Synthetic Phytosiderophore, Proline-2′-Deoxymugineic Acid, is E�ciently Utilized by Dicots

Purpose: Phytosiderophores (PS) from grasses solubilize sparingly soluble iron (Fe), and the resultant PS-Fe is an Fe source, even for dicots. Recently, the synthetic PS proline-2′-deoxymugineic acid (PDMA) has been developed as a moderately biodegradable Fe fertilizer for grasses. We aimed to investigate whether PDMA-Fe is also a good Fe source for dicots. Methods: The availability of PDMA-Fe to cucumber was evaluated in calcareous soil and hydroponic cultures under pH 7.0–9.0 by determining chlorophyll concentration, PSII activity, and Fe uptake. EDDHA-Fe, EDTA-Fe, and citrate-Fe were used as controls. The reducibility of Fe chelates by roots was measured to determine the mechanism underlying differences in availability. Expressions of Fe de�ciency-inducible genes (CsFRO1 and CsIRT1) were analyzed to estimate the Fe status in plants. Results: Application of PDMA-Fe and EDDHA-Fe to calcareous soil reduced Fe-de�cient chlorosis to a similar extent; however, shoot Fe concentration was higher in the PDMA-Fe treatment. In the hydroponic culture, PDMA-Fe had higher availability than the other chelates at every pH, which was con�rmed by higher PSII activity and lower expression of Fe de�ciency-inducible genes. The reducibility assay revealed that the reduction level of PDMA-Fe was greater than that of EDTA-Fe and citrate-Fe under alkaline pH. Conclusion: PDMA-Fe is utilized by cucumber roots more e�ciently than traditional synthetic chelates in both calcareous soil and hydroponic cultures. The higher availability of PDMA-Fe may be attributed to its higher reducibility. Our �ndings suggest that PDMA-Fe could be a good Fe fertilizer for dicots.


Introduction
Iron (Fe) is the third most abundant mineral in the Earth's crust; however, under aerobic conditions, the concentrations of Fe 3+ and Fe 2+ ions in soil solution are below 10 − 15 M at pH > 6, thus limiting plant growth at neutral or alkaline pH (Lindsay and Schwab 1982;Guerinot and Yi 1994). Therefore, plants have developed two mechanisms for Fe acquisition (called "Strategy I" and "Strategy II") to cope with this limitation (Marschner and Römheld 1986). Strategy II plants, which are mostly grasses, respond to Fe de ciency by secreting phytosiderophores (PS) into the rhizosphere which solubilize Fe(III) (Takagi 1976 (Eide et al. 1996). The acidi cation of the rhizosphere by H + release is also required to increase Fe solubility and to ensure Fe(III) reductase activity in this strategy (Römheld and Nikolic 2007). Based on the difference in mechanism, sparingly soluble Fe(III) is considered a substrate for the Strategy II system, while Fe(III) chelates are major Fe sources for the Strategy I system (Römheld and Marschner 1986).
To correct Fe de ciency in crops that insu ciently induce adaptive responses to Fe starvation, synthetic Fe chelates such as Fe-ethylenediamine-N,N,N',N'-tetraacetic acid complex (EDTA-Fe) and Feethylenediamine-N,N'-bis(2-hydroxy-phenyl acetic acid) complex (EDDHA-Fe) are supplied to the foliage or soil as Fe fertilizers (Lucena 2006; Römheld and Nikolic 2007). Among them, EDDHA-Fe is the most effective fertilizer for increasing soluble Fe in calcareous soil for uptake by the roots because of its higher stability constant at high pH (López-Rayo et al. 2009). However, the non-biodegradability of these chelates and their accumulation in the environment remains a concern (Nowack 2002;Hyvönen et al. 2003;Schenkeveld et al. 2012 Recently, a novel synthetic PS, proline-2′-deoxymugineic acid (PDMA), has been developed as a promising Fe fertilizer (Suzuki et al. 2021). The key structural difference between PDMA and natural 2′deoxymugineic acid (DMA), that is, substitution of l-proline for l-azetidine, contributes to moderate biodegradability and low synthesis cost. Application of PDMA with or without Fe to calcareous soil promoted Fe uptake in rice plants more effectively than EDDHA-Fe and EDTA-Fe. The higher availability of PDMA-Fe was attributed to the fact that PDMA-Fe can be directly taken up by YSL transporters. Since it has been suggested that dicot plants can utilize PS when intercropped with grasses (Zuo et al. 2000;Ma et al. 2003;Cesco et al. 2006;Ueno and Ma 2009), PDMA-Fe could also be a promising Fe source for dicots. Therefore, the present study aimed to evaluate the availability of PDMA-Fe to the Strategy I system using dicots. We investigated the effects of PDMA-Fe application on calcareous soil and pHdependent availability in hydroponic cultures, and revealed that PDMA-Fe could be a superior Fe fertilizer to the traditional synthetic chelates, even for Strategy I plants.

Preparation of Fe(III) chelates
Chemical synthesis of PDMA has been reported previously (Suzuki et al. 2021). To prepare the Fe(III) complex, 2.47 M FeCl 3 and 10 mM PDMA were mixed at the molar ratio of Fe:PDMA = 2:1. The pH of the solution was adjusted to 7.0 with 0.5 M NaOH to precipitate Fe 3+ as low-soluble Fe(III) oxide-hydroxide or Fe(III) oxide. The suspension was then incubated at 50 °C for 1 h with occasional mixing and then centrifuged at 15,000 rpm for 3 min. The supernatant containing PDMA-Fe was further ltered through a 0.22-μm syringe lter (Hawach Scienti c, Xi'an, China) to exclude precipitated Fe. Fe(III) citrate (Cit-Fe) was prepared similarly using citric acid (Cas No. 5949-29-1, Wako, Tokyo, Japan) instead of PDMA. Both Fe chelates were stored at −30 °C until use to avoid biodegradation. EDDHA-Fe (Dissolvine Q-Fe-6, Akzo Nobel, Amsterdam, the Netherlands) and EDTA-Fe (Cas No. 15708-41-5, Dojindo Laboratories, Kumamoto, Japan) were also used in the experiments.

Plant material and culture condition
The availability of PDMA-Fe to Strategy I plants was tested using cucumber (Cucumis sativus L., cv Hokushin, Takii, Kyoto, Japan). For the soil culture, seeds were germinated in moistened vermiculite at 27°C for 4 d. After germination, seedlings were transferred to pots (one plant per pot), which were 4.5-6 cm in diameter and 5 cm tall, lled with 100 g calcareous soil consisting of shelly fossils (pH 9.1, 15 g Fe kg -1 soil dry weight) purchased from Nihonkai Hiryo Co. Ltd., Japan. The soil was fertilized with N-P-K fertilizer  Laboratories]) for 1 h with occasional mixing at 25 °C in the dark. The solution was read at 535 nm using a spectrophotometer (V-630Bio, Jasco, Tokyo, Japan). After subtracting the A535 of the solution without the plant from that of the respective solution, the BPDS-Fe(II) concentration was calculated using the extinction coe cient of 22.14 mM -1 cm -1 . The fresh weight of the roots was recorded.
Measurement of O 2 -evolution rate O 2 exchange was monitored using a ROS Field Master (RFM) with a closed leaf-type chamber (Bunkoukeiki Co., Ltd, Tokyo, Japan). An RFM is a device that can simultaneously take a P700 absorption measurement and determine the oxygen evolution rate. The device consists of a measurement light, farred light, actinic red light, LED light source unit, closed chamber (including light detector, oxygen measurement sensor, and temperature/humidity/pressure sensor), signal processing unit, and touch panel display as the user interface. The device is powered by a 12V lithium-ion battery. The sample was irradiated with 16 × 16 mm light from the light guide path, and the transmitted light was received by a photodetector. In addition, the oxygen concentration in the closed chamber was measured using a galvanic oxygen sensor. The conversion of the sensor signal for oxygen measurement was calculated from the oxygen concentration in 1 mL of air and the amount of signal change, and the oxygen change was proportional to the measurement signal. In addition, the temperature, humidity, and atmospheric pressure inside the chamber were measured to compensate for the signal value of the oxygen sensor. The closed chamber has two doorways, one of which can be tted with a tube to allow human exhalation to saturate the interior of the chamber to a saturated CO 2 state. As a result, the inside of the closed chamber can be brought into a saturated CO 2 state, and the maximum photosynthetic activity can be measured. A leaf disc (2.5 cm 2 ) excised from the true leaves of seedlings cultured with Fe chelate (PDMA-Fe, EDDHA-Fe, or Cit-Fe) at pH 9.0 for 6 d was placed in the chamber. Actinic red light (660 nm) was illuminated from the top of the chamber, and the photon ux density (PFD) was adjusted to 1000 µmol photons m -2 s -1 . As the chamber is a closed system, CO 2 in the chamber was consumed during photosynthesis; therefore, additional CO 2 was supplied with expiratory air (assumed to be CO 2 saturated air).

Statistical analysis
Data were analyzed using Tukey's tests with BellCurve for Excel (Social Survey Research Information, Tokyo, Japan). Signi cant differences (P < 0.05) are indicated by different letters.

Effects of PDMA-Fe application to calcareous soil
To evaluate PDMA-Fe as an Fe source for Strategy I plants, we used cucumber and examined the effect of 30 μM PDMA-Fe application as a soil solution by comparing it with other Fe chelates. At 4 d after treatment, cucumber treated with Cit-Fe and without Fe treatment (control) showed Fe-de cient chlorosis, whereas plants treated with PDMA-Fe and EDDHA-Fe did not (Fig 1a). SPAD values in the Cit-Fe treatment and control groups were similar, ranging from 17 to 21 within 4 d (Fig. 1b). In contrast, the SPAD value linearly increased from 20 to 35 under the PDMA-Fe and EDDHA-Fe treatments (Fig. 1b).
For elemental analysis, shoots were divided into true leaves and other parts containing seed leaves and stems. Fe accumulated in the true leaves was mainly derived from the soil solution containing Fe chelates because Fe is rarely re-translocated from older leaves to younger leaves (Römheld and Nikolic 2007), while Fe accumulated in the other parts originated from both the seed and soil solutions. Thus, the true leaves were useful for estimating the rate of Fe uptake from Fe chelates in comparison with the whole shoot. The Fe concentration in the true leaves in the PDMA-Fe treatment was 34% higher than that in the EDDHA-Fe treatment, and more than twice as high as that in the Cit-Fe treatment and control (Fig.  2a). The Fe concentration in the other aerial parts was also signi cantly higher in the PDMA-Fe treatment than in the other treatments, but to a lesser degree (Fig. 2a). A greater effect of PDMA-Fe than EDDHA-Fe was also observed in pumpkin ( Supplementary Fig. S1). In the true leaves, Zn concentration was more than 1.5-times higher than that in the other treatments (Fig. 2b), whereas the Mn concentration was almost half that in the control and the Cit-Fe treatment but did not differ from that in the EDDHA-Fe treatment (Fig. 2c). In seed leaves and stems, both Zn and Mn concentrations showed similar tendencies to those in the true leaves but to a lesser extent (Figs. 2b, c). The Cu concentration did not differ signi cantly among the treatments (Fig. 2d). The higher shoot Fe concentration under the PDMA-Fe application suggests that PDMA-Fe is more available than the other Fe chelates in alkaline soil.

E cacy of PDMA-Fe to relieve Fe de ciency at various pH levels
To investigate the pH-dependent e cacy of PDMA-Fe to improve Fe chlorosis, we hydroponically applied Fe chelates under neutral-alkaline pH. Four days of exposure to PDMA-Fe improved Fe chlorosis more effectively than EDDHA-Fe at every pH (Fig. 3a). The SPAD value increased with time under the PDMA-Fe treatment and exhibited 1.4-, 2.1-, and 1.5-times higher rates at pH 7.0, 8.0, and 9.0, respectively, than that under the EDDHA-Fe treatment at 4 d (P < 0.01; Fig. 3b). In the Cit-Fe treatment, Fe-de cient chlorosis did not improve regardless of pH, and the SPAD value decreased with time and reached signi cantly lower levels than that in the EDDHA-Fe treatment at 4 d (P < 0.01; Figs. 3a, b). Recovery from Fe de ciency was also determined by analyzing the rate of O 2 -evolution as an index of PSII activity in true leaves of cucumber grown at pH 9.0. The rate of O 2 -evolution increased and reached a steady state at about 3 min in the PDMA-Fe and EDDHA-Fe treatments, while it did not change from a low rate in the Cit-Fe treatment (Fig. 4). The rate in the PDMA-Fe treatment was twice as high as that in the EDDHA-Fe treatment at the steady state (P < 0.01; Fig. 4).
The Fe concentration in true leaves was 1.6-and 5.5-times higher under the PDMA-Fe treatment than under the EDDHA-Fe and Cit-Fe treatments at pH 7.0, respectively (Fig. 5a). The concentration in the PDMA-Fe treatment decreased with elevated pH but was still more than twice as high as that in the other treatments (Fig. 5a). In seed leaves and stems, Fe concentration was also signi cantly higher under the PDMA-Fe treatment than under the other treatments at pH 7.0 and 8.0, but did not differ at pH 9.0 (Fig.  5b). In roots, the Fe concentration did not differ among the treatments at pH 7.0 or 8.0, but it was higher in the Cit-Fe treatment than in the PDMA-Fe treatment at pH 9.0 (Fig. 5c).

Effects of PDMA-Fe on expression of Fe de ciency-inducible genes
The higher availability of PDMA-Fe at various pH levels was further veri ed by analyzing the expression of Fe de ciency-inducible genes, CsFRO1 and CsIRT1, which have been demonstrated to be involved in ferric reduction and ferrous uptake in cucumber roots, respectively (Waters et al. 2007). At 72 h after Fe chelate application to Fe-de cient plants, CsFRO1 expression in the PDMA-Fe treatment was approximately one-fth of that in the Cit-Fe treatment and two-fths of that in the EDDHA-Fe treatment (Fig. 6a). CsIRT1 expression under the PDMA-Fe treatment was similar to that under the EDDHA-Fe treatment, but 41% lower than that under the Cit-Fe treatment (Fig. 6b).

Reducibility of PDMA-Fe
To determine the mechanism underlying the higher availability of PDMA-Fe, we assayed the reducibility of Fe from Fe chelates by cucumber roots. Colorless EDTA-Fe was used instead of EDDHA-Fe because EDDHA-Fe has a strong absorption band in the near region as BPDS-Fe(II) forms in this assay. The rate of PDMA-Fe reduction was 34% higher than that of EDTA-Fe at pH 7.0 (Fig. 7). The difference in reducibility increased with pH; the rates were 3.4-and 5.6-times higher at pH 8.0 and 9.0, respectively, in PDMA-Fe compared to EDTA-Fe. The reducibility of Cit-Fe was signi cantly lower than that of PDMA-Fe and EDTA-Fe at pH 7.0 and 8.0 but was similar to that of EDTA-Fe at pH 9.0.

Discussion
Grass-borne PS and microbial siderophores can solubilize Fe in soil (Takagi et al. 1988; Ahmed and Holmström 2014), and the resultant Fe complex can be utilized by dicots as substrates of the reductionbased Fe acquisition system (Römheld and Marschner 1986). In the rst study of synthetic PDMA (Suzuki et al. 2021), it was demonstrated that the PDMA-Fe complex can be directly taken up by grasses, leading to a higher availability than traditional chelates to the Strategy II system. Here, we provide evidence that PDMA-Fe could also be a good Fe source for Strategy I plants.
In the present study, PDMA-Fe provided more Fe than the other chelates in calcareous soil and hydroponic culture (Figs. 2,5). Consistent with Fe accumulation, chlorophyll concentration and PSII activity were recovered greatly by PDMA-Fe application (Figs. 1, 3, 4) because Fe is involved in chloroplast development and the electron transport chain (Broadley et al. 2012). Application of PDMA-Fe lowered the expression of Fe de ciency-inducible genes more than the other Fe chelates (Fig. 6) , the higher Fe uptake from PDMA-Fe may be attributed to higher reducibility (Fig. 7). In the case of EDDHA-Fe, its high availability is due to the formation of highly reducible species, which are induced by the lowering of pH by root H + release (Gómez-Gallego et al. 2005;Escudero et al. 2012). Therefore, decreased Fe uptake with increasing pH (Fig. 5) may be due to decreased formation of such species as well as inhibited reductase activity. Although the chemical mechanism underlying the higher reducibility of PDMA-Fe requires clari cation, the reduction of PDMA-Fe may be structurally less sensitive to high pH in comparison with that of traditional synthetic Fe chelates. Thus, PDMA-Fe is effective for crops relying on the reduction-based Fe uptake system.
Another possible explanation for the higher availability of PDMA-Fe is that a portion of this complex may be directly taken up by roots via YSL transporters. There is evidence that Strategy I plants can directly take up PS-Fe in intercropping systems. Xiong et al. (2013) reported that DMA secreted from intercropped maize was detected in peanut (Arachis hypogaea L.) roots in the same pot, and AhYSL1 expressed in the root epidermis showed transport activity for DMA-Fe in yeast. Recently, several reports have implied the secretion of PS from dicots, such as tomato (Astol et al. 2020) and grapevine (Marastoni et al. 2020). In addition, endogenous DMA has been reported to be present in the leaves and xylem of olive plants (Suzuki et al. 2016). These ndings suggest that dicots also utilize exogenous and endogenous PS for the uptake and translocation of Fe, supporting our hypothesis. The direct uptake system is thought to be less sensitive to alkaline pH than the reduction-based uptake system, which requires acidi cation of the soil. Therefore, PDMA-Fe may be more useful for dicot species that highly depend on the direct uptake of PS-Fe.
The rst study that compared the availability of PS-Fe and EDDHA-Fe in cucumber was reported by Römheld and Marschner (1986). In contrast to PDMA-Fe, the availability of PS-Fe was similar to or less than that of EDDHA-Fe. This discrepancy may be due to differences in the experimental design. In the previous study, PS secreted from Fe-de cient barley was supplied to the cucumber. Barley is known to secrete hydroxylated analogs of PS in addition to DMA (Ma et al. 1999). Although the capability of PDMA-Fe and DMA-Fe to be transported by YSL is similar (Suzuki et al. 2021), it is possible that the reducibility of PS-Fe is different between analogs, and the reducibility of PDMA-Fe could be equal to or higher than that of natural PS-Fe. For reduction-based uptake systems, highly reducible analogs can be a better Fe source. In fact, there is evidence that the synthetic microbial siderophore-Fe with high reducibility could provide more Fe than natural siderophore-Fe (Ueno et al. 2019).
In conclusion, PDMA-Fe is utilized by cucumber roots more e ciently than traditional synthetic chelates in both calcareous soil and hydroponic cultures. The pH-dependent tests showed that the higher availability of PDMA-Fe may be attributed to the higher reducibility at alkaline pH. Our ndings suggest that PDMA-Fe can be a good Fe fertilizer in alkaline soil for Strategy I plants.

Declarations
Funding: This work was nancially supported by the AICHI STEEL COPORATION, Cabinet O ce grant in aid, the Advanced Next-Generation Greenhouse Horticulture by IoP (Internet of Plants), Japan (grant Effects of PDMA-Fe(III) on Fe status Seedlings pre-cultured in the absence of Fe for 4 d were exposed to a nutrient solution (pH 9.0) containing 0.5 μM Fe chelates for 72 h. Relative expression levels of ferric reduction oxidase 1 (CsFRO1) and iron-regulated transporter 1 (CsIRT1) in the roots grown under the Cit-Fe(III) treatment are shown. Actin 7 was used as the internal control. Data are presented as the mean ± standard deviation (n = 3). Different letters indicate signi cant differences (P < 0.05) using Tukey's test Reducibility of Fe(III) from Fe chelates by cucumber roots Roots of cucumbers grown hydroponically without Fe for 5 d were used. Reducibility was examined under neutral-alkaline pH (7.0, 8.0, and 9.0) using the bathophenanthroline disulfonic acid assay. Data are presented as the mean ± standard deviation (n = 3). Different letters indicate signi cant differences (P < 0.05) using Tukey's test

Supplementary Files
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