OsYSL18 is a rice iron(III)-deoxymugineic acid transporter specifically expressed in reproductive organs and phloem of lamina joints

Abstract

Iron uptake and translocation in plants are important processes for both plant and human nutrition, whereas relatively little is known about the molecular mechanisms of iron transport within the plant body. Several reports have shown that yellow stripe 1 (YS1) and YS1-like (YSL) transporters mediate metal-phytosiderophore uptake and/or metal-nicotianamine translocation. Among the 18 YSL genes in rice (OsYSLs), OsYSL18 is predicted to encode a polypeptide of 679 amino acids containing 13 putative transmembrane domains. An OsYSL18-green fluorescent protein (GFP) fusion was localized to the plasma membrane when transiently expressed in onion epidermal cells. Electrophysiological measurements using Xenopus laevis oocytes showed that OsYSL18 transports iron(III)-deoxymugineic acid, but not iron(II)-nicotianamine, zinc(II)-deoxymugineic acid, or zinc(II)-nicotianamine. Reverse transcriptase PCR analysis revealed more OsYSL18 transcripts in flowers than in shoots or roots. OsYSL18 promoter-beta-glucuronidase (GUS) analysis revealed that OsYSL18 was expressed in reproductive organs including the pollen tube. In vegetative organs, OsYSL18 was specifically expressed in lamina joints, the inner cortex of crown roots, and phloem parenchyma and companion cells at the basal part of every leaf sheath. These results suggest that OsYSL18 is an iron-phytosiderophore transporter involved in the translocation of iron in reproductive organs and phloem in joints.

Fig. 1

Sequence characteristics of OsYSL18. a Phylogenetic relationships of YS1-like proteins in rice, maize, barley, and Arabidopsis. Circles represent the four subfamilies. Accession numbers: AB190912 for OsYSL1; AB126253 (AB164646) for OsYSL2; AB190913 for OsYSL3; AB190914 for OsYSL4; AB190915 for OsYSL5; AB190916 for OsYSL6; AB190917 for OsYSL7; AB190918 for OsYSL8; AB190919 for OsYSL9; AB190920 for OsYSL10; AB190921 for OsYSL11; AB190922 for OsYSL12; AB164644 for OsYSL13; AB164645 for OsYSL14; AB190923 for OsYSL15; AB190924 for OsYSL16; AB190925 for OsYSL17; AB190926 for OsYSL18; AF186234 for YS1; AB214183 for HvYS1; At4g24120 for AtYSL1; At5g24380 for AtYSL2; At5g53550 for AtYSL3; At5g41000 for AtYSL4; At3g17650 for AtYSL5; At3g27020 for AtYSL6; At1g65730 for AtYSL7; and At1g48370 for AtYSL8; b comparison of the putative amino acid sequences of OsYSL18 and YS1. White letters in black indicate identical amino acid residues. Lines indicate putative transmembrane domains of OsYSL18 and YS1. The boxed region represents the sequence determining structural properties and substrate specificity of YS1/YSL transporters as reported by Harada et al. (2007)

Fig. 2

Transporting activity of OsYSL18 analyzed by electrophysiological measurements using Xenopus laevis oocytes. The transport activities of Fe(III)–DMA, Fe(II)–NA, Zn(II)–DMA, and Zn(II)–NA were measured using the two-electrode voltage-clamp method. The oocytes were clamped at −80 mV, and steady-state currents in response to the addition of a metal–chelate complex (10 μl, 5 mM) were obtained. The data are mean ± SE of six independent oocytes injected with OsYSL18. The same number of water-injected oocytes was used as a control. N.D., not detected

Fig. 3

RT-PCR analysis of OsYSL18 expression. Total RNA from root (R), crown (C), shoot (S), or flower (F) grown under Fe-sufficient conditions was reverse-transcribed and amplified by PCR using primers specific to OsYSL18 or α-tubulin for the indicated number of cycles

Fig. 4

Subcellular localization of OsYSL18 expression. OsYSL18–GFP fusion protein (a–f) or GFP alone (g–i) was transiently expressed in onion epidermal cells and was observed by confocal laser scanning microscopy. (a, d, g) Overlay of confocal cross-sections and transmission images. PM, plasma membrane; T, tonoplast. (b, e, h) Confocal cross-sections. (c, f, i) Transmission images. (d–f) Magnified images of the boxed area shown in (a). Scale bars 20 μm

Fig. 5

Cellular localization of OsYSL18 expression in flowers and developing seeds during maturation as observed by histochemical staining of OsYSL18 promoter–GUS expression in transgenic rice plants. Before anthesis (a), just after fertilization (b), and 3 (c), 5 (d), 10 (e), 15 (f), and 30 (g) days after fertilization. (h, i) Magnified image just after fertilization. (j) Germinated pollen tube on in vitro culture medium. Shown are the results from a representative T₁ line stained overnight

Fig. 6

Cellular localization of OsYSL18 expression in vegetative organs as observed by histochemical staining of OsYSL18 promoter–GUS expression in transgenic rice plants. (a) Expression in lamina joints. (b) Magnified image of (a). (c) Expression in crown roots. (d) Longitudinal section of crown roots. (e) Expression in the crown. (f) Longitudinal section around the basal parts of the leaf sheath. Three arrows indicate the positions of the cross-sections shown in (g–j). (g) Cross-section at the upper arrow of (f). The boxed area shows the magnified area in (h). (h–j) Magnified images of cross sections at the three arrows of (f). (h), upper arrow; (i),middle arrow; and (j) lower arrow. Intervals between the images are 600 μm. (k) Magnified image of a cross-section of a vascular bundle. Arrow, phloem companion cell; Xy, xylem. Shown are the results from a representative T₁ line stained for 3 h for (a–d), 30 min for (e) and (f), and overnight for (g–k). Scale bars 1 mm for (f–j), and 50 μm for (k)