Functional expression of two pine glutamine synthetase genes in bacteria reveals that they encode cytosolic holoenzymes with different molecular and catalytic properties

Two glutamine synthetase isogenes, GS1a and GS1b, isolated from pine have been functionally expressed in E. coli and the characteristics of individual gene products compared. When bacteria were grown at 37 degrees C most pine GS1 protein was found in the insoluble fraction but lowering of the expression temperature increased yield of both GS1 polypeptide and activity in the soluble fraction. High levels of functionally active GS1a (309 + or - 35 nkat mg(-1)) and GS1b (1,166 + or - 65 nkat mg(-1)) enzymes were obtained by decreasing the expression temperature to 10 degrees C. Purification and characterization of recombinant products showed that pine GS1 polypeptides are assembled in octameric GS holoenzymes showing structural and kinetic differences. The results are discussed with regard to the specific localization of GS1a and GS1b in different cell types of pine seedlings. The isoform GS1a may control the assimilation of the high levels of ammonium released in photosynthetic tissues, whereas GS1b enzyme could mitigate oscillations in glutamate availability providing a constant flux of glutamine for nitrogen transport in vascular cells.     

Differences in structure of adventitious roots in Salix clones with contrasting characteristics of cadmium accumulation and sensitivity

Glutamine synthetase (GS) genes, GS1a and GS1b, in pine ( Pinus sylvestris L.) are differentially regulated in tissue specificity and during seedling development. To gain insight into the regulatory mechanisms controlling their expression, we have analysed the 5'-flanking sequences of the gene GS1a using a transient expression system in pine protoplasts. Structural analysis of this region revealed the presence of putative regulatory elements including two AT-rich elements and a poly CT consensus sequence. A series of 5'- and 3'-deletions of the untranslated region covering the three putative elements, −800 to −626, −626 to −427 and +118 to +177 were analysed to demonstrate the functional implications of these elements in gene regulation. An electrophoretic mobility-shift assay showed that nuclear proteins prepared from pine cotyledons interact with both AT-rich regions (−800 to −427). Interestingly, no protein binding was detected when the untranslated region (+118 to +177) was included, even if deletion of that region suppressed promoter activity in the transient expression experiments conducted. However, simultaneous deletion of both types of cis elements, A/T and CT, resulted in a recovery of promoter activity of 50%. These results suggest a key regulatory role of the CT box by the interaction with A/T stretches in the distal part of the promoter and possibly with the proximal region (−427 to −1).     

植物谷氨酰胺合成酶研究进展及其应用前景

氮素是制约作物产量的主要营养元素之一,谷氨酰胺合成酶(Glutamine synthase,GS;EC 6.3.1.2)是氮素代谢途径中的关键酶。目前,拟南芥、水稻、小麦和玉米等植物中的GS成员均已被分离鉴定。研究表明,超表达GS能够提高植物对氮素的利用效率,从而在植株的生长发育特别是产量形成过程中发挥重要作用,但是其功能在不同植物上并不完全一致,可能与GS基因受到转录和翻译后等水平的调控有关。以下综述了植物GS基因分类、QTL定位、对氮素代谢响应、组织表达特异性、生物学功能及其分子调控机制等方面的研究进展,并展望了植物GS基因的应用前景,以期为利用GS基因来提高植物氮素利用效率提供具有参考价值的信息。     

Characterization of transgenic poplar with ectopic expression of pine cytosolic glutamine synthetase under conditions of varying nitrogen availability

The present study addresses the hypothesis that enhanced expression of glutamine synthetase (GS) in transgenic poplar, characterized by the ectopic expression of pine cytosolic GS, results in an enhanced efficiency of nitrogen (N) assimilation and enhanced growth. Transgenic and control poplar were supplied with low and high N levels and the role of ectopic expression of the pine GS in growth and N assimilation was assessed by using amino acid analysis, (15)N enrichment, biochemical analyses, and growth measurements. While leaves of transgenic poplar contained 85% less (P < 0.01) free ammonium than leaves of nontransgenic control plants, leaves of transgenics showed increases in the levels of free glutamine and total free amino acids. Transgenic poplar lines also displayed significant increases in growth parameters when compared with controls grown under both low (0.3 mm) and high (10 mm) nitrate conditions. Furthermore, (15)N-enrichment experiments showed that 27% more (P < 0.05) (15)N was incorporated into structural compounds in transgenic lines than in nontransgenic controls. Using the methods described here, we present direct evidence for increased N assimilation efficiency and growth in GS transgenic lines.