| Glutamine synthetase (GS; EC 6.3.1.2) plays fundamental roles in plant N assimilation and is regarded as the'hinge'over which inorganic N is converted into its organic form. GS isoenzymes have demonstrated central effects to the enhancement of plant N use efficiency. According to their localization within the cell, GS isoenzymes in higher plants are classified into 2 groups, including GS1, the cytosolic forms that localize in cytosol responsible for assimilation of the NH4+ directly uptake from soil by roots and for re-assimilation of the NH4+ released from various metabolic pathways, and GS2, the plastid forms that function in cell plastids for incorporation of the NH4+ reduced from NO3- and that released from photorespiration process into glutamine.Not only the growth and development but also the ultimate yield and fruit quality of melons were reported significantly regulated by supplementation of N nutrition. However, our knowledge of N assimilation processes in melons is still very limited, and previous studies were basically contrained at the levels of plant physiology and yield and fruit quality, and reports on isolation and characterization of GS genes, the key player of plant N metabolism, in melons are not documented. With the aim of gaining new insights in this area, especially at molecular levels, in this study, we cloned and characterized the first cytosolic GS gene from melon, M-GS1 (GenBank accession No.: DQ851867), and comparatively analyzed at molecular levels the genomic copy number, gene products localizations within the cell, the GS activities of the corresponding recombinant proteins expressed and purified from E. coli, and the expression patterns of M-GS1 and M-GS2 (GenBank accession No.: AY773090), a GS2 gene previously cloned in melon by our group regulated, by various forms of N nutrients, and in addition, investigated at plant level the effects of M-GS1 over-expression in improving the nitrogen use efficiency of the transgenic Arabidopsis lines. The main results were indicated as follows: 1. The cloning and bioinformatics'analyses of M-GS1RACE-PCR techniques were employed to successfully clone the first GS1 gene from melon(Cucumis melo L. var. reticulatus Naud.) M-GS1 (GenBank accession No.: DQ851867). The full-length cDNA of M-GS1 contains 1494 nucleotides with an open reading frame (ORF) of 1068 nucleotides. The deduced 356 amino acid sequence showed high similarity with previously reported GS1 isoenzymes from various plant species. Sequence analysis revealed that the predicted protein contains a GS beta-Grasp domain, a GS catalytic domain, and the main conserved motifs characteristic of a plant GS1. The phylogenetic analysis displayed that M-GS1 is related most closely to the GS1 from Datisca glomerata evolutionarily. The predicted 3-D structure for M-GS1 indicated that it conforms structurally to the X-ray crystallogram of a typical GS subunit and requires the involvement of bi-valent metal ions including Mn2+ or Mg+, AMP and citric acid to fold into its active conformations.2. The genomic copy number of M-GS1 and M-GS2 in melonAs shown by Southern blot analysis, similarly but relatively simpler to the results found in other plants, melon genome contains 2-3 copies of M-GS1, the cytosol GS genes, but one copy of M-GS2, the chloroplastic GS gene.3. The sub-cellular localization analyses of M-GS1 and M-GS2In this experiment, we constructed two recombinant plant expression plasmids, in which both M-GS1 or M-GS2 was fused with a fluorescence protein YFP, the reporter gene, and the fusion was driven by a CamV 35S promoter. The recombinant plant expression plasmids were then transferred into the onion epidermal cells via the bombardment using a gene gun, and expressed transiently. Fluorescence emissing from the expressed products localizing in different cell organelles was visualized and analyzed using laser-scanning confocal microscope techniques. The results showed, that while M-GS1::YFP fusion proteins were localized in the cytosol, M-GS2::YFP fusion proteins were localized in plastids, confirming that both M-GS1 and M-GS2 indeed bears the sub-cellular localization characteristics of the corresponding GS group that they were assigned to as a member, respectively.4. M-GS1 and M-GS2 expression in E. coli and GS activity related analyses of the purified recombinant proteinsTo characterize the biochemical properties of M-GS1 and M-GS2, we constructed 3 recombinant expression plasmids to express the coding sequences of either M-GS1, M-GS2, or M-GS2 minus cTP(M-GS2-cTP) sequences in E. coli., respectively. As shown by SDS-PAGE and biochemical analyses, all the 3 recombinant proteins, purified using Ni-NTA affinity chromatography, were properly expressed, but only M-GS1 and M-GS2-cTP were found having GS activities. Activity analysis showed that M-GS1 has evidently higher affinity for NH4+ and higher synthetase and transferase activity than M-GS2-cTP but is less resistant to heat, and Mg2+ was found playing a significant role in enhancing the GS activities of both recombinant proteins. These results provided further evidences that the putative M-GS1 and M-GS2 genes cloned in melon were indeed GS genes that express GS isoenzymes of differential biochemical properties.5. The regulation of M-GS1 and M-GS2 expression by various forms of N nutrients in fertilizationReal-time quantitative PCR was performed to investigate the potential differential regulations of M-GS2 and M-GS1 expression by various forms of N nutrients in fertilization. Results showed that distinct forms of nitrogen (N) found in fertilizers transcriptionally regulated M-GS2 differently. Ammonium and nitrate feeding only significantly regulated M-GS2 transcripts in leaf; starving (0.75 mM) or moderate (3.75 mM) N levels dramatically increased M-GS2 transcripts for 1 day, decreasing to a constant low level after 2-3 days, while sufficient N level (7.5 mM) had a minor effect throughout 3 days compared to controls. Glutamate feeding, however, not only significantly regulated M-GS2 transcripts in leaf (decreased initially then increased to higher levels than controls), but also in root, where it was up-regulated continuously. M-GS1 was expressed in all plant tissues without evident tissue specificity, but with differential patterns when the melon plants were fed in hydroponic culture with different forms of N nutrient at differential N concentrations: ammonium dramatically enhanced the levels of M-GS1 transcripts in all tested tissues, while nitrate stimulated M-GS1 transcription only in the roots and leaves, but not in the stems; Glu (glutamate), however, depressed M-GS1 transcripts in the roots, but resulted in no significant change to the levels of M-GS1 transcripts in the stems and leaves. These results suggest that to enable sustained N assimilation under different conditions of N availability melon responded by differentially regulating the expression of GS genes into GS isoenzymes of differential physiological functions.6. The N related effects of M-GS1 over-expression on transgenic Arabidopsis linesThe coding sequence of M-GS1 cDNA was ligated properly with plant expression vector pEZT-NL to construct a plant expression vector driven by CAM 35S promoters that expresses a fusion protein M-GS1::EGFP. This expression vector was transferred into Arabidopsis via Agrobacterium tumefaciens EHA105 using the'Floral-dip'method. Ten independent transgenic plant lines were obtained through PPT screening and DNA PCR verification. RT-PCR analysis showed M-GS1 expression were detected in all independent transgenic lines. As shown in N-level-dependant growth experiments, the effects of M-GS1 over-expression were evident only when N was supplied at low levels. As indicated by biochemical and growth indicators, all transgenic lines displayed evidently higher rosette total GS activity, total soluble protein content and biomass than wild-type plants under low N supply. These results suggested that M-GS1 over-expression enabled the transgenic plants to sustain relatively higher levels of N assimilation and growth under N limited conditions than wild-type plants, and has the potentials in improving plant N use efficiency. |
