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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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Effects of MSX pre-treatment on the induction of AtIPT transcript accumulation by exogenous nitrogen compounds. Arabidopsis seedlings were grown under standard conditions for 2 weeks after germination and transferred to nitrogen-free medium for 5 d. The seedlings were pre-treated with 1 mM MSX for 2 h and then incubated for 2 h with nitrogen-free medium (–N), 50 mM glutamine (+Gln) or 10 mM KNO3 (+KNO3) solution containing 1 mM MSX. Total RNA prepared from roots was subjected to qPCR which was performed in triplicate. The amounts of transcripts were normalized to that of the Actin 2 transcript. The experiment was independently repeated twice. The values were normalized with respect to those obtained in the nitrogen-free medium, and mean values with the SD are shown. Asterisks indicate significant differences between the nitrogen-free treatment and the Gln or nitrate treatment, according to Student’s t-test (**P < 0.01; *P < 0.05).
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pct127-F9: Effects of MSX pre-treatment on the induction of AtIPT transcript accumulation by exogenous nitrogen compounds. Arabidopsis seedlings were grown under standard conditions for 2 weeks after germination and transferred to nitrogen-free medium for 5 d. The seedlings were pre-treated with 1 mM MSX for 2 h and then incubated for 2 h with nitrogen-free medium (–N), 50 mM glutamine (+Gln) or 10 mM KNO3 (+KNO3) solution containing 1 mM MSX. Total RNA prepared from roots was subjected to qPCR which was performed in triplicate. The amounts of transcripts were normalized to that of the Actin 2 transcript. The experiment was independently repeated twice. The values were normalized with respect to those obtained in the nitrogen-free medium, and mean values with the SD are shown. Asterisks indicate significant differences between the nitrogen-free treatment and the Gln or nitrate treatment, according to Student’s t-test (**P < 0.01; *P < 0.05).

Mentions: To see whether the glutamine-related regulation is conserved in Arabidopsis IPT regulation, we examined the effects of MSX pre-treatment followed by nitrate and glutamine exposure in Arabidopsis (Fig. 9). AtIPT3 expression in roots was induced by exogenous nitrate even after MSX pre-treatment. Glutamine application also increased the accumulation of AtIPT3 transcripts about 2-fold (Fig. 9). Although this increase seemed small compared with the nitrate response, it consistently occurred in two independent experiments (Fig. 9). Thus, the glutamine-related regulatory mechanism of IPT expression exists in rice as well as in Arabidopsis, although the nitrate-specific regulation is the major one in Arabidopsis.Fig. 9


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)

Effects of MSX pre-treatment on the induction of AtIPT transcript accumulation by exogenous nitrogen compounds. Arabidopsis seedlings were grown under standard conditions for 2 weeks after germination and transferred to nitrogen-free medium for 5 d. The seedlings were pre-treated with 1 mM MSX for 2 h and then incubated for 2 h with nitrogen-free medium (–N), 50 mM glutamine (+Gln) or 10 mM KNO3 (+KNO3) solution containing 1 mM MSX. Total RNA prepared from roots was subjected to qPCR which was performed in triplicate. The amounts of transcripts were normalized to that of the Actin 2 transcript. The experiment was independently repeated twice. The values were normalized with respect to those obtained in the nitrogen-free medium, and mean values with the SD are shown. Asterisks indicate significant differences between the nitrogen-free treatment and the Gln or nitrate treatment, according to Student’s t-test (**P < 0.01; *P < 0.05).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

pct127-F9: Effects of MSX pre-treatment on the induction of AtIPT transcript accumulation by exogenous nitrogen compounds. Arabidopsis seedlings were grown under standard conditions for 2 weeks after germination and transferred to nitrogen-free medium for 5 d. The seedlings were pre-treated with 1 mM MSX for 2 h and then incubated for 2 h with nitrogen-free medium (–N), 50 mM glutamine (+Gln) or 10 mM KNO3 (+KNO3) solution containing 1 mM MSX. Total RNA prepared from roots was subjected to qPCR which was performed in triplicate. The amounts of transcripts were normalized to that of the Actin 2 transcript. The experiment was independently repeated twice. The values were normalized with respect to those obtained in the nitrogen-free medium, and mean values with the SD are shown. Asterisks indicate significant differences between the nitrogen-free treatment and the Gln or nitrate treatment, according to Student’s t-test (**P < 0.01; *P < 0.05).
Mentions: To see whether the glutamine-related regulation is conserved in Arabidopsis IPT regulation, we examined the effects of MSX pre-treatment followed by nitrate and glutamine exposure in Arabidopsis (Fig. 9). AtIPT3 expression in roots was induced by exogenous nitrate even after MSX pre-treatment. Glutamine application also increased the accumulation of AtIPT3 transcripts about 2-fold (Fig. 9). Although this increase seemed small compared with the nitrate response, it consistently occurred in two independent experiments (Fig. 9). Thus, the glutamine-related regulatory mechanism of IPT expression exists in rice as well as in Arabidopsis, although the nitrate-specific regulation is the major one in Arabidopsis.Fig. 9

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