Glutamine rapidly induces the expression of key transcription factor genes involved in nitrogen and stress responses in rice roots

Abstract

Background: Glutamine is a major amino donor for the synthesis of amino acids, nucleotides, and other nitrogen-containing compounds in all organisms. In addition to its role in nutrition and metabolism, glutamine can also function as a signaling molecule in bacteria, yeast, and humans. By contrast, the functions of glutamine in nutrition and as a signaling molecule remain unclear in plants.

Results: We demonstrated that glutamine could effectively support the growth of rice seedlings. In glutamine-treated rice roots, the glutamine contents increased dramatically, whereas levels of glutamate remained relatively constant. Transcriptome analysis of rice roots revealed that glutamine induced the expression of at least 35 genes involved in metabolism, transport, signal transduction, and stress responses within 30 min. Interestingly, 10 of the 35 early glutamine responsive genes encode putative transcription factors, including two LBD37-like genes that are involved in the regulation of nitrogen metabolism. Glutamine also rapidly induced the expression of the DREB1A, IRO2, and NAC5 transcription factor genes, which are involved in the regulation of stress responses.

Conclusions: In addition to its role as a metabolic fuel, glutamine may also function as a signaling molecule to regulate gene expression in plants. The rapid induction of transcription factor genes suggests that glutamine may efficiently amplify its signal and interact with the other signal transduction pathways to regulate plant growth and stress responses. Thus, glutamine is a functional amino acid that plays important roles in plant nutrition and signal transduction.

Fig. 1

Effects of glutamine on the growth of rice seedlings. a 17-day-old rice seedlings grown in hydroponic solutions containing NH₄NO₃ or glutamine as the nitrogen source. Shoot length (b), root length (c), and chlorophyll contents (d) of 17-day-old rice seedlings grown in different concentrations of glutamine. Data are means ± SD (n = 40). Different letters indicate significant differences between treatments, tested by one-way ANOVA followed by Tukey’s test (P < 0.05). +N, + 1.43 mM NH₄NO₃; −N, no nitrogen

Fig. 2

Amino acid contents in the medium and in rice roots during the time course of glutamine treatment. a The hydroponic solution initially contained 2.5 mM glutamine and glutamine concentrations were measured over the 24-h time course. b-f Contents of Gln, Glu, Ala, Asp and Asn in the roots after 0–24 h of glutamine treatment. Fold change indicates the relative amounts of amino acids in 2.5 mM glutamine-treated roots (0.25–24 h) compared to those of control (0 h). Data are means ± SD (n = 4). Different letters indicate significant differences between treatments, tested by one-way ANOVA followed by Tukey’s test (P < 0.05)

Fig. 3

RT-PCR analyses of glutamine-responsive genes. Total RNA extracted from roots of 17-day-old glutamine-treated rice seedlings was used for RT-PCR analysis to verify the expression of 35 genes identified by microarray analysis. a Transcription factor genes. b Kinase or signal transducer genes. c Metabolic or transporter genes. d Stress response or unknown function genes. The expression levels of EF1α in the same samples are shown as a control

Fig. 4

Quantitative RT-PCR analyses of glutamine-responsive transcription factor genes. Seventeen-day-old rice seedlings grown in hydroponic solution without nitrogen were subsequently transferred to medium containing 2.5 mM glutamine, glutamate, or 1.43 mM ammonium nitrate for 0, 15′, 30′, 1, 4, and 24 h. Total RNA extracted from roots was used for quantitative RT-PCR to analyze the expression of ZOS5-02 (a), LBD37-like Os07g0589000 (b), LBD37-like Os03g0445700 (c), AP2/ERF106 (d), bHLH Os04g0301500 (e), IRO2 (f), DREB1A (g), MYB-like Os07g0119300 (h), NAC5 (i), and WRKY69 (j). The expression level of each gene in the control sample (0 h) was set at 1. Fold change indicates the relative expression of each gene as compared to that of control. Quantitative RT-PCRs were performed in triplicate for each sample in three independent experiments. All of the quantifications were normalized to the nuclear gene UBC3 (Os02g0634800)

Fig. 5

Glutamine may function as a signaling nutrient in plants. Glutamine entering the plant cell can be directly used for metabolism to support plant growth and development. The internal glutamine and nitrogen status may affect gene expression in the nucleus. In addition, plants may be able to sense the external glutamine and nitrogen status to regulate gene expression. GOGAT glutamine oxoglutarate aminotransferase, TCA tricarboxylic acid, Mt mitochondrion