Characterization of zinc uptake and translocation visualized with positron-emitting 65Zn tracer and analysis of transport-related gene expression in two Lotus japonicus accessions

Abstract

Background and aims: Zinc (Zn) is an essential element for humans and plants. However, Zn deficiency is widespread and 25 % of the world's population is at risk of Zn deficiency. To overcome the deficiency of Zn intake, crops with high Zn content are required. However, most crop-producing areas have Zn-deficient soils, therefore crops with excellent Zn uptake/transport characteristics (i.e. high Zn efficiency) are needed. Our objective was to identify the crucial factors responsible for high Zn efficiency in the legume Lotus japonicus.

Methods: We evaluated Zn efficiency by static and real-time visualization of radioactive Zn (65Zn) uptake/transport in two L. japonicus accessions, MG-20 and B-129, that differ in Zn efficiency. The combination of visualization methods verified the dynamics of Zn accumulation and transport within the plant. We compared gene expression under a normal Zn concentration (control) and Zn deficiency to evaluate genetic factors that may determine the differential Zn efficiency of the accessions.

Key results: The accession B-129 accumulated almost twice the amount of Zn as MG-20. In the static 65Zn images, 65Zn accumulated in meristematic tissues, such as root tips and the shoot apex, in both accessions. The positron-emitting tracer imaging system (PETIS), which follows the transport process in real time, revealed that 65Zn transport to the shoot was more rapid in B-129 than in MG-20. Many genes associated with Zn uptake and transport were more highly expressed in B-129 than in MG-20 under the control condition. These gene expression patterns under Zn deficiency differed from those under the control Zn condition.

Conclusions: PETIS confirmed that the real-time transport of 65Zn to the shoot was faster in B-129 than in MG-20. The high Zn efficiency of B-129 may be due to the elevated expression of a suite of Zn uptake- and transport-related genes.

Keywords: Lotus japonicus; Zinc; gene expression; positron emitting tracer imaging system; translocation; uptake.

Fig. 1.

Distribution of ⁶⁵Zn radioactivity after application for 2 d in L. japonicus accessions MG-20 and B-129 visualized by static radio-imaging. Plants were grown hydroponically for 1 month in 10 %-strength Hoagland’s solution and subjected to ⁶⁵Zn application. The ⁶⁵Zn radioactivity in the plants was detected with a BAS 1800-II Bio-Imaging Analyzer. Arrowheads indicate nodes, root tips and developing leaves. Scale bar = 1 cm.

Fig. 2.

Observation of ⁶⁵Zn uptake and translocation in L. japonicus accessions MG-20 and B-129 by PETIS. (A) Real-time images of ⁶⁵Zn distribution in MG-20 and B-129 obtained by PETIS. Radiation intensity is represented as blue when weak and red when strong, as shown in the colour bar. Black line at bottom left in the first panel of (A) is Scale bars = 5 cm. The original images acquired at 1-min intervals were integrated into composite images for 4-h intervals. Red and blue boxes indicate the region of interest for the time–activity curve in (B). (B) Time–activity curve of ⁶⁵Zn relative intensity in the shoot of the two accessions. PETIS was performed with three independent biological replicates. The time–activity curve shows the mean values of three replicates; error bars indicate the standard error. The dashed red lines marking the period from 8 to 16 h indicate the interval for which the inclination of each ⁶⁵Zn translocation (i, the difference in relative intensity per hour) was calculated. The values of i are the inclination of each curve.

Fig. 3.

Transcriptional expression of Zn transport-related genes in the root of L. japonicus accessions MG-20 and B-129 under normal and Zn-deficiency conditions. Total RNA was isolated from the roots of 1-month-old seedlings and the difference in transcriptional expression between the two accessions was detected by qPCR. The genes analysed were involved in (A) Zn uptake from the rhizosphere into root cells, (B) intercellular transport up to loading of xylem vessels, and (C) vacuolar sequestration. ACTIN7 was used as an internal reference gene. N, normal Zn concentration; L, Zn deficiency. Asterisks and daggers above the bars indicate significant differences in gene expression in B-129 compared with MG-20 under normal and Zn-deficiency conditions, respectively. *P < 0.05, **P < 0.01, ^†P < 0.05, ^††P < 0.01. Error bars indicate the standard deviation. Analyses were performed with four biological replicates.

Fig. 4.

Transcriptional expression of putative regulators for Zn efficiency in L. japonicus accessions MG-20 and B-129. (A) Amino acid alignment of A. thaliana AtIMA1 and L. japonicus MG-20 LjIMA1. The red box indicates the consensus motif of plant IMA1. (B) Expression analysis of the putative regulator of Zn efficiency LjPHR1 in the root and the shoot. (C) Expression of LjIMA1 in the root and the shoot. The inset shows a magnified graph of gene expression in the root. Error bars indicate the standard deviation. Analyses were performed with four biological replicates. **P < 0.01.