Arabidopsis thaliana ASN2 encoding asparagine synthetase is involved in the control of nitrogen assimilation and export during vegetative growth

Abstract

We investigated the function of ASN2, one of the three genes encoding asparagine synthetase (EC 6.3.5.4), which is the most highly expressed in vegetative leaves of Arabidopsis thaliana. Expression of ASN2 and parallel higher asparagine content in darkness suggest that leaf metabolism involves ASN2 for asparagine synthesis. In asn2‐1 knockout and asn2‐2 knockdown lines, ASN2 disruption caused a defective growth phenotype and ammonium accumulation. The asn2 mutant leaves displayed a depleted asparagine and an accumulation of alanine, GABA, pyruvate and fumarate, indicating an alanine formation from pyruvate through the GABA shunt to consume excess ammonium in the absence of asparagine synthesis. By contrast, asparagine did not contribute to photorespiratory nitrogen recycle as photosynthetic net CO2 assimilation was not significantly different between lines under both 21 and 2% O2. ASN2 was found in phloem companion cells by in situ hybridization and immunolocalization. Moreover, lack of asparagine in asn2 phloem sap and lowered 15N flux to sinks, accompanied by the delayed yellowing (senescence) of asn2 leaves, in the absence of asparagine support a specific role of asparagine in phloem loading and nitrogen reallocation. We conclude that ASN2 is essential for nitrogen assimilation, distribution and remobilization (via the phloem) within the plant.

Impact of the Disruption of ASN3-Encoding Asparagine Synthetase on Arabidopsis Development

“…Leaf fixation and 8-µm-section microtome preparations of Arabidopsis rosette leaves were carried out as described in [8]. Hybridization probes were prepared from cDNA strands synthesized from 2 µg total RNA using an Omniscript RT kit (Qiagen, GmbH, Germany).…”

“…Amplified sense and antisense DNA fragments (400 ng each) were reverse-transcribed with a Promega transcription kit (Madison WI, USA) using digoxigenin (DIG)-UTP and subjected to DNase digestion. In situ hybridization was carried out as described in [8] except for (not include in the steps) the incubation of leaf sections with secondary anti-DIG antibody conjugated to alkaline phosphatase. The visualization of ASN3 RNA by alkaline phosphatase activity was carried out with sealed slides and fluorescence observed using a Leica DMR microscope (Leica Microsystems, Wetzlar, Germany).…”

“…Denatured proteins were subjected to SDS-PAGE using 7% gels [11]. Proteins were transferred to a nitrocellulose membrane and probed with rabbit antibodies raised against Arabidopsis recombinant ASN2-encoded asparagine synthetase [8]. After hybridization with goat serum anti-rabbit antibodies conjugated with a peroxidase, ASN protein was detected by the peroxidase activity in the presence of 3.4 mM 4-chloro-1-naphtol and 0.01% (v/v) H 2 O 2 .…”

“…The GC-MS analysis was performed using an Agilent 7890A gas chromatograph coupled to an Agilent 5975C mass spectrometer as described in [8]. Peaks were identified using the AMDIS 32 software after retention index (RI) calibration with an alkane mix (C10, C12, C15, C19, C22, C28, C32, C36) injected during the course of the analysis.…”

“…Several lines of evidence indicate that ASN1-encoded asparagine synthetase plays a role in nitrogen export from source to sink organs via the phloem in the dark [7] and in the recapture of ammonium produced under biotic and abiotic stresses in Arabidopsis [7]. Loss-of-function studies of ASN2-deficient mutants provided evidence that ASN2-encoded asparagine synthetase is involved in leaf primary nitrogen assimilation during the vegetative stage [8] and in ammonium detoxification under abiotic stress [9].…”