Abstract
Iron deficiency is a serious agricultural problem, particularly in alkaline soils. Secretion of coumarins by Arabidopsis thaliana roots is induced under iron deficiency. An essential enzyme for the biosynthesis of the major Arabidopsis coumarins, scopoletin and its derivatives, is Feruloyl-CoA 6'-Hydroxylase1 (F6'H1), which belongs to a large enzyme family of the 2-oxoglutarate and Fe2+-dependent dioxygenases. We have functionally characterized another enzyme of this family, which is a close homologue of F6'H1 and is encoded by a strongly iron-responsive gene, At3g12900. We purified At3g12900 protein heterologously expressed in Escherichia coli and demonstrated that it is involved in the conversion of scopoletin into fraxetin, via hydroxylation at the C8 position, and that it thus functions as a scopoletin 8-hydroxylase (S8H). Its function in plant cells was confirmed by the transient expression of S8H protein in Nicotiana benthamiana leaves, followed by metabolite profiling and biochemical and ionomic characterization of Arabidopsis s8h knockout lines grown under various iron regimes. Our results indicate that S8H is involved in coumarin biosynthesis, as part of mechanisms used by plants to assimilate iron.
Highlights
Iron (Fe) is an essential micronutrient for all living organisms
When we conducted a double ip infiltration leading to the overexpression of S8H and F6’H in tobacco leaves, we detected a significantly higher level of fraxetin in comparison to leaves transiently expressing S8H alone r (p = 0.01) or leaves infiltrated with control (Figure 4). This result c is consistent with an increase of the scopoletin pool that occurs due to the F6’H activity, s which can be further transformed into fraxetin and confirms that At3g12900 functions as a u scopoletin 8-hydroxylase. an Characterization of independent s8h mutant alleles grown in Fe-depleted hydroponics M To get an insight into the physiological role of S8H, we identified two independent mutant d lines with non-functional S8H dioxygenase (s8h-1 and s8h-2) and investigated their behaviour in various types of culture
The targeted metabolite profiling of methanolic extracts from the plant roots grown in hydroponic systems showed a highly significant increase in scopolin (P < 0.01) and scopoletin (P < 0.01) concentration in roots grown in Fe-depleted conditions for both mutant lines and a significant increase of umbelliferon accumulation in s8h-1 compared with Col-0 plants (Figure 5C)
Summary
Iron (Fe) is an essential micronutrient for all living organisms. In plants, chloroplast and mitochondria have high Fe demand (Nouet et al, 2011). P As phosphate (Pi) and Fe deficiency interact with respect to Fe-induced coumarin secretion e (Ziegler et al, 2016), and our soil experiments found that Col-0 and s8h-1 phenotypes were c dependent on the P to Fe ratio (Figure S10, Table S6), another observation from the trace c elements analysis is the lower levels of P content in both mutants (significantly lower in s8h-1 Aline, P < 0.05), in comparison to Col-0 plants under Fe-depleted (0 μM Fe2+) conditions (Figure S11A). D Plants grown on Fe-deficient plates (10 μM Fe2+) under UV light showed that both s8h mutant lines te secreted to media an increased level of fluorescent compound (Figure S14A), which might be related to significantly higher amounts of scopoletin detected in s8h Fe-deficient liquid culture solution p (Figure 6B) This difference in fluorescence was not observed in the control conditions (50 μM Fe2+), e where mutant plants seemed to accumulate slightly more fluorescent compounds in roots compared Acc to WT plants (Figure S14B). Shoots of mutant lines seemed to be Aslightly brighter
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
