Full Length Transcriptome Highlights the Coordination of Plastid Transcript Processing

Plastid gene expression involves many post-transcriptional maturation steps resulting in a complex transcriptome composed of multiple isoforms. Although short-read RNA-Seq has considerably improved our understanding of the molecular mechanisms controlling these processes, it is unable to sequence full-length transcripts. This information is crucial, however, when it comes to understanding the interplay between the various steps of plastid gene expression. Here, we describe a protocol to study the plastid transcriptome using nanopore sequencing. In the leaf of Arabidopsis thaliana, with about 1.5 million strand-specific reads mapped to the chloroplast genome, we could recapitulate most of the complexity of the plastid transcriptome (polygenic transcripts, multiple isoforms associated with post-transcriptional processing) using virtual Northern blots. Even if the transcripts longer than about 2,500 nucleotides were missing, the study of the co-occurrence of editing and splicing events identified 42 pairs of events that were not occurring independently. This study also highlighted a preferential chronology of maturation events with splicing happening after most sites were edited.

NBS-LRR-containing class of salicylic acid-induced gene transcript in rye

NBS-LRR-type disease resistance gene-like cDNA, induced by salicylic acid (SA) was cloned from rye Secalecereale L. (2n = 14RR) var. Petkus, which has rust resistance genes such as Lr26, Sr31 andRr9. We designed primers based on the NBS region and performed PCR using Petkus genomic DNA as a template. Next, we TA-cloned a 532-bp DNA fragment containing five homologous amino acid sequences in the NBS region. The SA-treated rye showed strong expression of a transcript of approximately 3.5 knt in the Northern blots probed with the NBS fragment; however, no transcripts were observed with the untreated rye. We constructed a cDNA library of rye var. Petkus treated with SA, and then screened the cDNA library using the TA-cloned NBS fragments as a probe. The entire nucleotide sequence of a full length of rye NBS-LRR-containing class cDNA 3,446 bp was determined.

Assessing Transcriptional Responses to Light by the Dinoflagellate Symbiodinium

The control of transcription is poorly understood in dinoflagellates, a group of protists whose permanently condensed chromosomes are formed without histones. Furthermore, while transcriptomes contain a number of proteins annotated as transcription factors, the majority of these are cold shock domain proteins which are also known to bind RNA, meaning the number of true transcription factors is unknown. Here we have assessed the transcriptional response to light in the photosynthetic species Symbiodinium kawagutii. We find that three genes previously reported to respond to light using qPCR do not show differential expression using northern blots or RNA-Seq. Interestingly, global transcript profiling by RNA-Seq at LD 0 (dawn) and LD 12 (dusk) found only seven light-regulated genes (FDR = 0.1). qPCR using three randomly selected genes out of the seven was only able to validate differential expression of two. We conclude that there is likely to be less light regulation of gene expression in dinoflagellates than previously thought and suggest that transcriptional responses to other stimuli should also be more thoroughly evaluated in this class of organisms.

Downregulation of the Cl-/HCO3-Exchanger Pendrin in Kidneys of Mice with Cystic Fibrosis: Role in the Pathogenesis of Metabolic Alkalosis

Background/Aims: Patients with cystic fibrosis (CF) are prone to the development of metabolic alkalosis; however, the pathogenesis of this life threatening derangement remains unknown. We hypothesized that altered acid base transport machinery in the kidney collecting duct underlies the mechanism of impaired bicarbonate elimination in the CF kidney. Methods: Balance studies in metabolic cages were performed in WT and CFTR knockout (CF) mice with the intestinal rescue in response to bicarbonate loading or salt restriction, and the expression levels and cellular distribution of acid base and electrolyte transporters in the proximal tubule, collecting duct and small intestine were examined by western blots, northern blots and/or immunofluorescence labeling. Results: Baseline parameters, including acid-base and systemic vascular volume status were comparable in WT and CF mice, as determined by blood gas, kidney renin expression and urine chloride excretion. Compared with WT animals, CF mice demonstrated a significantly higher serum HCO3- concentration (22.63 in WT vs. 26.83 mEq/l in CF mice; n=4, p=0.013) and serum pH (7.33 in WT vs. 7.42 in CF mice; n=4, p=0.00792) and exhibited impaired kidney HCO3- excretion (urine pH 8.10 in WT vs. 7.35 in CF mice; n=7, p=0.00990) following a 3-day oral bicarbonate load. When subjected to salt restriction, CF mice developed a significantly higher serum HCO3- concentration vs. WT animals (29.26 mEq/L in CF mice vs. 26.72 in WT; n=5, p=0.0291). Immunofluorescence labeling demonstrated a profound reduction in the apical expression of the Cl-/HCO3- exchanger pendrin in cortical collecting duct cells and western and northern blots indicated diminished plasma membrane abundance and mRNA expression of pendrin in CF kidneys. Conclusions: We propose that patients with cystic fibrosis are prone to the development of metabolic alkalosis secondary to the inactivation of the bicarbonate secreting transporter pendrin, specifically during volume depletion, which is a common occurrence in CF patients.