Mitani N et al. (2009), Identification of maize silicon influx transpor...

XB-ART-38174

Plant Cell Physiol 2009 Jan 01;501:5-12. doi: 10.1093/pcp/pcn110.

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Identification of maize silicon influx transporters.

Mitani N , Yamaji N , Ma JF .

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Maize (Zea mays L.) shows a high accumulation of silicon (Si), but transporters involved in the uptake and distribution have not been identified. In the present study, we isolated two genes (ZmLsi1 and ZmLsi6), which are homologous to rice influx Si transporter OsLsi1. Heterologous expression in Xenopus laevis oocytes showed that both ZmLsi1 and ZmLsi6 are permeable to silicic acid. ZmLsi1 was mainly expressed in the roots. By contrast, ZmLsi6 was expressed more in the leaf sheaths and blades. Different from OsLsi1, the expression level of both ZmLsi1 and ZmLsi6 was unaffected by Si supply. Immunostaining showed that ZmLsi1 was localized on the plasma membrane of the distal side of root epidermal and hypodermal cells in the seminal and crown roots, and also in cortex cells in lateral roots. In the shoots, ZmLsi6 was found in the xylem parenchyma cells that are adjacent to the vessels in both leaf sheaths and leaf blades. ZmLsi6 in the leaf sheaths and blades also exhibited polar localization on the side facing towards the vessel. Taken together, it can be concluded that ZmLsi1 is an influx transporter of Si, which is responsible for the transport of Si from the external solution to the root cells and that ZmLsi6 mainly functions as a Si transporter for xylem unloading.

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Species referenced: Xenopus laevis
Genes referenced: actl6a

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	Fig. 1. Alignment of the amino acid sequences of ZmLsi1, ZmLsi6, OsLsi1 and OsLsi6 (A) and their phylogenetic relationship (B). The phylogenetic analysis was performed using CrustalW. NPA motifs were boxed and the aromatic/arginine selectivity. lter (H2, H5, LE1, and LE2) are indicated in blue and red letters, respectively. The red open box indicates the peptide sequence that the anti-ZmLsi1 and anti-ZmLsi6 antibody was raised against.
	Fig. 2. In. ux Si transport activity of ZmLsi1 and ZmLsi6 in Xenopus oocyte. The oocytes injected with cRNA of ZmLsi1, ZmLsi6, OsLsi1 (positive control) or water (negative control) were exposed to 1 mM silicic acid labeled with 68Ge for 30 min. Radioactivity in the oocytes were determined. Means ± SD of biological replicates (n = 3) are shown.
	Fig. 3. Expression of ZmLsi1 and ZmLsi6 in different tissuse of maize. Transcripts of ZmLsi1 and ZmLsi6 were detected by RT-PCR. Total RNA was isolated from the seminal roots including lateral roots (SR), crown roots (CR), leaf sheaths (LS) and leaf blades (LB) of 20-d-old seedlings grown hydroponically. Actin was used as an internal standard. Numbers in parentheses are PCR cycle.
	Fig. 4. Effect of Si supply on ZmLsi1 and ZmLsi6 expression. Seedlings (20-d-old) grown hydroponically were treated with or without 1.0 mM Si for different days and then relative expression levels of ZmLsi1 in the seminal roots (A) and ZmLsi6 in the crown roots (B) and leaf blades (C) were compared by quantitative RT-PCR. Means ± SD of biological replicates (n = 3) are shown.
	Fig. 5. Subcellular localization of ZmLsi1. A GFP-ZmLsi1 fusion (A) and GFP alone as control (B) were introduced by particle bombardment into onion epidermal cells.
	Fig. 6. Western blot analysis and subcellular localization. Expression of ZmLsi1 (A) and ZmLsi6 protein (B) in the roots. SDS-PAGE and Western blot analyses of root membrane protein were conducted using anti-ZmLsi1 or anti-ZmLsi6 antibody without (lane 1) or with pre-incubation of ZmLsi1 (lane 2) and ZmLsi6 (lane 3) peptide epitopes. (C–D) Heterologous expression of ZmLsi1 and ZmLsi6 in Xenopus oocytes. Oocytes expressed with ZmLsi1 (lane 1) or ZmLsi6 (lane 2) and without expression (lane 3) were detected with anti-ZmLsi1 (C) or anti-ZmLsi6 (D) antibody.
	Fig. 7. Immunostaining of ZmLsi1 protein in maize roots. The roots were stained with anti-ZmLsi1 polyclonal antibody. (A) Seminal root (30 mm from the tip); (B) lateral root; (C) crown root (15 mm from the tip). Ep, epidermis; Hy, hydrodermis. Scale bar = 100 μm.
	Fig. 8. Cellular localization of ZmLsi6 in maize. The roots and leaf blades and sheaths were stained with anti-ZmLsi6. (A) seminal root (30 mm from the tip); (B) lateral root; (C) crown root (15 mm from the tip); (D) leaf sheath; and (E) leaf blade. Asterisks in (E) indicate auto. uorescence from chloroplasts in mesophyll cells. Xylem and phloem are indicated as x and p, respectively in (D) and (E). Scale bar = 100 μm.

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