The Calcium Ion Is a Second Messenger in the Nitrate Signaling Pathway of Arabidopsis

Abstract

Understanding how plants sense and respond to changes in nitrogen availability is the first step toward developing strategies for biotechnological applications, such as improvement of nitrogen use efficiency. However, components involved in nitrogen signaling pathways remain poorly characterized. Calcium is a second messenger in signal transduction pathways in plants, and it has been indirectly implicated in nitrate responses. Using aequorin reporter plants, we show that nitrate treatments transiently increase cytoplasmic Ca(2+) concentration. We found that nitrate also induces cytoplasmic concentration of inositol 1,4,5-trisphosphate. Increases in inositol 1,4,5-trisphosphate and cytoplasmic Ca(2+) levels in response to nitrate treatments were blocked by U73122, a pharmacological inhibitor of phospholipase C, but not by the nonfunctional phospholipase C inhibitor analog U73343. In addition, increase in cytoplasmic Ca(2+) levels in response to nitrate treatments was abolished in mutants of the nitrate transceptor NITRATE TRANSPORTER1.1/Arabidopsis (Arabidopsis thaliana) NITRATE TRANSPORTER1 PEPTIDE TRANSPORTER FAMILY6.3. Gene expression of nitrate-responsive genes was severely affected by pretreatments with Ca(2+) channel blockers or phospholipase C inhibitors. These results indicate that Ca(2+) acts as a second messenger in the nitrate signaling pathway of Arabidopsis. Our results suggest a model where NRT1.1/AtNPF6.3 and a phospholipase C activity mediate the increase of Ca(2+) in response to nitrate required for changes in expression of prototypical nitrate-responsive genes.

Figure 1.

Nitrate treatments increase [Ca²⁺]_cyt in Arabidopsis roots. Wild-type plants expressing WT-AQ were grown hydroponically for 2 weeks with 1 mm ammonium as the only N source. Aequorin was reconstituted by incubating plant roots in 2.5 µm coelenterazine (CTZ) overnight in the dark. [Ca²⁺]_cyt levels were monitored in excised roots in response to 5 mm KNO₃ or KCl treatment (A) without pretreatment (B) or with pretreatment with the Ca²⁺ channel blocker lanthanum chloride (LaCl₃; C). Values plotted correspond to the means of at least three independent biological replicates of five plants per treatment ± sd.

Figure 2.

A PLC inhibitor blocks the increase in [Ca²⁺]_cyt and IP₃ levels in response to nitrate treatments in Arabidopsis roots. Wild-type plants expressing WT-AQ were grown hydroponically for 2 weeks with 1 mm ammonium as the only nitrogen source, and [Ca²⁺]_cyt and IP₃ levels were assayed as described in the text. [Ca²⁺]_cyt levels were monitored in excised roots pretreated with U73343 (nonfunctional analog; A) or U73122 (PLC inhibitor; B) after we treated with KNO₃ and KCl. C, Plants were pretreated with mock, U73122 (inhibitor of PLC), or U73343 (analogous no function), and we evaluated the IP₃ content in Arabidopsis roots in response to 5 mm KNO₃ or KCl. Values plotted correspond to the means of three independent biological replicates ± sd. Gray bars represent time zero (before treatment), white bars represent KCl treatment, and black bars represent KNO₃ treatment. The letters indicate means that significantly differ between control and treatment conditions (P < 0.05).

Figure 3.

NRT1.1/AtNPF6.3 is required for increases in [Ca²⁺]_cyt and IP₃ levels in response to nitrate treatments in Arabidopsis roots. Wild-type, chl1-5, and chl1-9 plants were grown hydroponically for 2 weeks with ammonium as the only nitrogen source, and [Ca²⁺]_cyt and IP₃ contents were assayed as described in the text. WT-AQ (A), chl1-5-AQ (B), and chl1-9-AQ (C) plants were reconstituted by incubating plant roots in 2.5 µm CTZ overnight in the dark. [Ca²⁺]_cyt levels were monitored over time. D, Wild-type (WT), chl1-5, and chl1-9 plants were treated with 5 mm KNO₃ and 5 mm KCl as control for 10 s, and then, we evaluated the IP₃ content. Values plotted correspond to the means of at least three independent biological replicates ± sd. Gray bars represent time zero (before treatment), white bars represent KCl treatment, and black bars represent KNO₃ treatment. The letters indicate means that significantly differ between control and treatment conditions (P < 0.05).

Figure 4.

Regulation of gene expression in response to nitrate treatments is mediated by NRT1.1/AtNPF6.3, a PLC activity, and Ca²⁺ in Arabidopsis roots. Col-0, chl1-5, and chl1-9 plants were grown for 15 d. Plants were pretreated with 5 mm LaCl₃ (A) or 10 µm U73122 or U73343 (B) and then, treated with 5 mm KNO₃ or KCl as control. Values plotted correspond to the means of three independent biological replicates ± sd. White bars represent KCl treatment, and black bars represent KNO₃ treatment. The ADAPTOR PROTEIN4 μ-ADAPTIN gene (At4g24550) was used as a normalization reference (Aceituno et al., 2008). The letters indicate means that significantly differ between control and pharmacological treatment (P < 0.05). WT, Wild type.

Figure 5.

A simplified model of the NRT1.1/AtNPF6.3 calcium-dependent and -independent nitrate signaling pathway. Nitrate is sensed by NRT1.1/AtNPF6.3 and activates a PLC activity that increases [Ca²⁺]_cyt. Increase in [Ca²⁺]_cyt activates gene expression of nitrate-responsive genes.