Optimization of ribosome profiling in plants including structural analysis of rRNA fragments.

Background: Ribosome profiling (or Ribo-seq) is a technique that provides genome-wide information on the translational landscape (translatome). Across different plant studies, variable methodological setups have been described which raises questions about the general comparability of data that were generated from diverging methodologies. Furthermore, a common problem when performing Ribo-seq are abundant rRNA fragments that are wastefully incorporated into the libraries and dramatically reduce sequencing depth.

Removal of the large inverted repeat from the plastid genome reveals gene dosage effects and leads to increased genome copy number.

The chloroplast genomes of most plants and algae contain a large inverted repeat (IR) region that separates two single-copy regions and harbours the ribosomal RNA operon. We have addressed the functional importance of the IR region by removing an entire copy of the 25.3-kb IR from the tobacco plastid genome.

Utilizing high-resolution ribosome profiling for the global investigation of gene expression in Chlamydomonas.

Ribosome profiling (Ribo-seq) is a powerful method for the deep analysis of translation mechanisms and regulatory circuits during gene expression. Extraction and sequencing of ribosome-protected fragments (RPFs) and parallel RNA-seq yields genome-wide insight into translational dynamics and post-transcriptional control of gene expression. Here, we provide details on the Ribo-seq method and the subsequent analysis with the unicellular model alga Chlamydomonas reinhardtii (Chlamydomonas) for generating high-resolution data covering more than 10 000 different transcripts.

Agrobacterium-Mediated Seedling Transformation to Measure Circadian Rhythms in Arabidopsis.

Circadian clocks are endogenous timing mechanisms that allow an organism to adapt cellular processes in anticipation of predictable changes in the environment. Luciferase reporters are well utilized as an effective, nondestructive method to measure circadian rhythms of promoter activity in Arabidopsis. Obtaining stable transgenic reporter lines can be laborious.

The calmodulin-like protein, CML39, is involved in regulating seed development, germination, and fruit development in Arabidopsis.

We show that the calcium sensor, CML39, is important in various developmental processes from seeds to mature plants. This study bridges previous work on CML39 as a stress-induced gene and highlights the importance of calcium signalling in plant development. In addition to the evolutionarily-conserved Ca sensor, calmodulin (CaM), plants possess a large family of CaM-related proteins (CMLs).

Transcript profiling indicates a widespread role for bacterial-type phosphoenolpyruvate carboxylase in malate-accumulating sink tissues.

Phosphoenolpyruvate carboxylase (PEPC) is an important regulatory enzyme situated at a key branch point of central plant metabolism. Plant genomes encode several plant-type PEPC (PTPC) isozymes, along with a distantly related bacterial-type PEPC (BTPC). BTPC is expressed at high levels in developing castor oil seeds where it tightly interacts with co-expressed PTPC polypeptides to form unusual hetero-octameric Class-2 PEPC complexes that are desensitized to allosteric inhibition by L-malate.