Nitrogen regulates CRY1 phosphorylation and circadian clock input pathways

ABSTRACT

The delayed flowering phenotype caused by nitrogen (N) fertilizer application has been known for a long time, but we know little about the specific molecular mechanism for this phenomenon before. Our study indicated that low nitrogen increases the NADPH/NADP⁺ and ATP/AMP ratios which affect adenosine monophosphate-activated protein kinase (AMPK) activity and phosphorylation and abundance of nuclear CRY1 protein. Then CRY1 acts in the N signal input pathway to the circadian clock. Here we further discuss: (1) the role of C/N ratio in flowering, (2) circadian oscillation of plant AMPK transcripts and proteins, (3) conservation of nutrition-mediated CRY1 phosphorylation and degradation, and (4) crosstalks between nitrogen signals and nitric oxide (NO) signals in flowering.

Cryptochrome 1 (CRY1) is a blue light receptor which works upstream of the photoperiod pathway and controls plants flowering through interaction with other light signaling components. CRY1 regulates the expression of flowering-related genes CONSTANS (CO) and FLOWERING LOCUS T (FT).^1-2 Here in our study, CRY1 has been identified as the pivotal gene in the nitrogen (N)-regulated flowering pathway.³ N mediates the central oscillator through the CRY1 input pathway.³ Carbohydrates (C) generate another nutrition signal, which also regulates flowering. A recent report found that sugars repress the expression of the morning-expressed gene PSEUDORESPONSE REGULATOR 7 (PRR7) and activate the key component the central oscillator CCA1.⁴ Similar to the high N condition, nitric oxide (NO) also causes the inhibition of floral transition through repressing circadian-clock output genes CO and GIGANTEA (GI).⁵ In summary, N, C and NO may influence the circadian clock through the input pathway, the central oscillator pathway and the output pathway, respectively (Fig. 1). Furthermore, circadian-clock output signals in turn give a feedback to the input pathway.

Figure 1.

Diagram of the effects of N, sugar, NO on circadian clock. The input pathway may also give a feedback to the input pathway.

C/N ratio may also affect flowering

C and N are indispensable elements among various nutrients and they are affected by many environmental cues such as abiotic and biotic stresses, atmospheric CO₂, circadian rhythm and so on.^6-10 The change of N must be accompanied by a change of C/N ratio. Therefore, our previous study did not rule out the side-effect of altered C/N ratios. However, both the low C condition (MS media without sucrose) and the high C condition (MS media with 5% sucrose) show late-flowering phenotypes (our unpublished data). Thus, C and N may regulate flowering through different pathways. Given that nitrogen levels regulate ferredoxin-NADP⁺-oxidoreductase and ATP synthesis rate (energy metabolism flow),^3,11 nitrogen metabolism and carbon metabolism may be tightly linked with each other. The crosstalk between C metabolite signals and N metabolite signals needs further investigations.

The role of AMPK in plant cells

AMPK is the key molecular in biological energy metabolism. AMPK is a heterotrimeric protein kinase consisting of a catalytic (α) subunit and 2 regulatory (β, γ) subunits.¹² Under conditions of hypoxia, exercise, ischemia, heat shock, and low glucose, AMPK is activated allosterically by rising cellular AMP and by phosphorylation of the catalytic α subunit.¹² In mammalian cells, AMPK plays a broader role in regulating whole-body energy metabolism and glucose homeostasis through the regulation of processes like muscle glucose uptake, insulin production and secretion, management of body lipids, and appetite.¹³ But its functions in plants were rarely reported. The SnRK family in Arabidopsis is the homologous to mammalian AMPK, which comprised of 3 distinct subfamilies.¹⁴ Arabidopsis AMPKα1 shows inhibitory responses to sugar metabolites, especially the trehalose-6-phosphate (T6P).¹⁵ But no clear correction between plant SnRK family proteins and sugar signaling or other nutritional signaling in plant cells has been identified. Here we find that the high level of AMP under the high N condition activates Arabidopsis AMPKα1.³ Similar to mammal AMPKs, the AMPKα1 protein in plant cell nuclei also shows a robust rhythm.³ Furthermore, nuclear activity of AMPKα1 was also higher in the day than at night. In mammal cells, circadian expression of AMPKβ2 transcript has been observed.¹⁶ Because the subunit composition of AMPK complexes regulates its localization, oscillating AMPKβ2 could diurnally regulate the nuclear import of AMPK.¹⁶ Circadian expression of AMPKβ2 mRNA and the diurnal AMPK import should be further verified in plant cells.

Conservation of AMPK-CRY1 pathway in nutritional signaling

Previous studies in mammalian cells have demonstrated that AMPK regulates CRY1 stability. Active nuclear AMPK phosphorylates cryptochromes, thus increasing their interaction with F-box and leucine-rich repeat protein 3 (FBXL3) and leading to ubiquitin-dependent proteasomal degradation.^16,17 However in Arabidopsis cells, blue-light-induced CRY1 phosphorylation is not accompanied by a decrease in its protein steady-state-level.¹⁸ Indeed, Arabidopsis CRY1 is a light-stable protein; while CRY2 is a light-labile protein.^18,19 Experiments in Arabidopsis demonstrated that the blue-light-dependent phosphorylation of CRY2 (but not CRY1) triggers its degradation.¹⁹ Different subcellular localization of Arabidopsis CRY1 between light conditions (mainly in the cytosol) and dark conditions (mainly in the nucleus) might be the reason.²⁰ In plant cells, nuclear AMPK induce nuclear CRY1 phosphorylation.³ While phosphorylated nuclear CRY1 promotes its ubiquitin-dependent degradation (the localization of ubiquitin-dependent proteasomes exist both in cytosol and the nuclear).^3,16 Thus, the nutritional status – AMPK – CRY1 – circadian clock pathway may represent a conserved mechanism in higher eukaryotes.^3,16

NO may participate in N-regulated flowering

Nitrate reductase (NR) is the key enzyme in N assimilation that catalytic reduction from nitrate to nitrite. NR produces NO from nitrite by NAD(P)H-dependent manner.^21-23 Therefore, our previous study cannot rule out the side-effect of NO generated by the N assimilation. NO is involved in photoperiod and autonomous flowering pathway (NO represses the amplitudes of output pathway components CO and GI).⁵ These correlations imply that N-regulated flowering pathways may be very complex. We should consider more about carbon metabolite signals and NO signals in the future studies.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by the Preeminent Youth Fund of Sichuan Province (grant number 2015JQO045) to S.Y.