Cyanobacteria’s resistance to highly salty environments has been triggering great research interest to reveal the mechanism. Some findings suggested that osmotic stress as a main factor could result in decreasing of water content in cytoplasm and an increase in intracelluar salt concentration . In the current study, we identified some up-regulated proteins in ASP-YZ at different salt concentrations by proteomics techniques, suggesting that up-regulation of the protein is a response to high salt environment. ASP-YZ acclimates to high salt environment probably through modulating osmotic regulation, because Glyceraldehyde-3-phosphate dehydrogenase (ORF3807, Table 1), the key enzyme of carbohydrate and amino acid metabolism, is detected. It has reported that generation of more trehalose is required to resist external osmotic pressure to protect the intracellular macromolecule stability . We speculate that salt-stress might result in the signal recognition particle (SRP) to synthesize more glucose, fructose and betaine. The synthesis of these energy substances can maintain intracellular pressure by balancing the osmotic pressure caused by salt-stress.
Under salt-stress conditions, oxygen free radicals in ASP-YZ cells are expected to accumulate rapidly, and are harmful to cells if they are not removed timely . We identified some up-regulated genes, at both protein and transcription levels, (Glutathione synthetase, ORF4938, Table 1) involved in glutathione synthetase in ASP-YZ, suggesting that salt-stress can enhance biosynthesis of intracellular glutathione, which in turn helps cells to clean oxygen free radicals, and to maintain the stability of intracellular environment .
The unsaturated fatty acid in thylakoid membrane of cyanobacteria has been found to play an important role in the process of adaptation to salt stress, by relieving the inhibition of synthesis and activity of Na+/H+ transporter caused by salt-stress . The up-regulation of 3-oxoacyl-[acyl-carrier protein] reductase (ORF1433, Table 1), at both protein and transcription levels, might promote the photosynthesis under salt-stress conditions, which accelerates the active expulsion of Na+ through Na+/H+ transporter and thus decreases ion poisoning [37, 38].
The transcription level of gene ORF4213 encoding signal recognition subunit SRP54 increased by >4-fold, which is in consistent with up-regulation of protein expression detected by 2-DE. The intracellular protein orientation transfer is an important link among protein quantity control, protein recognition and protein transport system, which is mediated by SRP . SRPs are RNA-protein complexes, which can identify existence of the signal peptide in the nascent polypeptide chain and thus can mediate the recognition and transport of the secretory and membrane proteins . Therefore, cell physiological metabolism and lesion are associated with protein targeting transport. It is predictable that expression of some SRP proteins (SRP54, ORF4213, Table 1) in response to salt-stress results from acceleration of the intracellular nascent peptide synthesis and enhancement of identification and transporter activity of the secretory and membrane proteins .
Under salt-stress conditions, many cyanobacteria can induce the expression of salt-stress proteins, in proportional increase to the tolerance to salt . The expression of stress proteins and molecular chaperone Dnak in ASP-YZ were also found to be up-regulated in the present study, making major contributions to promote the correct folding of intracellular protein and the tolerance to salt.
Under salt-stress, the up-regulated enzymes in ASP-YZ include peptidyl-prolylcis-trans isomerase, cyclophilin type (encoded by ORF2281), diaminopimelate epimerase (encoded by ORF4030), enoyl-[acyl-carrier protein] reductase (ORF5055), transcriptional regulator, abrB family (ORF1989) and FAD-dependent pyridine nucleotide-disulphide oxidoreductase (ORF3277). Of the above 5 genes, up-regulation of 4 genes (ORF-2281, –5055, –1989 and −3277) was observed at transcription level, while down-regulation of them at protein level. Obviously, the salt-tolerant mechanism of ASP-YZ is not solely mediated through up or down-regulation of genes, at transcription or translation level, in one metabolite or one metabolic pathway. It should be a perplexing result of the cross-regulation of many physiological, biochemical and metabolic pathways.
It is worthy to note that each protein spot in gel does not necessarily accord to one kind of protein. The above phenomenon lies in: (1) the same gene has different expression products, (2) the same protein can form different spots due to different structural modification, and (3) the same protein can produce multiple protein fragments due to degradation. Overall, the above factors can lead to several protein spots attributed to the same protein (the same DNA Open Read Frame). Nevertheless, the protein spot changes in 2-DE images imply the change of protein (subunits), revealing that this kind of change (gene expression diversity and protein post-translational modification) is unique advantage of 2-DE and proteomic techniques.
Some ASP-YZ genes, showing inconsistency between transcription and translation level, do not account for their independence on salt tolerance, and perhaps their major roles are regulatory effects including transcriptional regulation, differential processing of RNA transcript and differential translation of mRNA [43, 44]. Regulation of gene transcription and translation could be controlled by activation and transformation of gene structure, initiation of transcription, post-transcriptional processing and transport, mRNA degradation, translation and post-translational processing and protein degradation. The degradation of mRNA transcripts is an important reason to cause the inconsistency. Some inducible gene transcripts could be degraded immediately after translation and even in the course of translation . The inconsistency between transcriptional and translational levels is influenced by many factors, and thus the further verification is required to elucidate its mechanisms.
Additionally, we detected that seven genes with unassigned functions was up-regulated, at transcription level, to a remarkably high extent, for instance, up-regulated by 9-fold for ORF1198, 1×104-fold ORF3023, 2×104-fold for ORF5516, 15×104-fold for ORF273, 42×104-fold for ORF1251, and 2586×104 for ORF624 genes, respectively. It is of great importance to predict the role of these unknown candidate genes induced by salt-stress. The expression of some genes with unknown function was found to be down-regulated obviously, including ORF3277, ORF2281, ORF5055, ORF1251, ORF5516 and ORF3023. Therefore, it is necessary to study extensively the expression regulation mechanism of these genes at the transcriptional level under salt-stress conditions.
In a conclusion, we identified some interesting genes in response to salt-stress by proteomic analysis. To elucidate the salt-tolerant mechanism in ASP-YZ, the data in the present study will promote us to investigate further the expression regulation of these target genes.