Characterization of a glutamine synthetase gene DvGS1 from Dunaliella viridis and investigation of the impact on expression of DvGS1 in transgenic Arabidopsis thaliana

Abstract

A novel glutamine synthetase (GS) gene DvGS1 showing highest amino acid sequence identity of 78 % with the other homologous GS proteins from green algae, was isolated and characterized from Dunaliella viridis. Phylogenetic analysis revealed that DvGS1 occupied an independent phylogenetic position which was different with the GSs from higher plants, animals and microbes. Functional complement in E. coli mutant confirmed that the DvGS1 encoded functional GS enzyme. Real-time PCR analysis of DvGS1 in D. viridis cells under nitrogen starvation revealed that the mRNA level of DvGS1 was positively up-regulated in 12 h. The DvGS1 levels at the points of 12 and 24 h were separately twofold and fourfold of the level before nitrogen starvation. In order to investigate the potential application of DvGS1 in higher plants, the transgenic study of DvGS1 in Arabidopsis thaliana was carried out. Phenotype identification demonstrated that all three transgenic lines of T3 generation showed obviously enhanced root length (26 %), fresh weight (22-46 % at two concentrations of nitrate supplies), stem length (26 %), leaf size (29 %) and silique number (30 %) compared with the wild-type Arabidopsis. Biochemical analysis confirmed that all three transgenic lines had higher total nitrogen content, soluble protein concentration, total amino acid content and the leaf GS activity than the wild type plants. The free NH4 (+) and NO3 (-) concentration in fresh leaves of three transgenic lines were reduced by 17-26 % and 14-15 % separately (at two concentrations of nitrate supplies) compared with those of the wild types. All the results indicated that over-expression of DvGS1 in Arabidopsis significantly results in the improvement of growth phenotype and the host's nitrogen use efficiency.

The Effect of Poplar PsnGS1.2 Overexpression on Growth, Secondary Cell Wall, and Fiber Characteristics in Tobacco

“…In this study, a cytoplasmic GS1 type protein PsnGS1.2 , was isolated from P. simonii × P. nigra and characterized by overexpression in tobacco. We observed a number of phenotypic, biochemical, physiological, as well as anatomical changes in the PsnGS1.2 transgenic tobacco, which resembled the changes that were observed in other GS1s transgenic plants ( Man et al, 2005 ; Tabuchi et al, 2007 ; Zhu et al, 2014 ; Molina-Rueda and Kirby, 2015 ). At same time, we also found some distinct functions of PsnGS1.2 .…”

“…The deduced protein sequence comparisons demonstrated that the GS1 proteins are highly conserved in different species ( Figure 1A ). The conserved domain analysis revealed that PsnGS1.2 contained two conserved domains: Glutamine synthetase, beta-Grasp domain (IPR008147) and Glutamine synthetase, catalytic domain (IPR008146) ( Figure 1A ), which are typical structure characteristics of GS proteins ( Zhu et al, 2014 ). These results showed that the PsnGS1.2 belongs to the GS1 subfamily.…”

“…In contrast, the GS1 s that are primarily expressed in phloem tissue of stems may function in the translocation of the N compounds from roots to leaves, and thus their overexpression result in an increase of NH 4 + and NO 3 - contents in the leaves ( Lothier et al, 2011 ; Molina-Rueda and Kirby, 2015 ). In addition, there are also some GS1 s, which are constitutively expressed in leaves and play a primary role in the synthesis of Gln using NH 4 + that is converted from NO 3 - , and their overexpression causes both NH 4 + and NO 3 - contents decreased in transgenic plants ( Martin et al, 2006 ; Zhu et al, 2014 ). In addition, it is noteworthy that there are also some GS1.2 genes that do not have any effects on N assimilation and metabolism, and growth when they are overexpressed ( Ortega et al, 2001 ; Fei et al, 2003 ).…”

Dual RNA-seq of maize and H. seropedicae ZAE94 association, in different doses of nitrate, reveals novel insights into Plant-PGPB-environment relationship

“…Transgenic maize constitutively overexpressing GLN1-3 , which encodes a cytosolic GS, showed a 30% increase in grain number ( Martin et al., 2006 ), while GS-deficient maize mutants showed a reduction in grain yield ( Cañas et al., 2010 ). Additionally, overexpressing DvGS1 improved growth and N utilization in A. thaliana ( Zhu et al., 2014 ). Furthermore, overexpression of AspAT genes in rice led to altered N metabolism and increased amino acid and protein content in seeds.…”

Identification of candidate genes and residues for improving nitrogen use efficiency in the N-sensitive medicinal plant Panax notoginseng

“…NO 3 - are degraded to NH 4 + , then NH 4 + form amino acid through catalysis by N metabolism-related enzyme, as reflected by nitrite reductase (NiR), glutamine synthetase (GS) and glutamate dehydrogenase (GDH) [ 9 ]. Overexpression of DvGS1/2 and OsGS1 is positively associated with the biomass and NUE in the N-deficient Dunaliella viridis , Arabidopsis thaliana and O. sativa [ 12 – 14 ]. Meanwhile, the enhanced NO 3 - signal-sensing and transduction pathways might promote N uptake and utilization, and thus improve NUE [ 15 ].…”