Repercussion of mesophyll-specific overexpression of a soybean cytosolic glutamine synthetase gene in alfalfa (Medicago sativa L.) and tobacco (Nicotiana tabaccum L.)
Introduction
Alfalfa (Medicago sativa) is one of the most widely grown forage crop. Although closely associated with dairy production, many other livestock industries rely extensively on alfalfa as an important source of nutrition, and as such merits efforts to improve yields [1], [2]. A factor limiting crop yields and plant growth is the availability of the macronutrient nitrogen (N). Plants primarily acquire N from the soil in the form of nitrate (NO3−) which is sequentially reduced to ammonium (NH4+) in the leaves by the enzymes, nitrate reductase and nitrite reductase. Alfalfa and other legumes can also form symbiotic relationships with nitrogen fixing bacteria that reduce atmospheric dinitrogen (N2) to NH4+ in exchange for carbon (C) and energy [3]. While primary metabolism plays an important role in N nutrition, NH4+ can also be released by various secondary metabolic reactions within the plant such as photorespiration, protein turnover and synthesis of phenylpropanoids [3], [4].
The NH4+ taken up or released by the above processes is assimilated by glutamine synthetase (GS; EC 6.3.1.2), which catalyzes the ATP dependent condensation of NH4+ and glutamate (Glu) to yield glutamine (Gln). This is the first step in the incorporation of inorganic N into an organic N compound usable by plants. Glutamine synthetase is, therefore, the key enzyme of N assimilation [5]. Based on electron microscopic observations, plant GS, was originally thought to be an octamer, however, more recently GS has been shown to be decameric [6] with a native molecular weight of 440 kDa. Glutamine synthetase occurs as a number of isoenzyme forms and is present in either the cytosol (GS1) or chloroplast/plastid (GS2). GS1 isoforms are usually encoded by multigene families [7], [8]. Most of the terrestrial plants have single genes encoding GS2 [9]. However, some species may have multiple genes for GS2 [7], [10]. The two isoforms are differentially expressed and regulated throughout the growth and developmental stages in a plant's life cycle [4], [7], [8]. Both GS isoforms play important roles in N metabolism. The predominant isoform in the leaves is GS2, which functions in the assimilation of NH4+ released from the reduction of NO3− and the re-assimilation of photorespiratory NH4+ [9], [11]. Cytosolic glutamine synthetase is expressed primarily in the roots and nodules. In the roots, GS1 functions to assimilate NH4+ directly taken up from the soil or produced from the reduction of NO3−, while in the nodules, GS1 assimilates NH4+ produced by N2 fixation [12], [13], [14]. In addition, GS1 plays an important role in the remobilization of N during senescence, herbicide treatments, bacterial infections, and water stress [8], [15], [16]. The exclusive location of GS1 in the vasculature of shoots and leaves, also suggests that GS1 may be involved in the synthesis of Gln for transport [8], [17]. While there is ample evidence for transcriptional regulation of GS genes, there is also evidence for regulation at the level of transcript stability and translation initiation [18], posttranslational modification of the protein [19], [20], [21] and at the level of enzyme stability [22].
The coincidence of the positions of the QTLs for yield components and the genes for cytosolic GS in maize, rice, wheat and Arabidopsis genomes suggest that GS1 may represent a key component of nitrogen use efficiency and yield [23], [24], [25], [26]. This has prompted us and others in the field, to make attempts to alter GS1 activity in plants with the purpose of enhancing nitrogen use efficiency and plant performance [27], [28], [29], [30]. Some of these transformants showed an increase in GS1 polypeptide and GS activity and an improvement in plant growth. For example, Lotus japonicus transformants with an alfalfa GS1 gene (MsGS100) driven by the constitutive cauliflower mosaic virus (CaMV35S) promoter exhibited an increase in total protein and chlorophyll content, as well as an increase in total amino acid content in the leaves and stems [27]. Due to the significant increase in the level of Asn/Asp in the stem of the transformants, the authors attributed the increase in plant growth seen in the L. japonicus transformants to improved transport of assimilated NH4+. Other authors, however, have attributed improved performance in GS1 overexpressing plants to the presence of GS1 in the photosynthetic cells, functioning to enhance re-assimilation of NH4+ released during photorespiration and/or senescence [28], [29], [30], [31]. The levels of NH4+ released during photorespiration are very high and re-assimilation of this NH4+ is crucial for nitrogen-use efficiency [32], [33]. Moreover, as the leaves age, the roles of the two GS isoforms switch and GS1 becomes the predominant isoform which functions in the remobilization of N during senescence [8], [15], [34]. With the high levels of NH4+ produced in the leaf during these processes, increased levels of GS1 in the mesophyll cells may improve plant performance by re-assimilating NH4+ that may normally be lost.
The objective of this paper is to determine how alfalfa will respond to mesophyll-specific overexpression in comparison to constitutive overexpression of GS1. We have also included tobacco in this study to determine how alfalfa compares to tobacco with regards to overexpression of GS1. We have used the soybean GS1 (Gmglnβ1) gene as the transgene and since previous studies showed that the 3′UTR of the Gmglnβ1 gene plays a key role in regulating both transcript stability and translation initiation [18], we have used the Gmglnβ1 gene without its 3′UTR as the GS1 transgene. By taking a molecular, biochemical and physiological approach in the analysis of the two classes of transformants, this study casts light on how post-translational regulatory events affect the expression of the GS1 transgene and its eventual repercussions on physiological parameters.
Section snippets
Gene constructs
Standard procedures were used for all recombinant DNA manipulations [35]. The alfalfa RUB promoter was isolated from a pGEM-T Easy vector (Promega, Milwaukee, WI) as a 5′ Xba I and 3′ Bam HI fragment [36] and directionally cloned into the pBI101 vector (Clontech BD Biosciences, San Jose, CA; NCBI accession no. U12639) in front of the E. coli GUS coding region (uidA) producing the RUB-uidA-NOS3′ gene construct. Cloning and isolation of the Glycine max (soybean) Gmglnβ1 cDNA (NCBI accession no. AF301590
RuBisco promoter is active in the photosynthetic cells while the CaMV35S promoter is active in the mesophyll cells and the vascular bundles of both the leaves and the stem
The major objective of this paper is to determine how alfalfa plants will respond to mesophyll-specific overexpression in comparison to constitutive overexpression of GS1. To accomplish overexpression of GS1 in a mesophyll specific manner, we used the RuBisco SSU (RUB) promoter from alfalfa [36]. This promoter, along with the cauliflower mosaic virus 35S (CaMV35S) constitutive promoter, was placed in front of the GUS (uidA) coding region (Fig. 1A). The two constructs were introduced into
Discussion
Genetic approaches have identified the co-localization of QTLs for GS1 and QTLs for yield parameters in several plant species [23], [24], [25], [26], and since GS plays such a central role in N assimilation, it is logical to postulate that enhanced GS activity would lead to improved plant performance. However, the results obtained with the overexpression of GS1 genes in different plants has not been consistent [27], [28], [29], [37], [40], [41], [42], [43], [44]. This inconsistency can be
Acknowledgements
This work was supported by the National Institutes of Health (Grant Nos. GMO-8136-26, GMO-61222 and GMO-7667-25) and by the Agricultural Experiment Station at New Mexico State University. We would also like to thank the NSF program AGEP for fellowship support.
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