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

Abstract

Abstract:The aim of this study was to investigate the role of ASN3-encoded asparagine synthetase (AS, EC 6.3.5.4) during vegetative growth, seed development and germination of Arabidopsis thaliana. Phenotypic analysis of knockout (asn3-1) and knockdown (asn3-2) T-DNA insertion mutants for the ASN3 gene (At5g10240) demonstrated wild-type contents of asparagine synthetase protein, chlorophyll and ammonium in green leaves at 35 days after sowing. In situ hybridization localized ASN3 mRNA to phloem companion cells of vasculature. Young siliques of the asn3-1 knockout line showed a decrease in asparagine but an increase in glutamate. The seeds of asn3-1 and asn3-2 displayed a wild-type nitrogen status expressed as total nitrogen content, indicating that the repression of ASN3 expression had only a limited effect on mature seeds. An analysis of amino acid labeling of seeds imbibed with ( 15 N) ammonium for 24 h revealed that asn3-1 seeds contained 20% less total asparagine while 15 N-labeled asparagine ((2-15 N)asparagine, (4-15 N)asparagine and (2,4-15 N)asparagine) increased by 12% compared to wild-type seeds. The data indicate a fine regulation of asparagine synthesis and hydrolysis in Arabidopsis seeds.

ASN1‐encoded asparagine synthetase in floral organs contributes to nitrogen filling in Arabidopsis seeds

“…Soil culture was performed by sowing seeds and growing seedlings for 45 days in a growth chamber using nutrient solution under a 16‐h light (150 μmol photons m −2 sec −1 , 21°C)/8‐h dark (17°C) photoperiod with 80% relative humidity as described in (Gaufichon et al ., ). The experiments on reproductive organs were carried out on soil‐cultured plants at different stages of embryogenesis defined as follows: Floral organs: stage 0 (ovule stage/flower buds) at 1 to 2 h after fertilization; stage 1 (four‐cell to globular stage/50% opened flowers) at 1 day after pollination and stage 2 (globular to heart stage/2–3 mm long silique) at 2 days after pollination (Boyes et al ., ; Kleindt et al ., ).…”

“…Soil culture was performed by sowing seeds and growing seedlings for 45 days in a growth chamber using nutrient solution under a 16-h light (150 lmol photons m À2 sec À1 , 21°C)/8-h dark (17°C) photoperiod with 80% relative humidity as described in (Gaufichon et al, 2016b). The experiments on reproductive organs were carried out on soil-cultured plants at different stages of embryogenesis defined as follows:…”

“…The Arabidopsis genome harbours three loci encoding asparagine synthetase: ASN1, ASN2 and ASN3 (The Arabidopsis Genome Initiative, 2000).The physiological functions of asparagine synthetase in Arabidopsis have been investigated using molecular approaches by analysing ASN mRNA profiles. Changes of ASN1 and ASN2 expression by light and metabolites in seedlings occur in an opposite way (Tsai and Coruzzi, 1991;Lam et al, 1998Lam et al, , 2003, whereas ASN3 mRNA levels in different organs remain lower (Gaufichon et al, 2016b). Initially, ASN1 was thought to be a major gene involved in primary nitrogen assimilation in the dark for long-distance nitrogen transport because dark induction of ASN1 mRNA correlates with higher asparagine in Arabidopsis phloem (Coruzzi, 2003).…”

Special Issue: Nitrogen Transport and Assimilation in Plants

“…Dang) [7] and of the role of a gene encoding asparagine synthetase in Arabidopsis thaliana (L.) Heynh. [8], another model species, are included in two of the four research articles. The importance of N remobilization and recycling in two crops, namely Hordeum vulgare L. and oilseed rape (Brassica napus L.), is presented in the two other research papers, with particular emphasis on the role of senescence and autophagy [9,10].…”