Glutamine synthetase and glutamate dehydrogenase in triticale seeds: molecular cloning and genes expression

Abstract

In higher plants, glutamine synthetase (GS; EC 6.3.1.2) and glutamate dehydrogenase (GDH; EC 1.4.1.2) are the predominant enzymes in nitrogen metabolism. In this study, we cloned both the GS and GDH genes and analyzed their expression levels and variations in their activity in developing and germinating x Triticosecale (cv. Witon) kernels. The developing kernel samples were collected 3, 5, 7, 9, 13, 15, 20, 25, 30, 35, 40 and 45 days after flowering (DAF). The germinating kernel samples were collected after 8, 16, 24, 48 and 72 h of imbibition. There are two GS isoforms that are localized to different compartments: the cytosol (GS1) and the chloroplast (GS2). Five cDNAs encoding GS proteins in triticale plants were obtained using RT-PCR. We cloned the four genes encoding GS1, which we designated TsGS1-1, TsGS1-2, TsGS1-3 and TsGS1-4 and the only gene encoding GS2, which was designated TsGS2-1. We studied the changes in the enzymatic activity and the expression profiles of the GDH, GS1 and GS2 genes in both the developing and germinating seeds of triticale. Based on our results, there is likely cooperation between GDH and GS1 in the synthesis of glutamine and glutamate during the early stages of seed formation and in the scutella of kernels for up to 24 h of imbibition.

Transcriptional identification and characterization of differentially expressed genes associated with embryogenesis in radish (Raphanus sativus L.)

“…Additionally, several genes encoding enzymes involved in embryo development have been validated by qRT-PCR in the present study, including GST , GAT and GS1-4 with high expression at 15 DAP ( Fig. 6 ), which were involved in controlling seed development 59 60 . Similarly, in triticale, Grabowska et al demonstrated that during the early phase of seed development, the activity of glutamine synthetase with a high level was observed as early as 3 day after flowering (DAF) and increased and reached its maximum at 15 DAF 59 , consisting with our result.…”

Cytosolic Glutamine Synthetase Gln1;2 Is the Main Isozyme Contributing to GS1 Activity and Can Be Up-Regulated to Relieve Ammonium Toxicity

“…The fact that GS2 eluted before GS1, as also previously observed in Arabidopsis (Lothier et al, 2011), Trientalis europaea (Parry et al, 2000) and Pinus sylvestris (Elmlinger and Mohr, 1992), reflects differences in amino acid sequence and pI of the proteins. The GS2 protein can carry a chloroplast transient peptide that may be removed from the mature protein (Grabowska et al, 2012), leading to a reduction of the pI and the elution time relative to GS1. This presumption with respect to GS2 protein processing can also affect the elution of GS2 itself, resulting in the observed two separate peaks for GS2.…”

Molecular cloning and functional analysis of the second gene encoding glutamate dehydrogenase in triticale

“…This gene is the second GDH to be cloned in triticale. Previous studies carried out by Grabowska et al (2011Grabowska et al ( , 2012 analyzed the TsGDH1 gene. Sequence alignments of the previously cloned full-length TsGDH1 gene (Grabowska et al The soluble protein extracts of leaves were subjected to native PAGE followed by NAD-GDH in-gel deaminating activity staining.…”

“…Thereafter, the physiological functions of GDH have been the subject of extensive study. Many authors believe that NADH-GDH can act in vivo as an ammonium-assimilating enzyme only under certain conditions, for instance, in dark-stressed plants, under abiotic stress, and during the processes of natural plant aging and of seed formation (Grabowska et al 2012; Kwinta and Kolik 2006;Skopelitis et al 2006). Other authors have suggested that GDH plays a major role in amino acid catabolism during seed germination, plant senescence and nutrient remobilization (Aubert et al 2001;Grabowska et al 2011;Lehmann and Ratajczak 2008).…”