A plant-specific RNA-binding domain revealed through analysis of chloroplast group II intron splicing

TS. Kroeger, KP. Watkins, G. Friso, KJ van Wijk, A. Barkan. 2009.  A plant-specific RNA-binding domain revealed through analysis of chloroplast group II intron splicing.  Proceedings of the National Academy of Sciences of the United States of America 106: 4537-4532.

Domain of unknown function 860, DUF860, is a protein domain found only in land plants.  One of its proteins WTF1 (“what’s that factor?”), was thought to be required for and promoted the splicing of the introns with which it associates.  To determine which introns WTF1 associates with, the scientists used a genome-wide RNA coimmunoprecipitation assay (RIP-chip).  WTF1 was immunoprecipiated from stroma and combined with fluorescently labeled RNA and  then hybridized to the maize chloroplast genome.  These results were compared to those of the control experiment that used the OE16 antibody which does not bind to RNA.  The array elements that showed association with WTF1 were all from loci containing group II introns, including petB.  To furthur verify these findings, the RNA from the RNA-chip experiment was analyzed with slot-blot hybridization using WTF and OE16.  All of the introns that showed association in the RNA-chip assay, also positively hybridized with WTF1 in the slot-blot.  These experiements showed that the group II introns associated with WTF1 in chloroplasts. Then to determine whether WTF1 promotes splicing in vivo, the splicing of all group II introns was assayed in WTF1 mutants.  They analyzed splicing in WTF1 mutants with moderate ribosome loss, not expected to do much damage as well as with WTF1 mutants with severe ribosome loss.  These results wer compared with the control group and poisened-primer extension assays showed a reduced ratio of spliced to unspliced RNA in the group II introns.  This showed that WTF promotes splicing in the introns with which it associates.

Photosynthetic growth despite a broken Q-cycle

Malnoe, A, Wollman, FA, Vitry, C & Rappaport, F. 2011. Photosynthetic growth despite a broken Q-cycle. Nature Communications 2:301 DOI: 10.1038/ncomms1299

 
In chloroplast the petB gene is responsible for encoding the cytochrome b6f complex, which has a major function in the electron transport chain of photosynthesis and respiration. It along with cytochrome bc use the Q-cycle to increase the proton motive force, or the energy tha t is generated as protons or electrons are transferred across a membrane. Normally the Q-cycle is responsible for photosynthesis, but if it is knocked out, photosynthesis will not occur. Luckily for the plant this can be rectified. If there is a mutation of the H202Q region of the petB gene occurs it will cause the cytochrome b6f complex to be reduced in prevalence within the cell and this lacking can be corrected if the mutated H202Q region of the petB gene was to be combined with another mutant genetically. The other mutant is called R420C and together they have created a double mutant called QiKO that is capable of photosynthesis.

The Cytochrome bf Complex and Alteration of Gene Expression in Cyanobacteria

While land plants garner the most attention in regards to photosynthesis, cyanobacteria are perhaps the most important, especially considering they are responsible for the process of photosynthesis coming on the scene. The process of photosynthesis is a complex one that must be  tightly regulated and it is a constant chemical balancing act to keep it working properly. The cytochrome bf complex is critical in this balance as it “senses” the redox levels of the ETC (electron transport chain) and signals the re-distribution of phycobiliprotein light-harvesting molecules which allows the cyanobacterium to regulate its metabolism as needed in response to its environment. Its signaling capabilities are thought to also play a role in regulating gene expression, which is the hypothesis this paper regards. This experiment used mutants of  the cyanobacterium Synechococcus with mutations in the PetB region (mutated the low potential chain in cytochrome bf complex) and PetC1 region (high potential chain) to see the roles these redox chains play in signaling gene expression. The scientists used cDNA to map out RNA transcription start sites and a fluorescent dye to quantify the level of gene expression.

The results showed mutations  in both regions caused massive alteration of gene expression, with petB having the more dramatic affect. About 800 genes were either up or down-regulated in the petB mutant, 60 of which showed an 8(+)x difference in expression. They concluded that a disruption of either chain in the cytochrome bf complex (especially the low-potential affected by the petB mutation) can have great effect on the expression of many genes. Given the signaling role of the protein complex, mutations hindering the ETC  in this region could adversely affect the ability of an individual to metabolically adapt to its environment.

 

GL Weir, K Short, Dr. T Kallas. 2009. Global Gene Expression in Cyanobacterial Electron Transfer Mutants. Oshkosh Scholar Volume IV: Pages 17-27.

PrfB3 Important in Regulation of petB Transcript Stability in Arabidopsis Chloroplasts

PrfB3, a protein similar to ribosomal peptide chain release factor B (PfrB), is found localized in the stroma of chloroplasts in a complex that has petB RNA in vascular plants. PfrB3 is fundamental in photoautotrophic growth and is needed to manage the stability of chloroplast petB transcript in Arabidopsis. The 3’ processed petB transcripts are conserved by PrfB3 against exonucleolytic degradation, or the cleavage of a nucleotide chain near the end of the strand. In PrfB3 mutants, the only significantly reduced transcripts are petB transcripts that have been 3’ processed or spliced. Reduced splicing, loss of processing, or increased 3’ exonucleolytic attack were thought to cause the missing transcripts, but evidence contradicts the former two. After reviewing the minimal amounts of 3’ processed petB transcripts in the mutants, which are shorter at the 3’ end than in the wild type, they were determined to be possible intermediates produced by exonucleolytic attack.

Stoppel, R, L Lezhneva, S Schwenkert, S Torabi, S Felder, K Meierhoff, P Westhoff, and J Meurer. 2011. Recruitment of a Ribosomal Release Factor for Light- and Stress-Dependent Regulation of petB Transcript Stability in Arabidopsis Chloroplasts. The Plant Cell 23: 2680-2695.

Four Embryophyte Introns and psbB Operon Indicate Chlorokybus as a Basal Streptophyte Lineage

In the evolutionary history of life on Earth one of the most important events would arguably have to be the transition of aquatic algae into land plants (embryophytes). The goal of this study was to help paint a better picture of the evolution of algae to land plants. The gene cluster that includes psbB, psbT, psbH, petB and petD from the chloroplastic genome was examined in the five major lineages of charophytes.

It was found that the psbB gene cluster found in almost all charophytes and embryophytes except Spirogyra (Zygnematales) which lacks it due to intra-genomic rearrangement. All four introns are absent in Chlorokybus but present in some or all of the other four charophyte lineages (Klebsormidiales, Zygnematales, Coleochaetales, and Charales). Chlorokybus also has an unusually long spacer between psbH-petB, over 2 kb long. This result seems to show that Chlorokybus branched off before most of the introns were gained in the most of the charophytes but after the  psbB gene cluster was gained. PCR was used to clone the intron containing portion from total cellular DNA. But in a few of the species chloroplastic genomic DNA was used.

Jungho Lee and James R. Manhart. 2002. Four Embryophyte Introns and psbB Operon Indicate Chlorokybus as a Basal Streptophyte Lineage. Algae Vol. 17 (1): 53-58

Molecular Analysis of Stemona Plants Based on 4 Chloroplast DNA Regions

Fan, LL, Zhu, S, Chen, HB, Yang, DH, Cai, SQ and Katsuko, K. 2009. Molecular Analysis of Stemona Plants in China Based on Sequence of Four DNA Regions. Biological & Pharmaceutical Bulletin. Vol. 32, No. 8: 1439-1446.

 

There were 3 species thoroughly studied and analyzed from the genus Stemona: S. sessilifolia, S. japonica and S. tuberosa. These species are the orginal sources of Stemonae Radix specified in the Chinese Pharmacopoeia, and are linked with antitussive and insecticidal remedies. Antitussive activities were shown in a variety of degrees in citric acid-induced guinea pig cough models. The nucleotide sequences of chloroplast DNA trnL-trnF, trnH–psbA, petB-petD and trnK-rps16 regions of the species from the genus Stemona, S. parviflora, and the “counterfeits” of Stemonae Radix were determined to identify the said true sources of Stemonae Radix. The petB region was important during this analysis because it, along with the 3 others, were most informative, providing information necessary to distinguish the 3 Chinese Pharmacopoeia species from one another, as well as the imitations.

 

Complex RNA maturation in chloroplasts The psbB operon from spinach

In this study, hybrids of the plastids of spinach plants were lysed to isolate the RNA of the psbB operon (psbB, psbH, petB, and petD). The RNA was mapped using radiolabelled DNA. By inserting selected RNA strands into rabbit blood cells, the recombination of the operon could be observed by protein production.

It was found that the different RNA species of this operon are from the same DNA strand and are processed into their individual mRNAs after transcription. PetB and petD both contain an intron within their sequence and are dicistronic while psbB and psbH are monocistronic. This requires the transcribed RNA to be cleaved before translation can occur. By comparing samples that had been in light to those in dark, the presence of light does not affect the modification of these RNAs, but does affect translation.

WESTHOFF, P. and HERRMANN, R. G. (1988), Complex RNA maturation in chloroplasts. European Journal of Biochemistry, 171: 551–564. doi: 10.1111/j.1432-1033.1988.tb13824.x