The phenomenon of delayed flowering after the application of nitrogen (N) fertilizer has long been known in agriculture, but the detailed molecular basis for this phenomenon is largely unclear. Here we used a modified method of suppression-subtractive hybridization to identify two key factors involved in N-regulated flowering time control in Arabidopsis thaliana, namely ferredoxin-NADP + -oxidoreductase and the blue-light receptor cryptochrome 1 (CRY1). The expression of both genes is induced by low N levels, and their loss-offunction mutants are insensitive to altered N concentration. Low-N conditions increase both NADPH/NADP + and ATP/AMP ratios, which in turn affect adenosine monophosphate-activated protein kinase (AMPK) activity. Moreover, our results show that the AMPK activity and nuclear localization are rhythmic and inversely correlated with nuclear CRY1 protein abundance. Low-N conditions increase but high-N conditions decrease the expression of several key components of the central oscillator (e.g., CCA1, LHY, and TOC1) and the flowering output genes (e.g., GI and CO). Taken together, our results suggest that N signaling functions as a modulator of nuclear CRY1 protein abundance, as well as the input signal for the central circadian clock to interfere with the normal flowering process. T he transition from vegetative to reproductive development is a central event in the plant life cycle, which is coordinately regulated by various endogenous and external cues. In the model dicotyledonous plant species Arabidopsis thaliana, five distinct genetic pathways regulating flowering time have been established: the vernalization pathway, photoperiod pathway, gibberellin acid (GA) pathway, autonomous pathway, and endogenous (age) pathway (1). These pathways ultimately converge to regulate a set of floral integrator genes, FLOWERING LOCUS T (FT) and SUPPRESSOR OF CONSTANS 1 (SOC1), which in turn activate the expression of floral meristem identity genes to trigger the formation of flowers (2-4).Plants use the circadian clock as the timekeeping mechanism to measure day length and to ensure flowering at the proper season (5, 6). As a facultative long-day (LD) plant, Arabidopsis flowers earlier under LD conditions than under short-day (SD) conditions. Forward genetics in A. thaliana have identified the GI-CO-FT hierarchy as the canonical genetic pathway promoting flowering specifically under LD conditions (5,7,8). In this pathway, GI (GIGANTEA) can be considered the output point of the circadian clock to control flowering by regulating CONSTANS (CO) expression in the right phase, which activates expression of FT and TSF (TWIN SISTER OF FT) in the companion cells of the phloem within the vascular tissue (2, 9). FT and TSF proteins act as the long-sought florigens that move from leaves to the apical meristem to induce genes required for reproductive development (2-4). Both GI and CO are regulated by the circadian clock and by light signaling simultaneously and at both transcriptional and posttranscriptional levels, to en...

Nitric oxide (NO), an active signaling molecule in plants, is involved in numerous physiological processes and adaptive responses to environmental stresses. Under high-salt conditions, plants accumulate NO quickly, and reorganize Na + and K + contents. However, the molecular connection between NO and ion homeostasis is largely unknown. Here, we report that NO lowers K + channel AKT1-mediated plant K + uptake by modulating vitamin B6 biosynthesis. In a screen for Arabidopsis NO-hypersensitive mutants, we isolated sno1 (sensitive to nitric oxide 1), which is allelic to the previously noted mutant sos4 (salt overly sensitive 4) that has impaired Na + and K + contents and overproduces pyridoxal 5′-phosphate (PLP), an active form of vitamin B6. We showed that NO increased PLP and decreased K + levels in plant. NO induced SNO1 gene expression and enzyme activity, indicating that NO-triggered PLP accumulation mainly occurs through SNO1-mediated vitamin B6 salvage biosynthetic pathway. Furthermore, we demonstrated that PLP significantly repressed the activity of K + channel AKT1 in the Xenopus oocyte system and Arabidopsis root protoplasts. Together, our results suggest that NO decreases K + absorption by promoting the synthesis of vitamin B6 PLP, which further represses the activity of K + channel AKT1 in Arabidopsis. These findings reveal a previously unidentified pivotal role of NO in modulating the homeostasis of vitamin B6 and potassium nutrition in plants, and shed light on the mechanism of NO in plant acclimation to environmental changes.genetic approach | electrophysiological studies | potassium nutrition N itric oxide (NO) acts as a crucial signaling molecule in various physiological processes in plants, such as seed germination and dormancy (1, 2), root development (3), leaf senescence (4, 5), floral transition (6), stomatal movement (7, 8), iron homeostasis (9, 10), and hormone responses (11,12). NO production is altered when plants are subjected to abiotic or biotic stresses (13,14). High salt, a major environmental factor that limits agriculture yield, induces a quick endogenous NO accumulation in plants (15,16), and triggers enhanced Na + influx and reduced K + absorption in the root (17). Both endogenously produced NO and exogenously applied NO have been proposed to enhance plant salt tolerance (18-21) by attenuating high saltinduced increases in the Na + to K + ratio. Genetic analysis showed that K + nutrition, but not Na + , plays critical role in plant salt tolerance (22). However, the molecular basis of NO effect on K + or Na + content is elusive.K + levels in plant tissues are determined by K + uptake and translocation, which are mediated by a large number of transporters and channels. In Arabidopsis, the K + channel AKT1 (23, 24) and K + transporter AtHAK5 (25) are the two major molecular entities responsible for K + absorption from the environment (26-28). AKT1 contributes to K + acquisition over a wide range of external K + concentrations (10 μM-10 mM), whereas AtHAK5 mediates limited uptake capac...

Two cytochrome P450 enzymes, CYP97A3 and CYP97C1, catalyze hydroxylations of the β- and ε-rings of α-carotene to produce lutein. Chirality is introduced at the C-3 atom of both rings, and the reactions are both pro-3R–stereospecific. We determined the crystal structures of CYP97A3 in substrate-free and complex forms with a nonnatural substrate and the structure of CYP97C1 in a detergent-bound form. The structures of CYP97A3 in different states show the substrate channel and the structure of CYP97C1 bound with octylthioglucoside confirms the binding site for the carotenoid substrate. Biochemical assays confirm that the ferredoxin-NADP+reductase (FNR)–ferredoxin pair is used as the redox partner. Details of the pro-3Rstereospecificity are revealed in the retinal-bound CYP97A3 structure. Further analysis indicates that the CYP97B clan bears similarity to the β-ring–specific CYP97A clan. Overall, our research describes the molecular basis for the last steps of lutein biosynthesis.