 min after solubilization in HCl at pH 2.3 and B in KOH at pH 8.7, corresponding to the two lowest pKa of L-DOPA. The experiment was carried out three times. C, D UHPLC-MS analysis of L-DOPA extracted from roots of Arabidopsis plants. Two compounds with very close elution time and m/z were detected. E SR-FTIR analysis of the dark precipitate forming in nutrient solution containing L-DOPA and of L-DOPA, melanin and DOPA-melanin. The precipitate has absorption properties similar to melanin and DOPA-melanin but distinct from them
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