Computational protocol: The FBH family of bHLH transcription factors controls ACC synthase expression in sugarcane

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Protocol publication

[…] To investigate the phylogenetic relationship of sugarcane ScACS2 (NCBI accession ADZ96244.1), we retrieved ACS-related protein sequences from sorghum (Sorghum bicolor), maize (Zea mays), foxtail millet (Setaria italica), and switchgrass (Panicum virgatum) databases available on Phytozome ( and performed the alignment using the WebPRANK global alignment tool () with default parameters. Arabidopsis and tomato ACS were included to guide the classification of subgroups ( at JXB online). The phylogenetic tree was constructed in MEGA6 () software using the neighbor-joining method with 1000 bootstrap replicates. Orthologous groups were deduced by a bidirectional best hit approach. To classify sugarcane FBH proteins, the bHLH domains of Arabidopsis FBH1 (At1g35460), FBH2 (At4g09180), FBH3 (At1g51140), FBH4 (At2g42280), AtbHLH128 (At1g05805), AtbHLH129 (At2g43140), AMS (At2g16910), GL3 (At5g41315), ICE1 (At3g26744), PIF3 (At1g09530), PIF4 (At2g43010), SPT (At4g36930), and TT8 (At4g09820), and sorghum Sb02g028300, Sb03g04286, and Sb07g027810 proteins were aligned, and the phylogenetic tree was constructed as described. [...] To identify putative conserved regulatory motifs in the ScACS2 promoter, its sequence was compared with sequences of orthologous promoters of Sorghum bicolor (Sb03g03070), Zea mays (GRMZM2G163015), Setaria italica (Si001369m), and Panicum virgatum (Pavirv00040519m). Promoter sequences were selected by retrieving a 1 kb region upstream of the translational start site using Phytozome genomic data and alignments were performed by using BlastN, with the recommended parameters described by , LAGAN (), and MULAN () algorithms. Conserved promoter elements present in at least three different species, including sugarcane, were considered in the search for putative transcription factor binding sites (TFBS). We identified putative TFBS using the PlantPAN 2.0 database (), and the resulting cis-element sequences were manually double-checked against original references; elements containing inconsistencies were discarded. [...] The three-dimensional homology models of the bHLH domain of Arabidopsis and sugarcane FBHs were generated with the SWISS-MODEL workspace (; ), based on sequence alignment with the 4ATK structure deposited on Protein Data Bank ( The quality of the homology models was assessed by QMEAN6 Z-scores (0< |Z-score| <1) and a Ramachandran plot considering more than 90% of residues in the most favored regions. The models were superimposed and visualized using Chimera 1.11.2 software (; ). […]

Pipeline specifications

Databases Phytozome ExPASy PlantPAN
Applications Phylogenetics, Protein structure analysis, Nucleotide sequence alignment
Organisms Saccharomyces cerevisiae, Arabidopsis thaliana
Chemicals Sucrose