The identification of new cytosolic glutamine synthetase and asparagine synthetase genes in barley (Hordeum vulgare L.), and their expression during leaf senescence.

Glutamine synthetase and asparagine synthetase are two master enzymes involved in ammonium assimilation in plants. Their roles in nitrogen remobilization and nitrogen use efficiency have been proposed. In this report, the genes coding for the cytosolic glutamine synthetases (HvGS1) and asparagine synthetases (HvASN) in barley were identified. In addition to the three HvGS1 and two HvASN sequences previously reported, two prokaryotic-like HvGS1 and three HvASN cDNA sequences were identified. Gene structures were then characterized, obtaining full genomic sequences. The response of the five HvGS1 and five HvASN genes to leaf senescence was then studied. Developmental senescence was studied using primary and flag leaves. Dark-exposure or low-nitrate conditions were also used to trigger stress-induced senescence. Well-known senescence markers such as the chlorophyll and Rubisco contents were monitored in order to characterize senescence levels in the different leaves. The three eukaryotic-like HvGS1_1, HvGS1_2, and HvGS1_3 sequences showed the typical senescence-induced reduction in gene expression described in many plant species. By contrast, the two prokaryotic-like HvGS1_4 and HvGS1_5 sequences were repressed by leaf senescence, similar to the HvGS2 gene, which encodes the chloroplast glutamine synthetase isoenzyme. There was a greater contrast in the responses of the five HvASN and this suggested that these genes are needed for N remobilization in senescing leaves only when plants are well fertilized with nitrate. Responses of the HvASN sequences to dark-induced senescence showed that there are two categories of asparagine synthetases, one induced in the dark and the other repressed by the same conditions.

Analysis of gene expression in amyloplasts of potato tubers.

Gene expression in amyloplasts derived from potato tubers was analyzed at the levels of transcription, mRNA accumulation and formation of polysomes. Compared with chloroplasts, overall transcriptional activity is considerably reduced in amyloplasts. Nevertheless, several transcripts are synthesized in amyloplasts during growth of tubers. Among the transcribed amyloplast genes are the ribosomal operon and the psbA gene. Primer extension analysis provided evidence that in amyloplasts the plastid encoded RNA polymerase (PEP) is the principal RNA polymerase involved in the transcription of the rrn operon. Analysis of plastid steady state transcripts showed that there are only small differences in the levels of specific transcripts between amyloplasts and chloroplasts. With respect to the low transcription rate of the accumulating RNA-species in amyloplasts, a high stability of these transcripts is obvious. Though amyloplasts possess polysomes, specific mRNAs associated with such polysomes could not be detected. This suggests that translation could be impaired in amyloplasts, which, in turn, implies that these organelles are not suitable targets for the expression of transgenes introduced into the plastid genome by plastid transformation.