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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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Subcellular localization of GFP-tagged OsIPT proteins in Arabidopsis observed by confocal laser-scanning microscopy. The translational fusion genes OsIPT1-GFP (A), OsIPT2-GFP (B), OsIPT3-GFP (C), GFP-OsIPT1 (D), GFP-OsIPT2 (E) and GFP-OsIPT3 (F) were transiently expressed in root epidermal cells by particle bombardment. The fusion genes pGGPS6-DsRed2 (G), a control for mitochondrial localization, and OsIPT7-GFP (H) were co-introduced into a leaf mesophyll cell. (I) Merged image of (G) and (H). The fusion genes OsIPT4-GFP (J), OsIPT5-GFP (K) and OsIPT8-GFP (L) were introduced into leaf mesophyll cells and superimposed on Chl autofluorescence (red). The fusion gene AtFSD3-DsRed2 (M, P and S), a control for nucleoid localization, was co-introduced into root epidermal cells with either OsIPT4-GFP (N), OsIPT5-GFP (Q) or OsIPT8-GFP (T). (O, R and U) Merged images of (M) and (N), (P) and (Q), and (S) and (T), respectively. Scale bars, 20 µm (A–F) and 10 µm (G–U).
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pct127-F5: Subcellular localization of GFP-tagged OsIPT proteins in Arabidopsis observed by confocal laser-scanning microscopy. The translational fusion genes OsIPT1-GFP (A), OsIPT2-GFP (B), OsIPT3-GFP (C), GFP-OsIPT1 (D), GFP-OsIPT2 (E) and GFP-OsIPT3 (F) were transiently expressed in root epidermal cells by particle bombardment. The fusion genes pGGPS6-DsRed2 (G), a control for mitochondrial localization, and OsIPT7-GFP (H) were co-introduced into a leaf mesophyll cell. (I) Merged image of (G) and (H). The fusion genes OsIPT4-GFP (J), OsIPT5-GFP (K) and OsIPT8-GFP (L) were introduced into leaf mesophyll cells and superimposed on Chl autofluorescence (red). The fusion gene AtFSD3-DsRed2 (M, P and S), a control for nucleoid localization, was co-introduced into root epidermal cells with either OsIPT4-GFP (N), OsIPT5-GFP (Q) or OsIPT8-GFP (T). (O, R and U) Merged images of (M) and (N), (P) and (Q), and (S) and (T), respectively. Scale bars, 20 µm (A–F) and 10 µm (G–U).

Mentions: To examine the intracellular localization of OsIPT proteins, translational fusions were made with green fluorescent protein (GFP) at the C-termini (designated as OsIPT1–GFP to OsIPT8–GFP) under the control of the Cauliflower mosaic virus (CaMV) 35S promoter. The constructs were introduced into Arabidopsis cells by particle bombardment (Fig. 5). Since rice tissues are mechanically rigid and it was difficult to observe subcellular localizations, we used Arabidopsis as a heterologous system. GFP fluorescence of OsIPT4–GFP, OsIPT5–GFP and OsIPT8–GFP appeared as small dots on plastids (Fig. 5J–L). When we co-introduced DsRed2-fused pAtFSD3, a plastid nucleoid-associated protein (Myouga et al. 2008), the fluorescence signals fully overlapped (Fig. 5M–U). The GFP fluorescence of OsIPT7–GFP co-localized with DsRed2-tagged Arabidopsis geranylgeranyl diphosphate synthase 6 (GGPS6) (Okada et al. 2000), a control marker for mitochondria (Fig. 5G–I). These results strongly suggested that nitrogen-inducible OsIPT4, OsIPT5 and OsIPT8 are localized in plastids, whereas OsIPT7 is localized in mitochondria. On the other hand, the GFP fluorescence from OsIPT1–GFP, OsIPT2–GFP and OsIPT3–GFP and their translational fusions at the N-termini (GFP–OsIPT1, GFP–OsIPT2 and GFP–OsIPT3) were all observed in the cytoplasm (Fig. 5A–F).Fig. 5


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)

Subcellular localization of GFP-tagged OsIPT proteins in Arabidopsis observed by confocal laser-scanning microscopy. The translational fusion genes OsIPT1-GFP (A), OsIPT2-GFP (B), OsIPT3-GFP (C), GFP-OsIPT1 (D), GFP-OsIPT2 (E) and GFP-OsIPT3 (F) were transiently expressed in root epidermal cells by particle bombardment. The fusion genes pGGPS6-DsRed2 (G), a control for mitochondrial localization, and OsIPT7-GFP (H) were co-introduced into a leaf mesophyll cell. (I) Merged image of (G) and (H). The fusion genes OsIPT4-GFP (J), OsIPT5-GFP (K) and OsIPT8-GFP (L) were introduced into leaf mesophyll cells and superimposed on Chl autofluorescence (red). The fusion gene AtFSD3-DsRed2 (M, P and S), a control for nucleoid localization, was co-introduced into root epidermal cells with either OsIPT4-GFP (N), OsIPT5-GFP (Q) or OsIPT8-GFP (T). (O, R and U) Merged images of (M) and (N), (P) and (Q), and (S) and (T), respectively. Scale bars, 20 µm (A–F) and 10 µm (G–U).
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pct127-F5: Subcellular localization of GFP-tagged OsIPT proteins in Arabidopsis observed by confocal laser-scanning microscopy. The translational fusion genes OsIPT1-GFP (A), OsIPT2-GFP (B), OsIPT3-GFP (C), GFP-OsIPT1 (D), GFP-OsIPT2 (E) and GFP-OsIPT3 (F) were transiently expressed in root epidermal cells by particle bombardment. The fusion genes pGGPS6-DsRed2 (G), a control for mitochondrial localization, and OsIPT7-GFP (H) were co-introduced into a leaf mesophyll cell. (I) Merged image of (G) and (H). The fusion genes OsIPT4-GFP (J), OsIPT5-GFP (K) and OsIPT8-GFP (L) were introduced into leaf mesophyll cells and superimposed on Chl autofluorescence (red). The fusion gene AtFSD3-DsRed2 (M, P and S), a control for nucleoid localization, was co-introduced into root epidermal cells with either OsIPT4-GFP (N), OsIPT5-GFP (Q) or OsIPT8-GFP (T). (O, R and U) Merged images of (M) and (N), (P) and (Q), and (S) and (T), respectively. Scale bars, 20 µm (A–F) and 10 µm (G–U).
Mentions: To examine the intracellular localization of OsIPT proteins, translational fusions were made with green fluorescent protein (GFP) at the C-termini (designated as OsIPT1–GFP to OsIPT8–GFP) under the control of the Cauliflower mosaic virus (CaMV) 35S promoter. The constructs were introduced into Arabidopsis cells by particle bombardment (Fig. 5). Since rice tissues are mechanically rigid and it was difficult to observe subcellular localizations, we used Arabidopsis as a heterologous system. GFP fluorescence of OsIPT4–GFP, OsIPT5–GFP and OsIPT8–GFP appeared as small dots on plastids (Fig. 5J–L). When we co-introduced DsRed2-fused pAtFSD3, a plastid nucleoid-associated protein (Myouga et al. 2008), the fluorescence signals fully overlapped (Fig. 5M–U). The GFP fluorescence of OsIPT7–GFP co-localized with DsRed2-tagged Arabidopsis geranylgeranyl diphosphate synthase 6 (GGPS6) (Okada et al. 2000), a control marker for mitochondria (Fig. 5G–I). These results strongly suggested that nitrogen-inducible OsIPT4, OsIPT5 and OsIPT8 are localized in plastids, whereas OsIPT7 is localized in mitochondria. On the other hand, the GFP fluorescence from OsIPT1–GFP, OsIPT2–GFP and OsIPT3–GFP and their translational fusions at the N-termini (GFP–OsIPT1, GFP–OsIPT2 and GFP–OsIPT3) were all observed in the cytoplasm (Fig. 5A–F).Fig. 5

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