Os04g0390500

Yellow Stripe-Like (YSL) proteins belong to the oligopeptide transporter family and have been implicated in metal transport and homeostasis in different plant species. OsYSL6 is one of the rice (Oryza sativa) YSL members.

Annotated Information

Function

• Among 18 members of YSLs in rice, OsYSL6 forms a separate cluster with OsYSL5 . Knockout of OsYSL6 only resulted in increased sensitivity to high Mn concentration but did not affect the sensitivity to either deficiency or toxicity of other metals, including Fe, Cu, and Zn. These results revealed that, different from other members characterized, OsYSL6 is involved in the detoxification of excess Mn in rice.

• OsYSL6 protein is able to transport Mn-NA complex, but not Mn-DMA complex.

Mutation

• To investigate the role of OsYSL6, the researchers obtained a Tos- 17 insertion line (NC6189; osysl6). No transcript of OsYSL6 was detected in this line, indicating that this is a knockout line of OsYSL6. When the plants were subjected to different Mn concentrations, the growth of osysl6 did not differ from the wild-type rice at Mn concentrations up to 100 mM but was reduced at 1,000 mM (Fig. 1). At 1,000 mM Mn treatment, the fresh weight of the shoots and roots in the knockout line was 58% and 53%, respectively, of the wild type (Fig. 2). The older true leaf of the knockout line exhibited necrosis, a typical Mn toxicity symptom(Fig. 1F), which was not observed in the wild-type rice (Fig. 1E).

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) mM 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 mM MnCl2 for 3 weeks. Photographs were taken at harvest. .

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 mM 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. .

• To confirm that disruption of OsYSL6 was responsible for the increased Mn sensitivity in the osysl6 mutant observed above (Figs. 1 and 2), we conducted a complementation test by introducing a DNA fragment containing the OsYSL6 promoter region, the entire open reading frame, and the 3# untranslated region into the knockout line by Agrobacterium tumefaciensmediated transformation. Analysis with two independent transgenic lines (lines 1 and 2) showed that there was no difference in the growth among transgenic lines, the wild type, and the osysl6 mutant at 0.5 mM Mn (Fig. 3A). At 1,000 mM Mn, although the growth of all lines was inhibited, the inhibition was alleviated in the transgenic lines compared with the knockout line (Fig. 3B). These results indicated that OsYSL6 is a responsible gene for the phenotype observed in the mutant.

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 mM (A) or 1,000 mM (B) MnCl2 for 2 weeks. .

• The researchers compared the concentrations of Mn, Fe, Cu, and Zn in the roots and shoots of wild-type rice and the knockout line exposed to different Mn concentrations. The Mn concentrations in both the roots and shoots increased with increasing external Mn concentrations (Fig. 4, A and C), but there was no significant difference between wild-type rice and the knockout line at all Mn concentrations tested, although the growth of the knockout line was inhibited at 1,000 mM Mn (Figs. 1 and 2). The concentration of Fe tended to decrease with increasing Mn concentrations in the external solution in both the roots and shoots, but there was no difference between wild-type rice and the knockout line (Fig. 4, B and D). Differences in the concentrations of Cu and Zn were also not found.

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 mM 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. .

Expression

• The expression level of OsYSL6 in both the shoots and roots was determined by absolute quantitative real-time reverse transcription (RT)-PCR. OsYSL6 showed 9.1 3 103 and 7.1 3 103 copies ng21 RNA in the shoots and roots, respectively (Fig. 6A). A dose-response experiment with different Mn concentrations showed that OsYSL6 expression was not induced by either Mn excess or deficiency in both the roots and shoots (Fig. 6, B and C). The expression did not respond to the deficiency of Zn, Fe, Mn, and Cu in both the roots and shoots (Fig. 6, D and E). These trends were unchanged when the expression was normalized based on two other internal standards (Actin and Ubiquitin). These results indicate that OsYSL6 was constitutively expressed in the shoots and roots.

Figure 6. Expression pattern of OsYSL6. A, Copy number of OsYSL6 in roots and shoots of rice. .

• The expression of OsYSL6 in different leaves was further investigated in plants treated with 500 mM Mn. The expression level increased with leaf age: old leaf (leaf 1) showed higher expression, while young leaf (leaf 5) showed lower expression (Fig. 7A). This trend is similar to Mn concentration in the different leaves (Fig. 7B).

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 mM 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. .

• To investigate the tissue expression profile of OsYSL6, the reserachers introduced the OsYSL6 promoter region fused with GFP into wild-type rice (cv Nipponbare) by Agrobacterium-mediated transformation. The localization of GFP was examined by immunostaining using an anti-GFP antibody. In OsYSL6 promoter-GFP transgenic rice, the GFP signal was observed in all cells of both the roots and leaves (Fig. 8, A and C). No fluorescence signal was detected in the wild-type rice (Fig. 8, B and D).

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 mm. .

Evolution

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Labs working on this gene

• Institute of Plant Science and Resources, Okayama University, Kurashiki 710–0046, Japan