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Nitrogen-dependent regulation of de novo cytokinin biosynthesis in rice: the role of glutamine metabolism as an additional signal.

Kamada-Nobusada T, Makita N, Kojima M, Sakakibara H - Plant Cell Physiol. (2013)

Bottom Line: In this study, we have identified another regulatory system of cytokinin de novo biosynthesis in response to nitrogen status.In rice, OsIPT4, OsIPT5, OsIPT7 and OsIPT8 were up-regulated in response to exogenously applied nitrate and ammonium, with accompanying accumulation of cytokinins.In transgenic lines repressing the expression of OsIPT4, which is the dominant IPT in rice roots, the nitrogen-dependent increase of cytokinin in the xylem sap was significantly reduced, and seedling shoot growth was retarded despite sufficient nitrogen.

View Article: PubMed Central - PubMed

Affiliation: RIKEN Center for Sustainable Resource Science, Tsurumi, Yokohama, 230-0045 Japan.

ABSTRACT
Cytokinin activity in plants is closely related to nitrogen availability, and an Arabidopsis gene for adenosine phosphate-isopentenyltransferase (IPT), IPT3, is regulated by inorganic nitrogen sources in a nitrate-specific manner. In this study, we have identified another regulatory system of cytokinin de novo biosynthesis in response to nitrogen status. In rice, OsIPT4, OsIPT5, OsIPT7 and OsIPT8 were up-regulated in response to exogenously applied nitrate and ammonium, with accompanying accumulation of cytokinins. Pre-treatment of roots with l-methionine sulfoximine, a potent inhibitor of glutamine synthetase, abolished the nitrate- and ammonium-dependent induction of OsIPT4 and OsIPT5, while glutamine application induced their expression. Thus, neither nitrate nor ammonium, but glutamine or a related metabolite, is essential for the induction of these IPT genes in rice. On the other hand, glutamine-dependent induction of IPT3 occurs in Arabidopsis, at least to some extent. In transgenic lines repressing the expression of OsIPT4, which is the dominant IPT in rice roots, the nitrogen-dependent increase of cytokinin in the xylem sap was significantly reduced, and seedling shoot growth was retarded despite sufficient nitrogen. We conclude that plants possess multiple regulation systems for nitrogen-dependent cytokinin biosynthesis to modulate growth in response to nitrogen availability.

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Changes in the accumulation of OsIPT transcripts in response to nitrogen sources in roots (A) and shoots (B). Rice seedlings were hydroponically grown and treated with 1 mM NH4Cl (filled circles), 1 mM KNO3 (open circles) or 1 mM KCl (filled triangles) in the same manner as in Fig. 1, and roots and shoots were harvested at the indicated times. Total RNA prepared from the samples was subjected to qPCR. The amounts of transcripts were normalized to the value at 0 min. qPCR was performed in triplicate, and mean values with the SD are shown.
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pct127-F2: Changes in the accumulation of OsIPT transcripts in response to nitrogen sources in roots (A) and shoots (B). Rice seedlings were hydroponically grown and treated with 1 mM NH4Cl (filled circles), 1 mM KNO3 (open circles) or 1 mM KCl (filled triangles) in the same manner as in Fig. 1, and roots and shoots were harvested at the indicated times. Total RNA prepared from the samples was subjected to qPCR. The amounts of transcripts were normalized to the value at 0 min. qPCR was performed in triplicate, and mean values with the SD are shown.

Mentions: Since nucleotide precursors of cytokinins were increased by the nitrogen supplements, we examined the accumulation levels of OsIPT transcripts in roots and shoots (Fig. 2). Since OsIPT6 seems to be non-functional in the Nipponbare cultivar (Sakamoto et al. 2006), we excluded it from analysis. To discriminate between nitrate-specific and nitrogen-non-specific responses, genes for non-photosynthetic-type ferredoxin-NADP+ oxidoreductase (FNR) (Aoki and Ida 1994) and NADH-dependent glutamate synthase 1 (NADH-GOGAT1) (Hirose et al. 1997) were used as indicators for the two response types, respectively, in roots (Fig. 2A). The NR gene (Hamat et al. 1989) served as an indicator for nitrate-specific responses in shoots (Fig. 2B). In roots, the accumulation of OsIPT4 and OsIPT5 transcripts was clearly increased by both ammonium and nitrate (Fig. 2A). Ammonium had a more potent effect on the induction of OsIPT4 expression than nitrate. On the other hand, the level of the OsIPT7 transcript was decreased by both treatments. In shoots, the accumulation levels of OsIPT4, OsIPT7 and OsIPT8 transcripts were increased by both nitrogen sources, with ammonium having a stronger effect than nitrate (Fig. 2B). The expression patterns of the indicator genes did not suggest any nitrate effects caused by ammonium-containing media.Fig. 2


Nitrogen-dependent regulation of de novo cytokinin biosynthesis in rice: the role of glutamine metabolism as an additional signal.

Kamada-Nobusada T, Makita N, Kojima M, Sakakibara H - Plant Cell Physiol. (2013)

Changes in the accumulation of OsIPT transcripts in response to nitrogen sources in roots (A) and shoots (B). Rice seedlings were hydroponically grown and treated with 1 mM NH4Cl (filled circles), 1 mM KNO3 (open circles) or 1 mM KCl (filled triangles) in the same manner as in Fig. 1, and roots and shoots were harvested at the indicated times. Total RNA prepared from the samples was subjected to qPCR. The amounts of transcripts were normalized to the value at 0 min. qPCR was performed in triplicate, and mean values with the SD are shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814184&req=5

pct127-F2: Changes in the accumulation of OsIPT transcripts in response to nitrogen sources in roots (A) and shoots (B). Rice seedlings were hydroponically grown and treated with 1 mM NH4Cl (filled circles), 1 mM KNO3 (open circles) or 1 mM KCl (filled triangles) in the same manner as in Fig. 1, and roots and shoots were harvested at the indicated times. Total RNA prepared from the samples was subjected to qPCR. The amounts of transcripts were normalized to the value at 0 min. qPCR was performed in triplicate, and mean values with the SD are shown.
Mentions: Since nucleotide precursors of cytokinins were increased by the nitrogen supplements, we examined the accumulation levels of OsIPT transcripts in roots and shoots (Fig. 2). Since OsIPT6 seems to be non-functional in the Nipponbare cultivar (Sakamoto et al. 2006), we excluded it from analysis. To discriminate between nitrate-specific and nitrogen-non-specific responses, genes for non-photosynthetic-type ferredoxin-NADP+ oxidoreductase (FNR) (Aoki and Ida 1994) and NADH-dependent glutamate synthase 1 (NADH-GOGAT1) (Hirose et al. 1997) were used as indicators for the two response types, respectively, in roots (Fig. 2A). The NR gene (Hamat et al. 1989) served as an indicator for nitrate-specific responses in shoots (Fig. 2B). In roots, the accumulation of OsIPT4 and OsIPT5 transcripts was clearly increased by both ammonium and nitrate (Fig. 2A). Ammonium had a more potent effect on the induction of OsIPT4 expression than nitrate. On the other hand, the level of the OsIPT7 transcript was decreased by both treatments. In shoots, the accumulation levels of OsIPT4, OsIPT7 and OsIPT8 transcripts were increased by both nitrogen sources, with ammonium having a stronger effect than nitrate (Fig. 2B). The expression patterns of the indicator genes did not suggest any nitrate effects caused by ammonium-containing media.Fig. 2

Bottom Line: In this study, we have identified another regulatory system of cytokinin de novo biosynthesis in response to nitrogen status.In rice, OsIPT4, OsIPT5, OsIPT7 and OsIPT8 were up-regulated in response to exogenously applied nitrate and ammonium, with accompanying accumulation of cytokinins.In transgenic lines repressing the expression of OsIPT4, which is the dominant IPT in rice roots, the nitrogen-dependent increase of cytokinin in the xylem sap was significantly reduced, and seedling shoot growth was retarded despite sufficient nitrogen.

View Article: PubMed Central - PubMed

Affiliation: RIKEN Center for Sustainable Resource Science, Tsurumi, Yokohama, 230-0045 Japan.

ABSTRACT
Cytokinin activity in plants is closely related to nitrogen availability, and an Arabidopsis gene for adenosine phosphate-isopentenyltransferase (IPT), IPT3, is regulated by inorganic nitrogen sources in a nitrate-specific manner. In this study, we have identified another regulatory system of cytokinin de novo biosynthesis in response to nitrogen status. In rice, OsIPT4, OsIPT5, OsIPT7 and OsIPT8 were up-regulated in response to exogenously applied nitrate and ammonium, with accompanying accumulation of cytokinins. Pre-treatment of roots with l-methionine sulfoximine, a potent inhibitor of glutamine synthetase, abolished the nitrate- and ammonium-dependent induction of OsIPT4 and OsIPT5, while glutamine application induced their expression. Thus, neither nitrate nor ammonium, but glutamine or a related metabolite, is essential for the induction of these IPT genes in rice. On the other hand, glutamine-dependent induction of IPT3 occurs in Arabidopsis, at least to some extent. In transgenic lines repressing the expression of OsIPT4, which is the dominant IPT in rice roots, the nitrogen-dependent increase of cytokinin in the xylem sap was significantly reduced, and seedling shoot growth was retarded despite sufficient nitrogen. We conclude that plants possess multiple regulation systems for nitrogen-dependent cytokinin biosynthesis to modulate growth in response to nitrogen availability.

Show MeSH
Related in: MedlinePlus