Computational protocol: A Division in PIN-Mediated Auxin Patterning during Organ Initiation in Grasses

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Protocol publication

[…] Coding sequences from Phytozome (http://www.phytozome.org/), NCBI (http://www.ncbi.nlm.nih.gov/), and CoGe (http://genomevolution.org/CoGe/) were analyzed with Geneious (http://www.geneious.com/). We found that the third codon position was GC base rich in monocots (60% monocots, 45% eudicots) and biased phylogenetic analyses, thus after MUSCLE alignment, this position was removed. Analysis of GC normalized sequences (33% monocots, 30.2% eudicots) was performed with MrBayes 2.0.3 (http://mrbayes.sourceforge.net/) on GreenButton (geneious.greenbutton.net) using the Jukes-Cantor model of nucleotide evolution (selected using the AIC: MEGA 5.0, http://www.megasoftware.net/). Four chains, sampled every 200 generations, were run until convergence (1,013,000 generations, standard deviation of split frequencies below 0.01). After examination of the likelihood scores, 25% of trees were discarded as burnin. P. patens - Pp1s10_17V6.1 was used as the outgroup. The final tree is available for download on Treebase: http://purl.org/phylo/treebase/phylows/study/TB2:S15020. BLAST searches using both DNA and protein sequences of SoPIN1 members identified only PIN1 clade members in the Brassicaceae. To identify the SoPIN1 syntenic regions across the angiosperms the sequence for a gene neighboring SoPIN1 in Papaya was used in CoGe. [...] Images were captured on a Leica TCS-SP5 laser-scanning confocal equipped with a water-dipping 20× objective (NA 0.7) (http://www.leica-microsystems.com/) and processed with ImageJ (http://rsbweb.nih.gov/ij/). Citrine was excited at 514 nm and mRFP at 561 nm. The pinhole was set to one Airy unit. For each z-stack, transmitted light was detected and flattened using an extended-depth-of-field plugin (http://bigwww.epfl.ch/demo/edf/). Fluorescent channels were processed with a median filter to reduce noise and were recombined with the processed transmitted light image either as single z-planes or maximum projections. In most images maximum projections were limited to internal sections in order to reveal sub-epidermal localization and midvein development. Brightness and contrast were adjusted after fluorescence channels were pseudo-colored with look-up tables. PIN cellular polarity was observed through multiple confocal sections, and similar to previous work was defined by a characteristic arching shape , , , . Multiple samples were analyzed at each developmental stage, and ratios printed below each figure label reflect the number of times phenomena discussed in the text were observed out of the total images captured. FM4-64 staining was performed as described in . […]

Pipeline specifications

Software tools ImageJ, Extended Depth of Field
Application Microscopic phenotype analysis
Organisms Arabidopsis thaliana, Brachypodium distachyon