Nutrient signalling integrates and coordinates gene expression, metabolism and growth. However, its primary molecular mechanisms remain incompletely understood in plants and animals. Here we report novel Ca2+ signalling triggered by nitrate with live imaging of an ultrasensitive biosensor in Arabidopsis leaves and roots. A nitrate-sensitized and targeted functional genomic screen identifies subgroup III Ca2+-sensor protein kinases (CPKs) as master regulators orchestrating primary nitrate responses. A chemical switch with the engineered CPK10(M141G) kinase enables conditional analyses of cpk10,30,32 to define comprehensive nitrate-associated regulatory and developmental programs, circumventing embryo lethality. Nitrate-CPK signalling phosphorylates conserved NIN-LIKE PROTEIN (NLP) transcription factors (TFs) to specify reprogramming of gene sets for downstream TFs, transporters, N-assimilation, C/N-metabolism, redox, signalling, hormones, and proliferation. Conditional cpk10,30,32 and nlp7 similarly impair nitrate-stimulated system-wide shoot growth and root establishment. The nutrient-coupled Ca2+ signalling network integrates transcriptome and cellular metabolism with shoot-root coordination and developmental plasticity in shaping organ biomass and architecture.

To counteract¯uctuating nutrient environments, plants have evolved high-and low-af®nity uptake systems. These two systems were traditionally thought to be genetically distinct, but, recently, two Arabidopsis transporters, AtKUP1 and CHL1, were shown to have dual af®nities. However, little is known about how a dual-af®nity transporter works and the advantages of having a dual-af®nity transporter. This study demonstrates that, in the case of CHL1, switching between the two modes of action is regulated by phosphorylation at threonine residue 101; when phosphorylated, CHL1 functions as a high-af®nity nitrate transporter, whereas, when dephosphorylated, it functions as a low-af®nity nitrate transporter. This regulatory mechanism allows plants to change rapidly between high-and low-af®nity nitrate uptake, which may be critical when competing for limited nitrogen. These results demonstrate yet another regulatory role of phosphorylation in plant physiology.

Higher plants have both high-and low-affinity nitrate uptake systems. These systems are generally thought to be genetically distinct. Here, we demonstrate that a well-known low-affinity nitrate uptake mutant of Arabidopsis, chl1 , is also defective in high-affinity nitrate uptake. Two to 3 hr after nitrate induction, uptake activities of various chl1 mutants at 250 M nitrate (a high-affinity concentration) were only 18 to 30% of those of wild-type plants. In these mutants, both the inducible phase and the constitutive phase of high-affinity nitrate uptake activities were reduced, with the inducible phase being severely reduced. Expressing a CHL1 cDNA driven by the cauliflower mosaic virus 35S promoter in a transgenic chl1 plant effectively recovered the defect in high-affinity uptake for the constitutive phase but not for the induced phase, which is consistent with the constitutive level of CHL1 expression in the transgenic plant. Kinetic analysis of nitrate uptake by CHL1 -injected Xenopus oocytes displayed a biphasic pattern with a Michaelis-Menten K m value of ‫ف‬ 50 M for the high-affinity phase and ‫ف‬ 4 mM for the low-affinity phase. These results indicate that in addition to being a low-affinity nitrate transporter, as previously recognized, CHL1 is also involved in both the inducible and constitutive phases of high-affinity nitrate uptake in Arabidopsis. INTRODUCTIONNitrate uptake is a physiological process critical for plant growth. Plants have evolved a host of transport systems to accommodate external nitrate concentration levels that can vary up to 10,000 times (Jackson and Caldwell, 1993), and expression of these systems appears to be tightly regulated (Hoff et al., 1994;Crawford, 1995; Glass and Siddiqi, 1995;Wirén et al., 1997). Early experiments with kinetic measurements have identified at least three nitrate transport systems in higher plants. One operates at high nitrate concentrations (over ‫ف‬ 0.5 mM) and is constitutively expressed. It is usually referred to as the cLATS (for constitutive low-affinity transport system); at ‫ف‬ 0.5 mM nitrate, uptake is performed by two HATS (for high-affinity transport system). One is constitutive (cHATS) and the other is inducible (iHATS) (reviewed in, e.g., Larsson and Ingemarsson, 1989; Glass and Siddiqi, 1995). Extensive efforts have been directed in recent years toward cloning and characterizing the genes that are responsible for these transport systems (reviewed in Crawford and Glass, 1998; Daniel-Vedele et al., 1998). As a result, our knowledge about nitrate transporters of higher plants is rapidly accumulating, and it is now clear that the three-system (cLATS, iHATS, and cHATS) model derived from early physiological studies is overly simplified.For example, cloning and functional characterization of the Arabidopsis nitrate transporter gene CHL1 ( AtNRT1 ) (Tsay et al., 1993; Huang et al., 1996) have shown that CHL1 is responsible for an additional LATS that is nitrate inducible, that is, iLATS, which was not revealed by previous physiological char...

The Arabidopsis CHL1 ( AtNRT1 ) gene encodes an inducible component of low-affinity nitrate uptake, which necessitates a "two-component" model to account for the constitutive low-affinity uptake observed in physiological studies. Here, we report the cloning and characterization of a CHL1 homolog, AtNRT1:2 (originally named NTL1 ), with data to indicate that this gene encodes a constitutive component of low-affinity nitrate uptake. Transgenic plants expressing antisense AtNRT1:2 exhibited reduced nitrate-induced membrane depolarization and nitrate uptake activities in assays with 10 mM nitrate. Furthermore, transgenic plants expressing antisense AtNRT1:2 in the chl1-5 background exhibited an enhanced resistance to chlorate (7 mM as opposed to 2 mM for the chl1-5 mutant). Kinetic analysis of AtNRT1:2 -injected Xenopus oocytes yielded a K m for nitrate of ‫ف‬ 5.9 mM. In contrast to CHL1 , AtNRT1:2 was constitutively expressed before and after nitrate exposure (it was repressed transiently only when the level of CHL1 mRNA started to increase significantly), and its mRNA was found primarily in root hairs and the epidermis in both young (root tips) and mature regions of roots. We conclude that low-affinity systems of nitrate uptake, like high-affinity systems, are composed of inducible and constitutive components and that with their distinct functions, they are part of an elaborate nitrate uptake network in Arabidopsis. INTRODUCTIONIon uptake during rapid vegetative growth can consume up to 36% of total respiratory energy cost (ATP) (Werf et al., 1988). A significant portion of this demand is devoted to nitrogen acquisition, for which nitrate from soil is a major supplying source. Extensive studies have hence focused on the kinetics and, more recently, the molecular genetics of nitrate uptake (Glass and Siddiqi, 1995; Wirén et al., 1997; Crawford and Glass, 1998;Daniel-Vedele et al., 1998).Two kinetically distinct nitrate uptake systems, the highaffinity transport system (HATS) and the low-affinity transport system (LATS), have been identified. Depending on the plant species, the HATS has a K m of ‫ف‬ 5 to 100 M, whereas the LATS shows linear kinetics or K m values in the millimolar range (Doddema and Telkamp, 1979; Goyal and Huffaker, 1986;Lee and Drew, 1986;Siddiqi et al., 1990; Aslam et al., 1992;Meharg and Blatt, 1995). In general, the LATS has a larger capacity than does the HATS. For example, it was reported that in Arabidopsis, the uptake rate at 10 mM nitrate ( ‫ف‬ 24 mol hr Ϫ 1 g Ϫ 1 fresh weight) is 24 times higher than is the V max of the HATS ( ‫ف‬ 1 mol hr Ϫ 1 g Ϫ 1 fresh weight) (Touraine and Glass, 1997). Thus, whereas the HATS is important for nitrogen acquisition when external nitrate concentration is low, the LATS is required for mass amounts of nitrogen acquisition. The latter may be more important for growing crops, because nitrate is difficult to retain and its concentration fluctuates significantly in arable soil.In response to nitrate induction, the HATS can be further divided into two compo...

Higher plants have both high- and low-affinity nitrate uptake systems. These systems are generally thought to be genetically distinct. Here, we demonstrate that a well-known low-affinity nitrate uptake mutant of Arabidopsis, chl1, is also defective in high-affinity nitrate uptake. Two to 3 hr after nitrate induction, uptake activities of various chl1 mutants at 250 microM nitrate (a high-affinity concentration) were only 18 to 30% of those of wild-type plants. In these mutants, both the inducible phase and the constitutive phase of high-affinity nitrate uptake activities were reduced, with the inducible phase being severely reduced. Expressing a CHL1 cDNA driven by the cauliflower mosaic virus 35S promoter in a transgenic chl1 plant effectively recovered the defect in high-affinity uptake for the constitutive phase but not for the induced phase, which is consistent with the constitutive level of CHL1 expression in the transgenic plant. Kinetic analysis of nitrate uptake by CHL1-injected Xenopus oocytes displayed a biphasic pattern with a Michaelis-Menten Km value of approximately 50 microM for the high-affinity phase and approximately 4 mM for the low-affinity phase. These results indicate that in addition to being a low-affinity nitrate transporter, as previously recognized, CHL1 is also involved in both the inducible and constitutive phases of high-affinity nitrate uptake in Arabidopsis.

BackgroundTransient gene expression via Agrobacterium-mediated DNA transfer offers a simple and fast method to analyze transgene functions. Although Arabidopsis is the most-studied model plant with powerful genetic and genomic resources, achieving highly efficient and consistent transient expression for gene function analysis in Arabidopsis remains challenging.ResultsWe developed a highly efficient and robust Agrobacterium-mediated transient expression system, named AGROBEST (Agrobacterium-mediated enhanced seedling transformation), which achieves versatile analysis of diverse gene functions in intact Arabidopsis seedlings. Using β-glucuronidase (GUS) as a reporter for Agrobacterium-mediated transformation assay, we show that the use of a specific disarmed Agrobacterium strain with vir gene pre-induction resulted in homogenous GUS staining in cotyledons of young Arabidopsis seedlings. Optimization with AB salts in plant culture medium buffered with acidic pH 5.5 during Agrobacterium infection greatly enhanced the transient expression levels, which were significantly higher than with two existing methods. Importantly, the optimized method conferred 100% infected seedlings with highly increased transient expression in shoots and also transformation events in roots of ~70% infected seedlings in both the immune receptor mutant efr-1 and wild-type Col-0 seedlings. Finally, we demonstrated the versatile applicability of the method for examining transcription factor action and circadian reporter-gene regulation as well as protein subcellular localization and protein–protein interactions in physiological contexts.ConclusionsAGROBEST is a simple, fast, reliable, and robust transient expression system enabling high transient expression and transformation efficiency in Arabidopsis seedlings. Demonstration of the proof-of-concept experiments elevates the transient expression technology to the level of functional studies in Arabidopsis seedlings in addition to previous applications in fluorescent protein localization and protein–protein interaction studies. In addition, AGROBEST offers a new way to dissect the molecular mechanisms involved in Agrobacterium-mediated DNA transfer.

The Arabidopsis CHL1 (AtNRT1) gene encodes an inducible component of low-affinity nitrate uptake, which necessitates a "two-component" model to account for the constitutive low-affinity uptake observed in physiological studies. Here, we report the cloning and characterization of a CHL1 homolog, AtNRT1:2 (originally named NTL1), with data to indicate that this gene encodes a constitutive component of low-affinity nitrate uptake. Transgenic plants expressing antisense AtNRT1:2 exhibited reduced nitrate-induced membrane depolarization and nitrate uptake activities in assays with 10 mM nitrate. Furthermore, transgenic plants expressing antisense AtNRT1:2 in the chl1-5 background exhibited an enhanced resistance to chlorate (7 mM as opposed to 2 mM for the chl1-5 mutant). Kinetic analysis of AtNRT1:2-injected Xenopus oocytes yielded a K(m) for nitrate of approximately 5.9 mM. In contrast to CHL1, AtNRT1:2 was constitutively expressed before and after nitrate exposure (it was repressed transiently only when the level of CHL1 mRNA started to increase significantly), and its mRNA was found primarily in root hairs and the epidermis in both young (root tips) and mature regions of roots. We conclude that low-affinity systems of nitrate uptake, like high-affinity systems, are composed of inducible and constitutive components and that with their distinct functions, they are part of an elaborate nitrate uptake network in Arabidopsis.

Nitrate is an essential nutrient and signaling molecule for plant growth. Plants sense intracellular nitrate to adjust their metabolic and growth responses. Here we identify the primary nitrate sensor in plants. We found that mutation of all seven Arabidopsis NIN-like protein (NLP) transcription factors abolished plants’ primary nitrate responses and developmental programs. Analyses of NIN-NLP7 chimeras and nitrate binding revealed that NLP7 is derepressed upon nitrate perception via its amino terminus. A genetically encoded fluorescent split biosensor, mCitrine-NLP7, enabled visualization of single-cell nitrate dynamics in planta. The nitrate sensor domain of NLP7 resembles the bacterial nitrate sensor NreA. Substitutions of conserved residues in the ligand-binding pocket impaired the ability of nitrate-triggered NLP7 to control transcription, transport, metabolism, development, and biomass. We propose that NLP7 represents a nitrate sensor in land plants.