Sucrose-specific signalling represses translation of the Arabidopsis ATB2 bZIP transcription factor gene.

The Arabidopsis bZIP transcription factor gene ATB2 has been shown previously to be expressed in a light-regulated and tissue-specific way. Here we describe the precise localization of ATB2 expression, using transgenic lines containing an ATB2 promoter-GUS reporter gene construct. The observed expression pattern suggests a role for ATB2 in the control of processes associated with the transport or utilization of metabolites. Remarkably, expression of the ATB2-GUS reporter gene construct was specifically repressed by sucrose. Other sugars, such as glucose and fructose, alone or in combination, were ineffective. Repression was observed at external sucrose concentrations exceeding 25 mM. Transcript levels of both the endogenous ATB2 gene and the ATB2-GUS reporter gene were not repressed by sucrose, suggesting that sucrose affects mRNA translation. This translational regulation involves the ATB2 leader sequence because deletion of the leader resulted in loss of sucrose repression. Our results provide evidence for a sucrose-specific sugar sensing and signalling system in plants.

Inducible expression of heterologous genes targeted to a chromosomal platform in the cyanobacterium Synechococcus sp. PCC 7942.

High-level, inducible expression of heterologous genes in the cyanobacterium Synechococcus sp. strain PCC 7942 was obtained using the Escherichia coli trc promoter and lacI repressor. The petE gene of Anabaena sp. strain PCC 7937 encoding plastocyanin precursor protein and the E. coli uidA gene encoding beta-glucuronidase were initially placed under the control of the trc promoter and lacI repressor by cloning into the E. coli pTrc99C expression vector and were introduced into the chromosomal platform for integration in metF (PIM) of the Synechococcus R2-PIM9 recipient strain. These pTrc99C-derived constructs often gave rise to transformants that did not contain a complete insert gene, probably because of gene conversion events. Selection of the desired Synechococcus R2-PIM9 transformants was vastly improved using the new pTrcIS vector that contains the aadA gene encoding streptomycin resistance as an extra antibiotic resistance marker. The influence of IPTG concentration and induction time on gene expression with the E. coli trc/lacI system in Synechococcus was determined using beta-glucuronidase as a reporter. The Anabaena PCC 7937 petE gene in Synechococcus was expressed to a high level upon induction with IPTG as shown by RNA and immunoblot analysis. The general usability of pTrcIS as a cloning vector for inducible heterologous gene expression in Synechococcus was confirmed by the introduction of several more genes.

Expression of Anabaena PCC 7937 plastocyanin in Synechococcus PCC 7942 enhances photosynthetic electron transfer and alters the electron distribution between photosystem I and cytochrome-c oxidase.

The petE gene encoding plastocyanin precursor protein from the cyanobacterium Anabaena PCC 7937 was introduced in the cyanobacterial host strain Synechococcus PCC 7942. The host normally only uses cytochrome c553 as Photosystem I (PS I) donor. The heterologous gene was efficiently expressed using the inducible Escherichia coli trc promoter. Accumulation of plastocyanin protein depended on the presence of Cu2+. The protein was accurately targeted to the thylakoid lumen, from which it could be isolated in the mature form. Redox difference spectroscopy proved the presence of a Cu2+ ion in the holoenzyme. Isolated heterologous plastocyanin was functional in reconstitution of in vitro electron transfer to PS I. The presence of Anabaena plastocyanin in Synechococcus thylakoid membranes increased PS I electron transfer rate 2.5 times. Analysis of P700 redox and PS II fluorescence transients in vivo showed a faster electron transfer through PS I because of enhanced electron supply in the presence of plastocyanin. In addition, the distribution of electrons between photosynthetic and respiratory electron transfer changed. Plastocyanin preferentially donates electrons to PS I rather than to the respiratory cytochrome-c oxidase complex and is not functionally equivalent to cytochrome c553.

Iron-dependent protection of the Synechococcus ferredoxin I transcript against nucleolytic degradation requires cis-regulatory sequences in the 5' part of the messenger RNA.

We have previously reported that the ferredoxin I gene from Synechococcus sp. PCC 7942 is regulated by iron at the level of differential mRNA stability. To identify iron-responsive elements in the Synechococcus ferredoxin transcript, we have tested chimaeric constructs containing translational fusions between the Synechococcus and the Anabaena sp. PCC 7937 ferredoxin genes for iron-dependent expression in transgenic Synechococcus strains. This strategy was based on the observation that the level of the Anabaena ferredoxin mRNA did not increase upon iron addition in Synechococcus. Our results show that the presence of the first 207 nucleotides of the Synechococcus ferredoxin transcript is sufficient to confer iron responsiveness to the chimaeric transcripts. This iron responsiveness was accomplished by an increased stability of the chimaeric transcript in the presence of iron, as was found for the intact Synechococcus ferredoxin gene. Addition of the translation inhibitor chloramphenicol to the cultures led to a rapid stabilization, in low- and high-iron conditions, of the wild-type Synechococcus ferredoxin transcript as well as all chimaeric ferredoxin transcripts tested. These results suggest the existence of a constitutively expressed nuclease capable of degrading the ferredoxin transcripts. They further support the suggestion that the first 207 nucleotides of the Synechococcus transcript contain a specific sequence that is recognized by an iron-responsive factor and that this interaction leads to protection against degradation.

Iron-dependent stability of the ferredoxin I transcripts from the cyanobacterial strains Synechococcus species PCC 7942 and Anabaena species PCC 7937.

The effect of iron on ferredoxin I specific mRNA levels was studied in the cyanobacterial strains Synechococcus sp. PCC 7942 (Anacystis nidulans R2) and Anabaena sp. PCC 7937 (Anabaena variabilis ATCC 29413). In both strains addition of iron to iron-limited cells resulted in a rapid increase in ferredoxin mRNA levels. To investigate the possible role of the ferredoxin promoter in iron regulation, a vector for promoter analysis in Synechococcus PCC 7942 strain R2-PIM9 was constructed, which contains the ferredoxin promoter fused to the gene encoding beta-glucuronidase (GUS) as reporter. Neither the Synechococcus nor the Anabaena ferredoxin promoter was able to direct iron-regulated GUS activity in Synechococcus R2-PIM9, indicating that transcription initiation is not responsible for the iron-dependent ferredoxin mRNA levels. Determination of the half-life of the ferredoxin transcript in iron-supplemented and iron-limited cells revealed that, in both strains, the ferredoxin transcript is much more stable in iron-supplemented cells than in iron-limited cells. These results lead to the conclusion that in these strains, iron-regulated expression of the ferredoxin I gene is mediated via differential mRNA stability.

Transcriptional regulation of the plastocyanin and cytochrome c553 genes from the cyanobacterium Anabaena species PCC 7937.

The effect of copper on the levels of plastocyanin (PC) and cytochrome c553 (cyt-c)-specific transcripts from Anabaena sp. PCC 7937 was investigated. The addition of copper resulted in a marked increase in PC mRNA levels, and a decrease in cyt c mRNA levels. Thus the functional exchange between PC and cyt c seems to be regulated at the mRNA level. The copper-dependent increase in PC and decrease in cyt c mRNA levels was abolished when chloramphenicol was added to the cells. This suggests that de novo synthesis of at least one trans-acting element is required to regulate PC and cyt c mRNA levels. Both PC and cyt c mRNA stability was found to be unaltered under varying Cu2+ regimes. This leads to the conclusion that expression of both genes is regulated at the level of initiation of transcription.

Identification of replication and stability functions in the complete nucleotide sequence of plasmid pUH24 from the cyanobacterium Synechococcus sp. PCC 7942.

The complete nucleotide sequence is presented for pUH24, the small plasmid of Synechococcus sp. PCC 7942. pUH24 consists of 7835bp and has a G + C content of 59%. The distribution of translation start and stop codons in the sequence allows 36 open reading frames that potentially encode polypeptides of 50 or more amino acids. We postulate that eight of these open reading frames are actual coding sequences. A region has been identified, by experiment, that contains two functions, designated pmaA and pmaB, involved in the segregational stability of the plasmid. The minimal region of pUH24 fully capable of supporting autonomous replication consists of a 3.6kb DNA fragment, which is almost entirely occupied by two overlapping genes most likely coding for essential replication proteins (repA and repB).

Genomic integration system based on pBR322 sequences for the cyanobacterium Synechococcus sp. PCC7942: transfer of genes encoding plastocyanin and ferredoxin.

Synechococcus sp. PCC7942 recipient strains were constructed for the chromosomal integration of DNA fragments cloned in any pBR322-derived vector, which carries the ampicillin resistance (ApR) marker. The construction was based on the incorporation of specific recombination targets, the so-called 'integration platforms', into the chromosomal metF gene. These platforms consist of an incomplete bla gene (ApS) and the pBR322 ori separated from each other by a gene encoding an antibiotic (streptomycin or kanamycin) resistance (SmR or KmR). Recombination between a pBR322-derived donor plasmid and such a chromosomal platform results with high frequency in restoration of the bla gene and replacement of the chromosomal marker (SmR or KmR) by the insert of the donor plasmid. The integration into the platform depends on recombination between pBR322 ori and bla sequences only and is therefore independent of the DNA insert to be transferred. The desired recombinants are found by selection for a functional bla gene (ApR) and subsequent screening for absence of the chromosomal antibiotic marker. Gene transfer with this integration system was found to occur efficiently and reliably. Furthermore, the presence of the pBR322 ori in the platform allowed for 'plasmid rescue' of integrated sequences. The system was applied successfully for the transfer of the gene encoding plastocyanin (petE1) from Anabaena sp. PCC7937 and for the integration of an extra copy of the gene encoding ferredoxin I (petF1) from Synechococcus sp. PCC7942 itself.

Genes encoding ferredoxins from Anabaena sp. PCC 7937 and Synechococcus sp. PCC 7942: structure and regulation.

The gene encoding ferredoxin I (petF1) from the filamentous cyanobacterium Anabaena sp. PCC 7937 (Anabaena variabilis ATCC 29413) was cloned by low stringency hybridization with the ferredoxin cDNA from the higher plant Silene pratensis. The petF1 gene from the unicellular cyanobacterium Synechococcus sp. PCC 7942 (Anacystis nidulans R2) was cloned by low stringency hybridization with the petF1 gene from Anabaena sp. PCC 7937. One copy of the petF genes was detected in both organisms, and a single transcript of about 630 b was found for Synechococcus sp. PCC 7942. Both the Synechococcus sp. PCC 7942 and the Anabaena sp. PCC 7937 petF1 genes contain a 297 bp open reading frame coding for a small acidic protein, consisting of 98 amino-acid residues, with a molecular mass of about 10.5 kDa.The ferredoxin content of Synechococcus sp. PCC 7942 is strongly reduced under ironlimited growth conditions. The slight decrease in the amount of ferredoxin transcript found under iron limitation does not account for the more severe reduction in ferredoxin protein observed. The main regulation of the ferredoxin content probably is effected at the level of translation and/or degradation. Although ferredoxin expression can be strongly reduced by iron stress, the ferredoxin function seems to be indispensable, as Synechococcus sp. PCC 7942 appeared refractory to yield mutants lacking the petF1 gene.