Isolation of photorespiratory mutants from Lotus japonicus deficient in glutamine synthetase

Abstract

A mutagenesis programme using ethyl methanesulphonate (EMS) was carried out on Lotus japonicus (Regel) Larsen cv. Gifu in order to isolate photorespiratory mutants in this model legume. These mutants were able to grow in a CO2-enriched atmosphere [0.7% (v/v) CO2] but showed stress symptoms when transferred to air. Among them, three mutants displayed low levels of glutamine synthetase (GS; EC 6.3.1.2) activity in leaves. The mutants accumulated ammonium in leaves upon transfer from 0.7% (v/v) CO2 to air. F1 plants of back crosses to wild type were viable in air and F2 populations segregated 3 : 1 (viable in air : air-sensitive) indicative of a single Mendelian recessive trait. Complementation tests showed that the three mutants obtained were allelic. Chromatography on DEAE-Sephacel used to separate the cytosolic and plastidic GS isoenzymes together with immunological data showed that: (1) mutants were specifically affected in the plastidic GS isoform, and (2) in L. japonicus the plastidic GS isoform eluted at lower ionic strength than the cytosolic isoform, contrary to what happens in most plants. The plastidic GS isoform present in roots of wild type L. japonicus was also absent in roots of the mutants, indicating that this plastidic isoform from roots was encoded by the same gene than the GS isoform expressed in leaf tissue. Viability of mutant plants in high-CO2 conditions indicates that plastidic GS is not essentially required for primary ammonium assimilation. Nevertheless, mutant plants did not grow as well as wild type plants in high-CO2 conditions.

Transcriptomic and Metabolic Changes Associated with Photorespiratory Ammonium Accumulation in the Model Legume Lotus japonicus

“…Previous studies showed that photorespiratory mutants of L. japonicus that are deficient in plastidic GS 2 accumulated NH 4 + at least during the first 24 h after exposure to normal CO 2 atmosphere (Orea et al, 2002). We aimed now to study the time course process of NH 4 + accumulation and to address the effect of an impaired photorespiratory cycle on an extended time scale.…”

“…In our laboratory, the first photorespiratory mutants of legumes were isolated from the model legume Lotus japonicus (Orea et al, 2002;Márquez, 2005;Márquez et al, 2005). These mutants, deficient in plastidic GS 2 , were further characterized at the molecular level (Betti et al, 2006) as well as nodule function (García-Calderón et al, 2012).…”

“…These mutants, deficient in plastidic GS 2 , were further characterized at the molecular level (Betti et al, 2006) as well as nodule function (García-Calderón et al, 2012). Under CO 2-enriched atmosphere, the mutants did not show any visible phenotype and only a slightly lower growth rate (Orea et al, 2002). However, the mutants accumulated high levels of NH 4 + when transferred from CO 2 -enriched to normal CO 2 atmosphere and showed severe stress symptoms when grown for long periods of time under these conditions.…”

“…In this work, we carried out a comparative study of the response to active photorespiration in wild-type and Ljgln2-2 mutant plants that were previously grown under conditions where the rate of photorespiration is very low (0.7% [v/v] CO 2 ; Orea et al, 2002). After the transfer to active photorespiratory conditions, a concerted regulation of the photorespiratory genes, together with massive transcriptomic changes and a perturbation of central metabolism, were observed in the case of the Ljgln2-2 mutant.…”

Identification and Expression Analysis of Glutamine Synthetase Genes in Ramie (Boehmeria nivea L. Gaud)

“…In rice, OsGS1;2 is localized in the surface cells of roots and is important in the primary assimilation of ammonium ions that are taken up by the roots, with OsGS1;1 in the roots being unable to compensate for the functions of OsGS1;2 [40]. 4 + that were released during photorespiration were very high and that the re-assimilation of this ammonium in the plant leaves was crucial for the efficiency of nitrogen use [42,43]. In our study, BnGS2 was expressed abundantly in the leaves, particularly during the seedling emergence and fiber development stages.…”