Both phyA and phyB mediate light-imposed repression of PHYA gene expression in Arabidopsis.

The negatively photoregulated PHYA gene has a complex promoter structure in Arabidopsis, with three active transcription start sites. To identify the photoreceptors responsible for regulation of this gene, and to assess the relative roles of the three transcription start sites, we analyzed the changes in PHYA transcript levels in wild-type and photoreceptor mutant seedlings under various irradiation conditions. Continuous far-red or red light exposures each induced a significant decline in transcript levels in wild-type etiolated seedlings. Analysis of mutants specifically lacking either phyA or phyB protein demonstrated that these phytochromes are required for the negative regulation induced by far-red and red light, respectively. Ribonuclease protection experiments showed further that this negative regulation is confined almost exclusively to the shortest, most abundant PHYA transcript, and occurs predominantly in shoots. By contrast, both of the other minor transcripts in shoots, and all three transcripts in roots, exhibit near constitutive expression. This complex expression pattern indicates that the PHYA gene is subject to regulation by multiple signals, including environmental, developmental, and organ-specific signals.

Expression analysis of a cytosolic glutamine synthetase gene in cotyledons of Scots pine seedlings: developmental, light regulation and spatial distribution of specific transcripts.

The expression of a cytosolic glutamine synthetase (GS1; EC 6.3.1.2) gene was examined in cotyledons of Scots pine seedlings. Light strongly stimulated GS1 mRNA accumulation during development. Similarly, steady-state levels of GS1 transcripts increased in dark-grown seedlings transferred to light and decreased in dark-adapted seedlings. Light/dark adaptation affected rbcS and lhcb2 mRNA levels and chlorophyll contents in the same manner. Light-grown seedlings in the presence of the herbicide norflurazon showed a drastic decrease in mRNA for GS and photosynthetic proteins, whereas the effect of the herbicide on mitochondrial beta-ATP synthase mRNA was limited. These results indicate that factors associated with developing chloroplasts could be required for maximal GS1 gene expression during seedling development. The level of GS polypeptide, determined by immunoblot, was up-regulated during seedling development in the light or dark. However, the levels of the polypeptide detected were unaltered by the light/dark adaptation treatments. The analysis of GS1 mRNA association with polysomes indicated that the discrepancies between GS protein and mRNA levels are not a result of a differential translational rate of the transcript in darkness relative to light. Two GS isoproteins with different isoelectric point were resolved by two-dimensional PAGE in light- and dark-germinated plants. The relative abundance of one of them was markedly affected by light and correlated with the observed changes in GS mRNA, suggesting that the other form is not a product derived from the detected transcript. In situ hybridization of cotyledon sections showed the presence of GS1 mRNAs in mesophyll and phloem cells confirming gene expression in photosynthetic tissues. High levels of transcript were detected also in meristematic cells of apical primordia. These data suggest a dual role for the GS1 gene associated with chloroplast development/activity and glutamine biosynthesis for nitrogen mobilization during early growth of Scots pine.

High-level expression of Pinus sylvestris glutamine synthetase in Escherichia coli. Production of polyclonal antibodies against the recombinant protein and expression studies in pine seedlings.

In a previous work we reported the molecular characterization of a glutamine synthetase (GS; EC 6.3.1.2.) complementary DNA from a woody plant (Cantón et al. (1993) Plant Mol. Biol. 22, 819-828). The isolated cDNA (pGSP114) encoding a Scots pine (Pinus sylvestris) cytosolic subunit, has been subcloned into the expression vector pET3c to overproduce the GS polypeptide in Escherichia coli cells. The recombinant GS protein showed the same molecular size as a native Scots pine GS subunit. Antibodies against the pET3c-GSP114 encoded protein were raised in rabbits by injecting purified preparations and specificity was determined by immunoprecipitation of GS activity present in pine crude extracts. In spite of the antibodies were able to recognize both cytosolic and chloroplastic GS in tomato plants, they were unable to immunodetect chloroplastic GS in green cotyledons of pine seedlings and cytosolic GS was the unique recognized polypeptide. Unlike to that found in other plant species, cytosolic GS was strongly expressed in green tissues as determined by protein and Northern analysis. Our results suggest a key role for cytosolic GS in photosynthetic tissues of conifers.

Expression of ferredoxin-dependent glutamate synthase in dark-grown pine seedlings.

Pine seedlings are able to accumulate chlorophylls and develop green plastids in a light-independent manner. In this work, we have characterized ferredoxin-dependent glutamate synthase (EC 1.4.7.1; Fd-GOGAT), a key enzyme in nitrogen interconversion during this process. Fd-GOGAT has been purified about 170-fold from cotyledons of maritime pine (Pinus pinaster). As occurs in angiosperms, the native enzyme is a single polypeptide with an apparent molecular mass of 163-168 kDa that is confined to the chloroplast stroma. Polyclonal antibodies generated against the purified enzyme were used to immunoscreen a lambda gt11 expression library from Scots pine (Pinus sylvestris) seedlings and partial cDNA clones were isolated and characterized. The clone with the longest cDNA insert (pGOP44) contained the codification for the C-terminal (550 amino acids) of the pine Fd-GOGAT polypeptide. Immunological cross-reactivity and comparative amino sequence analysis revealed that Fd-GOGAT is a well conserved protein in higher plants. Western blot analyses showed that protein was expressed in chloroplast-containing pine tissues and this expression pattern was not affected by exogenously supplied nitrogen. Fd-GOGAT mRNA, polypeptide and enzyme activity accumulated in substantial amounts in dark-grown pine seedlings. The presence of a functional Fd-GOGAT may be important to provide the required glutamate for the biosynthesis of nitrogen compounds during chloroplast biogenesis in the dark.

Molecular characterization of a cDNA clone encoding glutamine synthetase from a gymnosperm, Pinus sylvestris.

A full-length cDNA clone (pGSP114) encoding glutamine synthetase was isolated from a lambda gt11 library of the gymnosperm Pinus sylvestris. Nucleotide sequence analysis showed that pGSP114 contains an open reading frame encoding a protein of 357 amino acid residues with a calculated molecular mass of 39.5 kDa. The derived amino acid sequence was more homologous to cytosolic (GS1) (78-82%) than to chloroplastic (GS2) (71-75%) glutamine synthetase in angiosperms. The lack of N-terminal presequence and C-terminal extension which define the primary structure of GS2, also supports that the isolated cDNA encodes cytosolic GS. Southern blot analysis of genomic DNA from P. sylvestris and P. pinaster suggests that GS may be encoded by a small gene family in pine. GS mRNA was more abundant in cotyledons and stems than in roots of both Scots and maritime pines. Western blot analysis in P. sylvestris seedlings showed that only one GS polypeptide, similar in size to GS1 in P. pinaster, could be detected in several different tissues. Our results suggest that cytosolic GS is mainly responsible for glutamine biosynthesis in pine seedlings.

Accumulation of glutamine synthetase during early development of maritime pine (Pinus pinaster) seedlings.

Seedlings of Pinus pinaster Alton accumulated chlorophyll (Chl) when grown in complete darkness. Contents of Chl a and Chl b increased during germination, reaching similar levels in light- and dark-grown plants. Glutamine-synthetase (GS; EC 6.3.1.2) activity was detected in the embryo and its level increased markedly in cotyledons of dark-germinated seedlings. Similar levels of GS activity were observed when the seeds were germinated in the presence of white light. Only one GS form, which eluted at about 0.1 M KCl, was found by ion-exchange chromatography. A predominant GS polypeptide of 43 kDa was detected in cotyledons, and its steady-state level increased with development in a lightindependent fashion. In roots and needles, a related GS polypeptide of 43 kDa was the unique species detectable by western blot analysis. Immunoblots of soluble proteins from isolated chloroplasts showed low abundance of GS protein, indicating that glutamine synthesis in pine cotyledons occurs mainly in the cytosol. Nitrogen-feeding experiments carried out with detached shoots indicated that neither NO 3 (-) nor NH 4 (+) regulate GS levels and the polypeptide pattern. Our results indicate that environmental factors, such as light and nitrogen supply, have a limited role in GS accumulation during pine development.