The Arabidopsis nitrate transporter NRT2.5 plays a role in nitrate acquisition and remobilization in nitrogen-starved plants.

Nitrogen is a key mineral nutrient playing a crucial role in plant growth and development. Understanding the mechanisms of nitrate uptake from the soil and distribution through the plant in response to nitrogen starvation is an important step on the way to improve nitrogen uptake and utilization efficiency for better growth and productivity of plants, and to prevent negative effects of nitrogen fertilizers on the environment and human health. In this study, we show that Arabidopsis NITRATE TRANSPORTER 2.5 (NRT2.5) is a plasma membrane-localized high-affinity nitrate transporter playing an essential role in adult plants under severe nitrogen starvation. NRT2.5 expression is induced under nitrogen starvation and NRT2.5 becomes the most abundant transcript amongst the seven NRT2 family members in shoots and roots of adult plants after long-term starvation. GUS reporter analyses showed that NRT2.5 is expressed in the epidermis and the cortex of roots at the root hair zone and in minor veins of mature leaves. Reduction of NRT2.5 expression resulted in a decrease in high-affinity nitrate uptake without impacting low-affinity uptake. In the background of the high-affinity nitrate transporter mutant nrt2.4, an nrt2.5 mutation reduced nitrate levels in the phloem of N-starved plants further than in the single nrt2.4 mutants. Growth analyses of multiple mutants between NRT2.1, NRT2.2, NRT2.4, and NRT2.5 revealed that NRT2.5 is required to support growth of nitrogen-starved adult plants by ensuring the efficient uptake of nitrate collectively with NRT2.1, NRT2.2 and NRT2.4 and by taking part in nitrate loading into the phloem during nitrate remobilization.

The Arabidopsis nitrate transporter NRT2.4 plays a double role in roots and shoots of nitrogen-starved plants.

Plants have evolved a variety of mechanisms to adapt to N starvation. NITRATE TRANSPORTER2.4 (NRT2.4) is one of seven NRT2 family genes in Arabidopsis thaliana, and NRT2.4 expression is induced under N starvation. Green fluorescent protein and ß-glucuronidase reporter analyses revealed that NRT2.4 is a plasma membrane transporter expressed in the epidermis of lateral roots and in or close to the shoot phloem. The spatiotemporal expression pattern of NRT2.4 in roots is complementary with that of the major high-affinity nitrate transporter NTR2.1. Functional analysis in Xenopus laevis oocytes and in planta showed that NRT2.4 is a nitrate transporter functioning in the high-affinity range. In N-starved nrt2.4 mutants, nitrate uptake under low external supply and nitrate content in shoot phloem exudates was decreased. In the absence of NRT2.1 and NRT2.2, loss of function of NRT2.4 (triple mutants) has an impact on biomass production under low nitrate supply. Together, our results demonstrate that NRT2.4 is a nitrate transporter that has a role in both roots and shoots under N starvation.

Chloroplast acetyl-CoA carboxylase activity is 2-oxoglutarate-regulated by interaction of PII with the biotin carboxyl carrier subunit.

The PII protein is a signal integrator involved in the regulation of nitrogen metabolism in bacteria and plants. Upon sensing of cellular carbon and energy availability, PII conveys the signal by interacting with target proteins, thereby modulating their biological activity. Plant PII is located to plastids; therefore, to identify new PII target proteins, PII-affinity chromatography of soluble extracts from Arabidopsis leaf chloroplasts was performed. Several proteins were retained only when Mg-ATP was present in the binding medium and they were specifically released from the resin by application of a 2-oxoglutarate-containing elution buffer. Mass spectroscopy of SDS/PAGE-resolved protein bands identified the biotin carboxyl carrier protein subunits of the plastidial acetyl-CoA carboxylase (ACCase) and three other proteins containing a similar biotin/lipoyl-binding motif as putative PII targets. ACCase is a key enzyme initiating the synthesis of fatty acids in plastids. In in vitro reconstituted assays supplemented with exogenous ATP, recombinant Arabidopsis PII inhibited chloroplastic ACCase activity, and this was completely reversed in the presence of 2-oxoglutarate, pyruvate, or oxaloacetate. The inhibitory effect was PII-dose-dependent and appeared to be PII-specific because ACCase activity was not altered in the presence of other tested proteins. PII decreased the V(max) of the ACCase reaction without altering the K(m) for acetyl-CoA. These data show that PII function has evolved between bacterial and plant systems to control the carbon metabolism pathway of fatty acid synthesis in plastids.

PII is induced by WRINKLED1 and fine-tunes fatty acid composition in seeds of Arabidopsis thaliana.

The PII protein is an integrator of central metabolism and energy levels. In Arabidopsis, allosteric sensing of cellular energy and carbon levels alters the ability of PII to interact with target enzymes such as N-acetyl-l-glutamate kinase and heteromeric acetyl-coenzyme A carboxylase, thereby modulating the biological activity of these plastidial ATP- and carbon-consuming enzymes. A quantitative reverse transcriptase-polymerase chain reaction approach revealed a threefold induction of the AtGLB1 gene (At4g01900) encoding PII during early seed maturation. The activity of the AtGLB1 promoter was consistent with this pattern. A complementary set of molecular and genetic analyses showed that WRINKLED1, a transcription factor known to induce glycolytic and fatty acid biosynthetic genes at the onset of seed maturation, directly controls AtGLB1 expression. Immunoblot analyses and immunolocalization experiments using anti-PII antibodies established that PII protein levels faithfully reflected AtGLB1 mRNA accumulation. At the subcellular level, PII was observed in plastids of maturing embryos. To further investigate the function of PII in seeds, comprehensive functional analyses of two pII mutant alleles were carried out. A transient increase in fatty acid production was observed in mutant seeds at a time when PII protein content was found to be maximal in wild-type seeds. Moreover, minor though statistically significant modifications of the fatty acid composition were measured in pII seeds, which exhibited decreased amounts of modified (elongated, desaturated) fatty acid species. The results obtained outline a role for PII in the fine tuning of fatty acid biosynthesis and partitioning in seeds.

Metabolite regulation of the interaction between Arabidopsis thaliana PII and N-acetyl-l-glutamate kinase.

The metabolic control of the interaction between ArabidopsisN-acetyl-l-glutamate kinase (NAGK) and the PII protein has been studied. Both gel exclusion and affinity chromatography analyses of recombinant, affinity-purified PII (trimeric complex) and NAGK (hexameric complex) showed that NAGK strongly interacted with PII only in the presence of Mg-ATP, and that this process was reversed by 2-oxoglutarate (2-OG). Furthermore, metabolites such as arginine, glutamate, citrate, and oxalacetate also exerted a negative effect on the PII-NAGK complex formation in the presence of Mg-ATP. Using chloroplast protein extracts and PII affinity chromatography, NAGK interacted with PII only in the presence of ATP-Mg(2+), and this process was antagonized by 2-OG. These results reveal a complex metabolic control of the PII interaction with NAGK in the chloroplast stroma of higher plants.