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 FeCl3 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 Fe3+ 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 filtered through a 0.22-μm syringe filter (Hawach Scientific, 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, filled 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 (15-15-10; Chiyodakasei, SunAgro, Toyama, Japan) at 3 g kg-1 soil dry weight and watered daily with distilled water to saturation level (35 mL/100 g soil dry weight/pot). Seedlings were grown for 10 d until true leaves expanded. Seedlings with true leaves showing chlorosis were selected, and the soil was supplemented with or without PDMA-Fe, EDDHA-Fe, or Cit-Fe. The concentration of each Fe chelate in 35 mL soil solution was adjusted to 30 μM (0.586 mg Fe kg-1 soil dry weight). Plants were grown in a controlled growth chamber (14 h of light at 27 °C / 10 h of dark at 22 °C; light intensity 75–100 μmol photons m-2 s-1; relative humidity 50–60%) for 4 d. SPAD values in expanded true leaves were analyzed daily using a chlorophyll meter (SPAD-502Plus, Konica Minolta, Tokyo, Japan). This experiment was also performed on pumpkin (Cucurbita moschata L., cv YūYūikki white type, Saitama Gensyu Ikuseikai, Kuki, Japan).
For the hydroponic culture, seeds were germinated on moistened filter paper in Petri dishes at 25–27 °C for 1–2 d in the dark. Germinated seedlings were transferred to a net floated on 0.5 mM CaCl2 and incubated for 2–3 d. Then, seedlings were pre-cultured with a 1/5 Hoaglang nutrient solution (pH 5.8) containing the following macroelements (in mM): KNO3 (1), Ca(NO3)2 (1), MgSO4 (0.4), and (NH4)H2PO4 (0.2), and microelements (in μM): H3BO3 (3), MnCl2 (0.5), ZnSO4 (0.4), CuSO4 (0.2), and (NH4)6Mo7O24 (0.2). Fe was not added to the nutrient solution to induce Fe-deficient chlorosis. To investigate the effect of PDMA-Fe on relieving Fe deficiency, seedlings grown for a week were exposed to a treatment solution containing macroelements and 0.5 μM PDMA-Fe, EDDHA-Fe, or Cit-Fe. The treatment solution was buffered with 1 mM piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES)-NaOH (pH 7.0), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (EPPS)-NaOH (pH 8.0), or N-cyclohexyl-2-aminoethanesulfonic acid (CHES)-NaOH (pH 9.0). The solution was continuously aerated and replenished daily, and the pH was adjusted twice per day. The SPAD value in expanded true leaves was recorded daily during the 4 d of treatment. The availability of PDMA-Fe was also compared with that of a synthetic chelate with high stability in the high pH range (7.5–12), N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED)-Fe(III) (Adob, Poznań, Poland) (Ma et al. 1994); a natural microbial siderophore, deferoxamine B (DFOB)-Fe(III) (Sigma-Aldrich); and a synthetic microbial siderophore with high reducibility, tris[2-{(N-acetyl-N-hydroxy)glycylamino}ethyl]amine (TAGE)-Fe(III) (Matsumoto et al. 2001; Ueno et al. 2019) at pH 8.0.
After the treatments, plants were divided into roots, true leaves, and the other aerial parts (seed leaves and stems), washed twice with distilled water, blotted, and subjected to element concentration measurements.
Determination of element concentrations
Harvested samples were dried at 70 °C, weighed, digested with 60% (v/v) HNO3 at 140 °C, and then diluted with distilled water to appropriate concentrations. The concentrations of Fe, Zn, Mn, and Cu in the digested solution were determined using atomic absorption spectrometry (AA-6800, Shimadzu, Kyoto, Japan).
Quantitative RT-PCR
Expression of Fe deficiency-responsive genes, ferric reduction oxidase 1 (CsFRO1) (Acc. No. AY590765.1, https://www.ncbi.nlm.nih.gov/) and iron-regulated transporter 1 (CsIRT1) (Acc. No. XM_004145406.3), were compared among Fe chelate treatments to estimate differences in availability. Seedlings were pre-cultured in the absence of Fe for 4 d, then exposed to a nutrient solution (pH 9.0, 1 mM CHES-NaOH) containing 0.5 μM PDMA-Fe, EDDHA-Fe, or Cit-Fe for 72 h. The pH of the nutrient solution was adjusted three times per day. The nutrient solution was replenished 48 h after the Fe treatment. Total RNA was extracted from roots with the ISOSPIN Plant RNA kit (Nippon Gene, Tokyo, Japan), treated with DNase I (Toyobo, Osaka, Japan), and converted to cDNA using ReverTra Ace (Toyobo). Gene expression was measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR) with the primers 5′-TGTGGGCAACAACTATTCCTC-3′ and 5′-AGGAGATGCCAACATGGAAG-3′ for CsFRO1, and 5′-CTCATTGCGAGTGTCATTGG-3′ and 5′-GAATGATACCTGCTGCGAAAG-3′ for CsIRT1. Actin 7 (Acc. No. XM_011659465.2), used as an internal control, was analyzed using the primers 5′-TTGCAGACAGGATGAGCAAG-3′ and 5′-ACCCTCCAATCCAAACACTG-3′. qRT-PCR was carried out using a KOD SYBR qPCR mix (Toyobo) on a Prism 7300 Real Time PCR System (Applied Biosystems, Foster City, CA, USA).
Reducibility assay
The reducibility of Fe(III) chelates by plant roots was analyzed according to Romera et al. (1996), with some modifications. Roots of cucumber grown without Fe for 5 d were exposed to 10 mL of assay solution (0.2 mM CaSO4, 5 mM PIPES at pH 7.0, EPPS at pH 8.0, or CHES at pH 9.0; 0.1 mM PDMA-Fe, EDTA-Fe, or Cit-Fe; and 0.2 mM bathophenanthroline disulfonic acid [BPDS] [Cas. No. 98645-86-4, Dojindo 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 coefficient of 22.14 mM-1 cm-1. The fresh weight of the roots was recorded.
Measurement of O2-evolution rate
O2 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, far-red 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 fitted with a tube to allow human exhalation to saturate the interior of the chamber to a saturated CO2 state. As a result, the inside of the closed chamber can be brought into a saturated CO2 state, and the maximum photosynthetic activity can be measured. A leaf disc (2.5 cm2) 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 flux density (PFD) was adjusted to 1000 µmol photons m-2 s-1. As the chamber is a closed system, CO2 in the chamber was consumed during photosynthesis; therefore, additional CO2 was supplied with expiratory air (assumed to be CO2 saturated air).
Statistical analysis
Data were analyzed using Tukey’s tests with BellCurve for Excel (Social Survey Research Information, Tokyo, Japan). Significant differences (P < 0.05) are indicated by different letters.