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. 2011 Dec;157(4):1832-40.
doi: 10.1104/pp.111.186031. Epub 2011 Oct 10.

OsYSL6 Is Involved in the Detoxification of Excess Manganese in Rice

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Free PMC article

OsYSL6 Is Involved in the Detoxification of Excess Manganese in Rice

Akimasa Sasaki et al. Plant Physiol. .
Free PMC article

Abstract

Yellow Stripe-Like (YSL) proteins belong to the oligopeptide transporter family and have been implicated in metal transport and homeostasis in different plant species. Here, we functionally characterized a rice (Oryza sativa) YSL member, OsYSL6. Knockout of OsYSL6 resulted in decreased growth of both roots and shoots only in the high-manganese (Mn) condition. There was no difference in the concentration of total Mn and other essential metals between the wild-type rice and the knockout line, but the knockout line showed a higher Mn concentration in the leaf apoplastic solution and a lower Mn concentration in the symplastic solution than wild-type rice. OsYSL6 was constitutively expressed in both the shoots and roots, and the expression level was not affected by either deficiency or toxicity of various metals. Furthermore, the expression level increased with leaf age. Analysis with OsYSL6 promoter-green fluorescent protein transgenic rice revealed that OsYSL6 was expressed in all cells of both the roots and shoots. Heterogolous expression of OsYSL6 in yeast showed transport activity for the Mn-nicotianamine complex but not for the Mn-mugineic acid complex. Taken together, our results suggest that OsYSL6 is a Mn-nicotianamine transporter that is required for the detoxification of excess Mn in rice.

Figures

Figure 1.
Figure 1.
Phenotypic analysis of the OsYSL6 knockout line in response to Mn. Both wild-type rice (left) and knockout line osysl6 (right) were grown in a nutrient solution containing 0.05 (A), 0.5 (B), 100 (C), or 1,000 (D) μm MnCl2 for 3 weeks. E and F show close-up views of the oldest true leaves of wild-type rice (E) and osysl6 (F) exposed to 1,000 μm MnCl2 for 3 weeks. Photographs were taken at harvest.
Figure 2.
Figure 2.
Effects of different Mn concentrations on the growth of the OsYSL6 knockout line. Both wild-type rice (WT) and the knockout line (osysl6) were cultivated in a nutrient solution containing 0.05, 0.5, 100, or 1,000 μm MnCl2. After 3 weeks, the shoots (A) and roots (B) were harvested and their fresh weights were recorded. Data are means of three biological replicates. An asterisk above a bar indicates a significant difference (P < 0.05) between the wild type and the knockout line by Tukey’s test.
Figure 3.
Figure 3.
Complementation test of the OsYSL6 knockout line. A construct consisting of promoter and genomic DNA of OsYSL6 was transformed into knockout line osysl6. The wild-type rice (WT), two independent transgenic lines, and osysl6 were grown in a nutrient solution containing 0.5 μm (A) or 1,000 μm (B) MnCl2 for 2 weeks.
Figure 4.
Figure 4.
Concentrations of Mn and Fe in the shoots and roots. A knockout line of OsYSL6 (osysl6) and its wild-type rice (WT) were cultivated in a nutrient solution containing 0.05, 0.5, 100, or 1,000 μm MnCl2 for 3 weeks. The concentrations of Mn (A and C) and Fe (B and D) in the shoots (A and B) and roots (C and D) were determined by atomic absorption spectrometry. Data are means of three biological replicates.
Figure 5.
Figure 5.
Mn concentrations of apoplastic and symplastic solutions. The wild-type rice (WT) and the knockout line (osysl6) were cultivated in a nutrient solution containing 500 μm Mn for 7 d. The oldest true leaf was used for extraction of apoplastic (A) and symplastic (B) solutions. Mn concentration was determined by atomic absorption spectrometry. Data are means of three biological replicates. An asterisk above a bar indicates a significant difference (P < 0.05) between the wild type and the knockout line by Tukey’s test.
Figure 6.
Figure 6.
Expression pattern of OsYSL6. A, Copy number of OsYSL6 in roots and shoots of rice. The seedlings (cv Nipponbare) were exposed to a normal nutrient solution for 1 week. B and C, Effects of different Mn concentrations on OsYSL6 expression. The seedlings were exposed to a nutrient solution containing 0.05, 0.5, 100, or 1,000 μm MnCl2 for 3 weeks. D and E, Response of OsYSL6 expression to metal deficiency. Wild-type rice was cultivated in a nutrient solution with or without Zn, Fe, Mn, or Zn for 1 week. The copy number (A) was determined by absolute real-time RT-PCR. The expression levels of shoots (B and D) and roots (C and E) were determined by real-time RT-PCR. Histone H3 was used as an internal standard. Expression levels relative to 0.5 μm MnCl2 are shown in B to E. Data are means of three biological replicates.
Figure 7.
Figure 7.
Expression levels of OsYSL6 and Mn concentrations in different leaves. A, Expression pattern of OsYSL6 in leaves 1, 3, and 5 (numbered from the bottom). B, Mn concentrations in different leaves. Seedlings (cv Nipponbare) were grown in a nutrient solution containing 500 μm MnCl2 for 3 weeks. The expression level was determined by real time RT-PCR and Mn by atomic absorption spectrometry. Expression levels relative to leaf 5 are shown. Data are means of three biological replicates.
Figure 8.
Figure 8.
Tissue expression profile of OsYSL6. A and B, Immunostaining of the roots of the OsYSL6 promoter-GFP transgenic line (A) and wild-type rice (B). C and D, Immunostaining of the leaves of the OsYSL6 promoter-GFP transgenic line (C) and wild-type rice (D). Immunostaining was performed by using an antibody against GFP. Bars = 100 μm.
Figure 9.
Figure 9.
Transport activity of OsYSL6 in yeast. Time-dependent uptake of Mn into yeast cells is shown. Yeast strain smf1 transformed with empty vector pYES2 or OsYSL6 was cultivated in the presence of 2% Gal (A and C) or Glc (B) for 2 h and then exposed to a solution containing 5 μm Mn-NA (A and B) or Mn-DMA (C) for 20, 40, 60, or 120 min. Mn concentration was determined by atomic absorption spectrometry. Data are means of three biological replicates.

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