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. 2014 May 1;5:179. doi: 10.3389/fpls.2014.00179

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Copyright © 2014 Bedon, Ziolkowski, Walford, Dennis and Llewellyn.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Phylogenetic analysis, chromosomal location, gene expression of the cotton MML genes in wild type and transgenic G. hirsutum, and transactivation of the GhMML3 promoter in cotton protoplasts. (A) Phylogenetic analysis of the G. raimondii (Gr, filled diamonds) MML proteins and their putative G. hirsutum (Gh, empty diamonds) D-genome homologs. Four clades (Sg9-1 to 4) are indicated by shading. The rooted Neighbour–Joining tree was obtained in MEGA 5.0 with Clustal W alignment using the full length amino acid sequences (details in Supplementary data 1). Arabidopsis thaliana (At) MYB3R4 and Populus tremula × P. Tremuloides (Ptt) MYBR3 are R1R2R3-MYBs used as outgroups and the other AtMYB and Anthirrinum majus (Am) sequences are landmarks of Subgroup 9. (B) Schematic of the chromosomal distribution of the 10 GrMML genes indicated by the filled triangles, while the unfilled triangle represents a fragment of a MML gene. The directions of the triangle indicate the coding strand of the transcripts and the numbers under the triangle the particular GrMML gene. Adjacent triangles are tandemly arranged genes. (Chromosomes are only approximately to scale). The heat maps visualize the transcript level differences between the G. hirsutum homologues of the GrMML genes in: (C) cotton fibers, ovules and selected other plant organs, (D) three dissected tissues (OI, II and N) from wild type (G. hirsutum) ovules collected the day of anthesis [DPA(0)], (E) dissected OI of wild type ovules collected from 4 days before anthesis [DPA(−4)] to 2 days after anthesis [DPA(+2)], (F) OI dissected from G. hirsutum ovules silenced by RNAi for GhMYB25Like (ie., MYBML3) and GhMYB25 (i.e., MYBML7) and their respective controls (ie., null segregant plants) collected at DPA(−4), (−2), (0), and (+2) (F). Heat maps were made using Expander software based on gene expression relative to the cotton ubiquitin gene and normalized for each MML gene and separate experiment (details in Supplementary data 2). Primers used detect both the A- and D-genome homoeologues of each MML gene. (G) Transactivation assay of the GhMML3/GhMYB25 promoter-Luciferase reporter by GhMYB25Like and/or GhHD-1 in cotton cotyledon protoplasts (details in Supplementary data 3).