The clock-associated LUX ARRHYTHMO regulates high-affinity nitrate transport in Arabidopsis roots

Abstract

The circadian clock organizes physiological processes in plants to occur at specific times of the day, optimizing efficient use of resources. Nitrate is a crucial inorganic nitrogen source for agricultural systems to sustain crop productivity. However, because nitrate fertilization has a negative impact on the environment, it is important to carefully manage nitrate levels. Understanding crop biological rhythms can lead to more ecologically friendly agricultural practices. Gating responses through the circadian clock could be a strategy to enhance root nitrate uptake and to limit nitrate runoff. In Arabidopsis, the NITRATE TRANSPORTER 2.1 (NRT2.1) gene encodes a key component of the high-affinity nitrate transporter system. Our study reveals that NRT2.1 exhibits a rhythmic expression pattern, with daytime increases and nighttime decreases. The NRT2.1 promoter activity remains rhythmic under constant light, indicating a circadian regulation. The clock-associated transcription factor LUX ARRHYTHMO (LUX) binds to the NRT2.1 promoter in vivo. Loss-of-function of LUX leads to increased NRT2.1 transcript levels and root nitrate uptake at dusk. This supports LUX acting as a transcriptional repressor and modulating NRT2.1 expression in a time-dependent manner. Furthermore, applying nitrate at different times of the day results in varying magnitudes of the transcriptional response in nitrate-regulated genes. We also demonstrate that a defect in the high-affinity nitrate transport system feeds back to the central oscillator by modifying the LUX promoter activity. In conclusion, this study uncovers a molecular pathway connecting the root nitrate uptake and circadian clock, with potential agro-chronobiological applications.

Keywords: circadian clock; high‐affinity nitrate transport; nitrogen assimilation and metabolism; root uptake; transcription regulation.

Figure 1

Relative transcript levels of NRT2.1 in wild‐type, lux‐4, and LUXOX lines. (a) Col‐0 Root relative transcript level of NRT2.1 gene over a 24 h cycle was obtained by RT‐qPCR and normalized to the IPP2 gene. The Col‐0 genotype was grown in vitro for 10 days on 1 mM KNO₃ under a 12:12 LD photoperiod. Roots were harvested every 4 h for 1 day (ZT = 0, 4, 8, 12, 16, 20, 24). Values are means ± Standard Error of the means (SEM) of 4 technical replicates; three biological replicates were performed. (b) Seedlings of two homozygous independent pNRT2.1::LUC lines were entrained in 12:12 LD cycles for 10 days and grown in vitro on 1 mM KNO₃. Bioluminescence with values representing mean ± SEM. (c) Phase values were calculated using FFT‐NLLS, with values (mean ± SEM) (n = 12). (d) CAB2‐LUC and Col‐0 were used as WT for lux‐4 and LUXOX, respectively. These genotypes were grown in vitro for 10 days on 1 mM KNO₃ under a 12:12 LD photoperiod. Roots were harvested at ZT 16. Relative transcript levels were normalized to the IPP2 gene and shown in comparison to wild‐type (WT) (primers used are listed in Table S1). Values are means ± SEM of 4 technical replicates; three biological replicates were performed. Differences between WT and mutant or WT and LUXOX lines are statistically significant at ***P < 0.001 by two‐tailed t‐test. The white and black rectangles describe the light and dark periods, respectively. ZT, Zeitgeber Time.

Figure 2

NRT2.1 transcriptional regulation by LUX. (a) Promoter schematic of AtNRT2.1 with LUX binding site position. LBS are positioned upstream of the NRT2.1 transcription starting site at −936, −1316, −3541, and −3600 bp. (b) Root ChIP assays. Seedlings were grown in a 12:12 LD photoperiod. Root tissues of lux‐4 (negative control) and 35S::LUX:YFP were collected at ZT12 and were processed for ChIP using an anti‐GFP antibody. Relative enrichments of LUX‐YFP protein were analyzed by RT‐qPCR at two regions (−1050 to −900 bp and −1419 to −1282 bp from the start codon) of the NRT2.1 promoter and within the CDS (as a negative control, +1273 to +1454 bp) with specific primers (primers used are listed in Table S1). Values were normalized to the input DNA. Data represent the mean ± SEM of 3 technical replicates (n = 2 independent experiments). CDS, coding sequence. (c, d) Transactivation assays in N. benthamiana leaves. The effectors 35S::GFP as control and 35S::LUX:GFP were co‐expressed with the pNRT2.1::LUC reporter construct. Bioluminescence was measured 2 days post infiltration in 12:12 LD cycles. Data represent the mean ± SEM of 12 technical replicates, the experiment was performed three times. ZT, Zeitgeber Time. (e) Root ${\mathrm{NO}}_3^{-}$ influx measured at the external concentration of 100 μM ¹⁵ ${\mathrm{NO}}_3^{-}$ at ZT 12. Col‐0, lux‐4 and nrt2.1‐2 were grown in vitro on 10 mM KNO₃ for 5 days, then transferred on 100 μM KNO₃ for 16 days. Values are the means of 6 replicates ± SEM, n = 3 independent experiments. Asterisks indicate statistically significant differences between lux‐4 and LUXOX lines (b) or between WT and lux‐4 or nrt2.1‐2 (e). *P < 0.05; ***P < 0.001; by two‐tailed t‐test.

Figure 3

Impact of ${\mathrm{NO}}_3^{-}$ supply at different time of day on NRT2.1, NIA1, NIR, and NRT1.1 gene expression levels in wild‐type. Relative transcript levels of NRT2.1 (a), NIA1 (b), NIR (c), and NRT1.1 (d) genes were quantified in wild‐type roots by RT‐qPCR and normalized to the IPP2 gene (primers used are listed in Table S1). Col‐0 was grown for 10 days on 1/2 MS medium containing 0.5 mM ammonium succinate under a 12:12 LD photoperiod. A treatment of 1 h containing 1 mM KCl or KNO₃ was applied every 4 h during a 24 h cycle (ZT = 0, 4, 8, 12, 16, 20), then the roots were harvested. Arrows indicate the time of treatment. Values represent Means ± SEM of 4 technical replicates; the experiment was performed twice. The white and black rectangles describe the light and dark periods, respectively.

Figure 4

pLUX::LUC activity in WT and nrt2.1‐2 mutant. (a) Luminescence of pLUX::LUC rhythms in WT and in nrt2.1‐2 mutant. Seedlings grown on 1/2MS supplied with 10 mM KNO₃ for 7 days, entrained on 12:12 LD photoperiod, were transferred on 0.5 mM KNO₃ and in LL. Data represent the mean ± SEM. Circadian period (b) and amplitude (c) of pLUX::LUC in WT and nrt2.1‐2 were calculated using FFT‐NLLS, with values (mean ± SEM) (n = 12). (d) Phase response curve. Seedlings were grown in vitro for 10 days on 1/2 MS containing 10 mM KNO₃ under a 12:12 LD photoperiod, followed by 24 h in LL. Subsequently, seedlings were transferred to either 0.1 or 10 mM KNO₃ every hour across a 24‐h cycle (ZT = 0, 4, 8, 12, 16, and 20). Bioluminescence was measured hourly with the LL photoperiod maintained throughout the measurement period. Phase analysis was calculated using FFT‐NLLS, with values (mean ± SEM) (n = 12), and the phase response curve was calculated comparing phases between the control and depletion treatments (transfer to 10 or 0.1 mM KNO₃, respectively). Positive phase shifts correspond to phase advances. *P < 0.05; **P < 0.01; ***P < 0.001; by two‐tailed t‐test. The white and gray rectangles represent the subjective day and night, respectively.

Figure 5

Model of NRT2.1 transcriptional regulation by LUX. Nitrate transport is higher during the day than night. The NRT2.1 transcript levels increase during the day to peak at dusk, and progressively decrease during the night. LUX binds to the LBS within NRT2.1 promoter and represses its expression. Subsequently, the ${\mathrm{NO}}_3^{-}$ uptake is repressed during the night. Nitrate transport feeds back to the central oscillator through LUX.