Plant Chloroplast Genes Research Articles

Evidence from simulation studies for selective constraints on the codon usage of the Angiosperm psbA gene.

The codon usage of the Angiosperm psbA gene is atypical for flowering plant chloroplast genes but similar to the codon usage observed in highly expressed plastid genes from some other Plantae, particularly Chlorobionta, lineages. The pattern of codon bias in these genes is suggestive of selection for a set of translationally optimal codons but the degree of bias towards these optimal codons is much weaker in the flowering plant psbA gene than in high expression plastid genes from lineages such as certain green algal groups. Two scenarios have been proposed to explain these observations. One is that the flowering plant psbA gene is currently under weak selective constraints for translation efficiency, the other is that there are no current selective constraints and we are observing the remnants of an ancestral codon adaptation that is decaying under mutational pressure. We test these two models using simulations studies that incorporate the context-dependent mutational properties of plant chloroplast DNA. We first reconstruct ancestral sequences and then simulate their evolution in the absence of selection on codon usage by using mutation dynamics estimated from intergenic regions. The results show that psbA has a significantly higher level of codon adaptation than expected while other chloroplast genes are within the range predicted by the simulations. These results suggest that there have been selective constraints on the codon usage of the flowering plant psbA gene during Angiosperm evolution.

Host Plant Signal Persistence in the Gut of the Brown Marmorated Stink Bug (Hemiptera: Pentatomidae).

Determining the host range of an invasive insect in a new environment is a key step in the development of management strategies. As the brown marmorated stink bug, Halyomorpha halys Stål, expands into agricultural regions of North America, efforts to elucidate its dietary habits on a landscape scale rely on intensive sampling of potential host plants. Although this approach yields useful information, results can be biased toward common and easily sampled plant species; important hosts can be missed if sampling them is impractical or limited in scope. Here we lay the groundwork for the application of gut content analysis to the feeding ecology of H. halys by investigating the persistence of host plant DNA in the digestive tracts of insects with known feeding histories. Adult H. halys were fed bean seedlings (Phaseolus lunatus L.) for 7 d, followed by a forced host switch to carrot (Daucus carota L.). Insect guts were dissected out at 0, 1, 3, 7, and 14 d following the switch, and host plant chloroplast genes (trnF and trnL) were amplified via polymerase chain reaction. Amplicons were identified using high-throughput sequencing and analyzed for Phaseolus DNA. The original host remained detectable at 3 d (trnF) and 14 d (trnL) in substantial quantities. The proportion of total reads identified as Phaseolus rapidly decreased with time; a concomitant increase in Daucus reads was observed. Our results indicate that high-throughput sequencing of gut contents has great potential for exploring the dietary histories of field-caught H. halys and other phytophagous insects.

Chloroplast gene markers detect diatom DNA in a drowned mice establishing drowning as a cause of death.

Diatoms are unicellular microalgae with cell wall made up of rigid silica found in all open water bodies. They thus resist degradation and hence are important tool to diagnose cause of death in drowned bodies. The nitric acid digestion method practiced conventionally in forensic science laboratories has limitations due to manual error. Plant chloroplast genes found in diatoms such as ribulose-1,5-bisphosphate carboxylase oxygenase (rbcL-3P) and rbcL, universal plastidic amplicon (UPA), and photosynthesis I P700 apoprotein chlorophyll Al (psaA), which play an important role in photosystems I and II of photosynthesis, are tested to diagnose drowning in experimental mice. It was seen that psaA-2 showed amplification at 150bp in all biological samples. The sequences of psaA-2 gene marker showed 100% proximity to Thalassiosira weissflogii and rbcL-3P showed 99% resemblance to Pseudo-nitzschia multiseries. On the other hand, in postmortem drowned biological samples, the chloroplast-based gene marker failed to show any amplification.

Systematic analysis reveals cis and trans determinants affecting C-to-U RNA editing in Arabidopsis thaliana

BackgroundC-to-U RNA editing is prevalent in the mitochondrial and chloroplast genes in plants. The biological functions of a fraction of C-to-U editing sites are continuously discovered by case studies. However, at genome-wide level, the cis and trans determinants affecting the occurrence or editing levels of these C-to-U events are relatively less studied. What is known is that the PPR (pentatricopeptide repeat) proteins are the main trans-regulatory elements responsible for the C-to-U conversion, but other determinants especially the cis-regulatory elements remain largely uninvestigated.ResultsBy analyzing the transcriptome and translatome data in Arabidopsis thaliana roots and shoots, combined with RNA-seq data from hybrids of Arabidopsis thaliana and Arabidopsis lyrata, we perform genome-wide investigation on the cis elements and trans-regulatory elements that potentially affect C-to-U editing events. An upstream guanosine or double-stranded RNA (dsRNA) regions are unfavorable for editing events. Meanwhile, many genes including the transcription factors may indirectly play regulatory roles in trans.ConclusionsThe 5-prime thymidine facilitates editing and dsRNA structures prevent editing in cis. Many transcription factors affect editing in trans. Although the detailed molecular mechanisms underlying the cis and trans regulation remain to be experimentally verified, our findings provide novel aspects in studying the botanical C-to-U RNA editing events.

Reduced C-to-U RNA editing rates might play a regulatory role in stress response of Arabidopsis

C-to-U RNA editing is prevalent in the mitochondrial and chloroplast genes in plants. The C-to-U editing rates are constantly very high. During genome evolution, those edited cytidines are likely to be replaced with thymidines at the DNA level. C-to-U editing events are suggested to be designed for reversing the unfavorable T-to-C DNA mutations. Despite the existing theory showing the importance of editing mechanisms, few studies have investigated the genome-wide adaptive signals of the C-to-U editome or the potential function of C-to-U editing events in the stress response.By analyzing the transcriptome and translatome data of normal and heat-shocked Arabidopsis thaliana and the RNA-seq from cold-stressed plants, combined with genome-wide comparison of mitochondrial/chloroplast genes and nuclear genes from multiple aspects, we present the conservational and translational features of each gene and depict the dynamic mitochondrial/chloroplast C-to-U RNA editome.We found that the tAI (tRNA adaptation index) and basic translation levels are lower for mitochondrial/chloroplast genes than for nuclear genes. Interestingly, although we found adaptive signals for the global C-to-U RNA editome in mitochondrial/chloroplast genes, the C-to-U (T) alteration would usually cause a reduction in the codon tAI value. Moreover, the C-to-U editing rates are significantly reduced under heat or cold stress when compared to the normal condition. This reduction is irrelevant to the temperature-sensitive RNA structures. Several cases have illustrated that under heat stress, the reduced C-to-U editing rates alleviate ribosome stalling and consequently facilitate the local translation.Our study reveals that in Arabidopsis thaliana the mitochondrial/chloroplast C-to-U RNA editing rates are reduced under heat or cold stress. This reduction is associated with the alleviation of decreased tAI/translation rate of edited codons. The regulation of C-to-U editing rates could be the tradeoff between quantity and quality. We profile the dynamic change of C-to-U RNA editome under heat stress and propose a potential role of editing sites in the heat response. Our work should be appealing to the plant physiologists as well as the RNA editing community.

Cytosine deaminase as a negative selectable marker for the microalgal chloroplast: a strategy for the isolation of nuclear mutations that affect chloroplast gene expression.

Negative selectable markers are useful tools for forward-genetic screens aimed at identifying trans-acting factors that are required for expression of specific genes. Transgenic lines harbouring the marker fused to a gene element, such as a promoter, may be mutagenized to isolate loss-of-function mutants able to survive under selection. Such a strategy allows the molecular dissection of factors that are essential for expression of the gene. Expression of individual chloroplast genes in plants and algae typically requires one or more nuclear-encoded factors that act at the post-transcriptional level, often through interaction with the 5′ UTR of the mRNA. To study such nuclear control further, we have developed the Escherichia coli cytosine deaminase gene codA as a conditional negative selectable marker for use in the model green alga Chlamydomonas reinhardtii. We show that a codon-optimized variant of codA with three amino acid substitutions confers sensitivity to 5-fluorocytosine (5-FC) when expressed in the chloroplast under the control of endogenous promoter/5′ UTR elements from the photosynthetic genes psaA or petA. UV mutagenesis of the psaA transgenic line allowed recovery of 5-FC-resistant, photosynthetically deficient lines harbouring mutations in the nuclear gene for the factor TAA1 that is required for psaA translation. Similarly, the petA line was used to isolate mutants of the petA mRNA stability factor MCA1 and the translation factor TCA1. The codA marker may be used to identify critical residues in known nuclear factors and to aid the discovery of additional factors required for expression of chloroplast genes.

Contradiction between Plastid Gene Transcription and Function Due to Complex Posttranscriptional Splicing: An Exemplary Study of ycf15 Function and Evolution in Angiosperms

Plant chloroplast genes are usually co-transcribed while its posttranscriptional splicing is fairly complex and remains largely unsolved. On basis of sequencing the three complete Camellia (Theaceae) chloroplast genomes for the first time, we comprehensively analyzed the evolutionary patterns of ycf15, a plastid gene quite paradoxical in terms of its function and evolution, along the inferred angiosperm phylogeny. Although many species in separate lineages including the three species reported here contained an intact ycf15 gene in their chloroplast genomes, the phylogenetic mixture of both intact and obviously disabled ycf15 genes imply that they are all non-functional. Both intracellular gene transfer (IGT) and horizontal gene transfer (HGT) failed to explain such distributional anomalies. While, transcriptome analyses revealed that ycf15 was transcribed as precursor polycistronic transcript which contained ycf2, ycf15 and antisense trnL-CAA. The transcriptome assembly was surprisingly found to cover near the complete Camellia chloroplast genome. Many non-coding regions including pseudogenes were mapped by multiple transcripts, indicating the generality of pseudogene transcriptions. Our results suggest that plastid DNA posttranscriptional splicing may involve complex cleavage of non-functional genes.

The two largest chloroplast genome-encoded open reading frames of higher plants are essential genes.

The chloroplast genomes of most higher plants contain two giant open reading frames designated ycf1 and ycf2. In tobacco, ycf1 potentially specifies a protein of 1901 amino acids. The putative gene product of the ycf2 reading frame is a protein of 2280 amino acids. In an attempt to determine the functions of ycf1 and ycf2, we have constructed several mutant alleles for targeted disruption and/or deletion of these two reading frames. The mutant alleles were introduced into the tobacco plastid genome by biolistic chloroplast transformation to replace the corresponding wild-type alleles by homologous recombination. Chloroplast transformants were obtained for all constructs and tested for their homoplastomic state. We report here that all transformed lines remained heteroplastomic even after repeated cycles of regeneration under high selective pressure. A balanced selection was observed in the presence of the antibiotic spectinomycin, resulting in maintenance of a fairly constant ratio of wild-type versus transformed genome copies. Upon removal of the antibiotic and therewith release of the selective pressure, sorting out towards the wild-type plastid genome occurred in all transplastomic lines. These findings suggest that ycf1 and ycf2 are functional genes and encode products that are essential for cell survival. The two reading frames are thus the first higher plant chloroplast genes identified as being indispensable.

Rates of synonymous substitution do not indicate selective constraints on the codon use of the plant psbA gene.

The psbA gene of the flowering plant chloroplast genome has a pattern of codon bias that differs from all other angiosperm chloroplast genes. In psbA, unlike all other chloroplast genes, the third-codon-position composition does not reflect the general genome compositional bias of a high A+T content. Instead, in specific synonymous groups, the codon use of psbA more closely corresponds to the tRNA population available for translation. Since it requires a composition unlike the genome composition bias, this pattern of codon use is likely to be the result of selection. Selective constraints on codon use are expected to result in decreased rates of synonymous substitution, and it has been observed that psbA has the lowest rate of synonymous substitution among plant chloroplast genes. In the present study, this is examined further by testing whether or not those synonymous groups that specifically have an atypical codon use in psbA have correspondingly low rates of silent substitution. An analysis of synonymous substitution rate, performed separately for different degeneracy classes of amino acids, shows that, contrary to the expectation, those sites that are presumed, based on their codon use, to be under selective constraint in psbA do not show low rates of substitution and are not responsible for the overall low rate of synonymous substitution in this gene. Instead, they actually show increased rates of substitution relative to other chloroplast genes. Two hypotheses concerning the role of selection in psbA are advanced to explain the results.

Selection on the codon bias of Chlamydomonas reinhardtii chloroplast genes and the plant psbA gene.

Plant chloroplast genes have a codon use that reflects the genome compositional bias of a high A+T content with the single exception of the highly translated psbA gene which codes for the photosystem II D1 protein. The codon usage of plant psbA corresponds more closely to the limited tRNA population of the chloroplast and is very similar to the codon use observed in the chloroplast genes of the green alga Chlamydomonas reinhardtii. This pattern of codon use may be an adaptation for increased translation efficiency. A correspondence between codon use of plant psbA and Chlamydomonas chloroplast genes and the tRNAs coded by the chloroplast genome, however, is not observed in all synonymous codon groups. It is shown here that the degree of correspondence between codon use and tRNA population in different synonymous groups is correlated with the second codon position composition. Synonymous groups with an A or T at the second codon position have a high representation of codons for which a complementary tRNA is coded by the chloroplast genome. Those with a G or C at the second position have an increased representation of codons that bind a chloroplast tRNA by wobble. It is proposed that the difference between synonymous groups in terms of codon adaptation to the tRNA population in plant psbA and Chlamydomonas chloroplast genes may be the result of differences in second position composition.

Using bacteria to analyze sequences involved in chloroplast gene expression

The expression of higher plant chloroplast genes in prokaryotic cells has been used to examine organelle sequences involved in promoter recognition by RNA polymerase, and protein translocation through membranes. The similarity in sequence structure between Escherichia coli promoters and the maize chloroplast atpB promoter has been investigated using deletion and single base pair substitution mutants. The atpB mutants were mainly isolated by a selection system in E. coli, and then used as templates for the analysis of transcription using chloroplast RNA polymerase. It was found that both the bacterial and chloroplast RNA polymerases behaved in a similar fashion with the wild-type and mutant promoters, indicating that the sequences involved in promoter recognition share a considerable degree of homology. Signal peptide recognition of pea cytochrome f has also been examined in E. coli. This signal peptide, which is probably responsible for insertion of the protein into the thylakoid membrane, is efficiently recognized in E. coli leading to the inner membrane insertion of petA::lacZ fusion proteins. This process requires the bacterial SecA protein and points to a general similarity in the mechanisms of protein translocation within chloroplasts and bacteria.