Nitrogen and phosphorus are among the most widely used fertilizers worldwide. Nitrate (NO3−) and phosphate (PO43−) are also signaling molecules whose respective transduction pathways are being intensively studied. However, plants are continuously challenged with combined nutritional deficiencies, yet very little is known about how these signaling pathways are integrated. Here we report the identification of a highly NO3−-inducible NRT1.1-controlled GARP transcription factor, HRS1, document its genome-wide transcriptional targets, and validate its cis-regulatory-elements. We demonstrate that this transcription factor and a close homolog repress primary root growth in response to P deficiency conditions, but only when NO3− is present. This system defines a molecular logic gate integrating P and N signals. We propose that NO3− and P signaling converge via double transcriptional and post-transcriptional control of the same protein, HRS1
Root NO 3 À efflux to the outer medium is a component of NO 3 À net uptake and can even overcome influx upon various stresses. Its role and molecular basis are unknown. Following a functional biochemical approach, NAXT1 (for NITRATE EXCRETION TRANSPORTER1) was identified by mass spectrometry in the plasma membrane (PM) of Arabidopsis thaliana suspension cells, a localization confirmed using a NAXT1-Green Fluorescent Protein fusion protein. NAXT1 belongs to a subclass of seven NAXT members from the large NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER family and is mainly expressed in the cortex of mature roots. The passive NO 3 À transport activity (K m ¼ 5 mM) in isolated root PM, electrically coupled to the ATPdependant H þ -pumping activity, is inhibited by anti-NAXT antibodies. In standard culture conditions, NO 3 À contents were altered in plants expressing NAXT-interfering RNAs but not in naxt1 mutant plants. Upon acid load, unidirectional root NO 3 À efflux markedly increased in wild-type plants, leading to a prolonged NO 3 À excretion regime concomitant with a decrease in root NO 3 À content. In vivo and in vitro mutant phenotypes revealed that this response is mediated by NAXT1, whose expression is upregulated at the posttranscriptional level. Strong medium acidification generated a similar response. In vitro, the passive efflux of NO 3 À (but not of Cl À ) was strongly impaired in naxt1 mutant PM. This identification of NO 3 À efflux transporters at the PM of plant cells opens the way to molecular studies of the physiological role of NO 3 À efflux in stressed or unstressed plants.
Nitrogen (N) and phosphorus (P) are key macronutrients sustaining plant growth and crop yield and ensuring food security worldwide. Understanding how plants perceive and interpret the combinatorial nature of these signals thus has important agricultural implications within the context of (1) increased food demand, (2) limited P supply, and (3) environmental pollution due to N fertilizer usage. Here, we report the discovery of an active control of P starvation response (PSR) by a combination of local and long-distance N signaling pathways in plants. We show that, in Arabidopsis (Arabidopsis thaliana), the nitrate transceptor CHLORINA1/NITRATE TRANSPORTER1.1 (CHL1/NRT1.1) is a component of this signaling crosstalk. We also demonstrate that this crosstalk is dependent on the control of the accumulation and turnover by N of the transcription factor PHOSPHATE STARVATION RESPONSE1 (PHR1), a master regulator of P sensing and signaling. We further show an important role of PHOSPHATE2 (PHO2) as an integrator of the N availability into the PSR since the effect of N on PSR is strongly affected in pho2 mutants. We finally show that PHO2 and NRT1.1 influence each other's transcript levels. These observations are summarized in a model representing a framework with several entry points where N signal influence PSR. Finally, we demonstrate that this phenomenon is conserved in rice (Oryza sativa) and wheat (Triticum aestivum), opening biotechnological perspectives in crop plants.
We developed a method to characterize different classes of membrane proteins within a single experiment and using simple matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis. After membrane solubilization with the nondenaturing detergent n-dodecyl-beta-D-maltoside, proteins were separated successively by gel filtration and ion-exchange chromatography and finally by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This procedure allowed to characterize 70 proteins from a membrane fraction enriched in plant vacuolar membrane (Arabidopsis), including integral proteins like the V0 complex of the H(+)-ATPase, the H(+)-pyrophosphatase or the glutathione S-conjugate ATPase AtMRP1, and peripheral proteins like the subunits of the catalytic V1 complex of the H(+)-ATPase. Approximately 60% of identified proteins were predicted to possess at least two trans-membrane domains. Furthermore, proteins, with molecular masses ranging between 20 and 200 kDa were distributed into two populations with maximum frequencies at pI 5.3 and 8.9. Finally, this procedure appeared to allow the identification of proteins known to be minor in whole-cell extracts like signaling or vesicular trafficking proteins. Almost 50% of the proteins identified were functionally unknown whereas the others confirmed that the plant vacuole is a multipurpose compartment.
In most plants, NO(3)(-) constitutes the major source of nitrogen, and its assimilation into amino acids is mainly achieved in shoots. Furthermore, recent reports have revealed that reduction of NO(3)(-) translocation from roots to shoots is involved in plant acclimation to abiotic stress. NPF2.3, a member of the NAXT (nitrate excretion transporter) sub-group of the NRT1/PTR family (NPF) from Arabidopsis, is expressed in root pericycle cells, where it is targeted to the plasma membrane. Transport assays using NPF2.3-enriched Lactococcus lactis membranes showed that this protein is endowed with NO(3)(-) transport activity, displaying a strong selectivity for NO(3)(-) against Cl(-). In response to salt stress, NO(3)(-) translocation to shoots is reduced, at least partly because expression of the root stele NO(3)(-) transporter gene NPF7.3 is decreased. In contrast, NPF2.3 expression was maintained under these conditions. A loss-of-function mutation in NPF2.3 resulted in decreased root-to-shoot NO(3)(-) translocation and reduced shoot NO(3)(-) content in plants grown under salt stress. Also, the mutant displayed impaired shoot biomass production when plants were grown under mild salt stress. These mutant phenotypes were dependent on the presence of Na(+) in the external medium. Our data indicate that NPF2.3 is a constitutively expressed transporter whose contribution to NO(3)(-) translocation to the shoots is quantitatively and physiologically significant under salinity.
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