Members of the MYBMIXTA-like transcription factors may orchestrate the initiation of fiber development in cotton seeds

from the diploid G. raimondii (Gr), an extant species most closely related to the D-genome progenitor of tetraploid cotton, may help draw a more complete picture about the evolution of the MML gene subgroup in cotton and their apparent expansion and recruitment to specialized functions in epidermal seed fiber development.

MYBMIXTA-like (MML) transcription factors form the subgroup 9 of R2R3-MYBs (Stracke et al., 2001) whose first characterized member was MIXTA from Antirrhinum majus. Various MML genes have been shown to be important regulators of epidermal cell differentiation in different plant species, including specifying cell shape in petals, vegetative trichome initiation and branching and seed fiber initiation (Martin et al., 2002;Machado et al., 2009;Walford et al., 2011). Indeed, the conical cells of petals look very much like young fibers shortly after they protrude from the epidermal surface of the cotton seed and begin to elongate, so it is likely there is some commonality in cellular regulation between the different tissue types. In tetraploid cotton, Gossypium hirsutum L. (Gh), the silencing or over-expression of two MML genes (GhMYB25 and GhMYB25Like) expressed predominantly in the ovule epidermis during fiber initiation affect the initiation or timing of expansion of fiber initials (Machado et al., 2009;Walford et al., 2011). Based on silencing phenotypes and gene expression data, GhMYB25Like may be one of the most upstream genes in a regulatory cascade currently known to involve GhMYB25, GhMYB109 (an R2R3 MYB from subgroup 15, Pu et al., 2008) and other types of transcription factors, such as the homeodomain leucine zipper (HDzip) factor GhHD-1, that may act in a protein complex with GhMYB25 (Zhang et al., 2010;Walford et al., 2012). The recent release of the genome sequence from the diploid G. raimondii (Gr), an extant species most closely related to the D-genome progenitor of tetraploid cotton, may help draw a more complete picture about the evolution of the MML gene subgroup in cotton and their apparent expansion and recruitment to specialized functions in epidermal seed fiber development.

GENOMIC ORGANIZATION AND PHYLOGENY OF COTTON MMLs
The Gr genome contains over 200 R2R3 MYBs (Paterson et al., 2012), but 10 (GrMML1-10, Figure 1A) cluster with MIXTA, and all of these contain the signature protein motif AQWESARxxAExRLxRES previously indicated to be unique to subgroup 9 (Stracke et al., 2001). This number is considerably greater than the three MML genes found in Arabidopsis: AtMYB16, proposed to control the shape of petal epidermal cells (Baumann et al., 2007), AtMYB17 a putative regulator of early inflorescence development and seed germination (Zhang et al., 2009) and AtMYB106/NOK a negative regulator of trichome branching (Jakoby et al., 2008). The MML factors are distinct from the AtGL1-like MYBs (AtMYB0, AtMYB23, AtMYB66) of subgroup 15, involved in trichome and root hair initiation and development, that are often suggested as the likely models for regulators of seed fiber development in cotton. Subgroup 15 in Gr appears to have only one member, Gorai.012G061800/GrMYB109 (Paterson et al., 2012) that is a homolog of GhMYB109 with a demonstrated role in fiber elongation rather than initiation (Pu et al., 2008). Gr has very close homologs of GhMYB25 (Gorai.012G186500, GrMML7) and GhMYB25Like (Gorai.008G179600, GrMML3) as well as eight other novel MML genes, each with a pair of very close homologs in the Aand D-genomes of tetraploid Gh ( Figure 1A, Supplementary data 1). The 10 MML proteins fall into 4 distinct clades supported by elevated bootstrap scores. One of these, Sg9-2, includes GrMML3/GhMYB25Like, GrMML7/GhMYB25, Gr/GhMML9 and 10, but no Arabidopsis or Antirrhinum MML MYBs ( Figure 1A). In the Gr genome the 10 MML genes are distributed across 6 of the 13 diploid cotton chromosomes. However, on chromosomes Chr08 and Chr13 there are clusters of two (GrMML3 and 4 and a fragment of another MML gene) and three genes (GrMML8, 9, and 10), respectively, in tandem arrangements (Paterson et al., 2012; Figure 1B). This suggests that they may have evolved from gene duplications of ancestral MML genes and subsequently evolved new functions and in some cases different expression patterns.

MML GENE EXPRESSION DURING THE EARLY EVENTS OF FIBER INITIATION AND DEVELOPMENT
The 10 MML genes are nearly all expressed predominantly in early fiber development in whole ovules/seeds of tetraploid cotton from −2 to +2 day post anthesis (DPA) ( Figure 1C) with little expression in other cotton tissues. A few (GhMML5, 6, 10) are also expressed in leaves and/or cotyledons which are rich in either hair or glandular trichomes that are structurally related to seed fibers and share some common regulators like GhHD-1 (Walford et al., 2012). MML gene expression levels were also investigated using handmicrodissected tissues (Bedon et al., 2013) from whole ovules and young seeds to focus just on the fiber initiation process occurring in the epidermis. This circumvents the transcript dilution or confounding with expression from the other layers of ovule tissues. Six of the GhMML genes were preferentially expressed at 0 DPA in outer-integuments (OI; GhMML2, 3, 4, 7, 8, and 9), two in inner-integument (II; GhMML6 and 10) and two in the nucellus (N; GhMML1 and 5) (heat map in Figure 1D). Based on their differences in relative expression levels in the OI, three groups can be classified as having; high (GhMML2, 3 and 7), low to medium (GhMML 4,6,8,and 9), and very low (GhMML1, 5, and 10) expression (chart in Figure 1D). Further expression analysis in the OI was performed from −4 to +2 DPA which covered the stages of ovule epidermal cell differentiation (−4 to −1 DPA), fiber initiation (i.e., ballooning of fiber initials above the epidermis surface at 0 DPA), and early fiber elongation (+1 to +2 DPA) ( Figure 1E). There are four different patterns with some GhMML genes having their highest expression from −4 to −2 DPA (GhMML1, 2, 5, 6); genes peaking at −1 DPA (GhMML3 and 4); genes peaking at 0 to +1 DPA (GhMML7,8,9); and one member with highest expression at +2 DPA (GhMML10) (Figure 1E). These expression patterns support the specialization of different sets of MML genes for specific aspects of epidermal cell differentiation, although they may still have some roles in other tissues.

MML REGULATORY CASCADES AND INTERACTIONS
To unravel the potential transcriptional networks among the different GhMML members their expression levels were assessed in dissected OI from transgenic tetraploid cotton silenced (through RNAi) for GhMML3/GhMYB25Like (Walford et al., 2011) or GhMML7/GhMYB25 (Machado et al., 2009) compared to the transcript levels from the corresponding null segregant plants as controls ( Figure 1F, Supplementary data 2). In the GhMYB25Like silenced plants Figure 1F (upper panel), the transcript level of GhMYB25Like/GhMML3 was decreased at 0 and +2 DPA, as previously reported (Walford et al., 2011). Transcripts of GhMML7/GhMYB25, GhMML8, and 9 were completely abolished at 0 and +2 DPA, and GhMML4 was decreased to a lesser extent, indicating that they may all be downstream of GhMYB25-like. A significant increase in GhMML2 and 6 transcripts were observed at 0 and +2 DPA (Figure 1F), suggesting that they may be repressed by GhMYB25-like. A slight decrease in GhMML1 at −4 DPA was also seen (see chart representation in Supplementary data 2). In the GhMYB25 silenced plants (Figure 1Flower panel), the transcript levels of GhMML7/GhMYB25 was decreased at 0 and +2 DPA as previously reported by Machado et al. (2009). Transcript levels of GhMML3/GhMYB25Like, GhMML4 and 8, and to a lesser extent GhMML9, were increased at 0 DPA (Supplementary data 2), suggesting there may be some feedback regulation of GhMYB25Like by GhMYB25. The direct requirement of GhMYB25Like/GhMML3 for expression of GhMML7/GhMYB25 was assessed using a transient assay in cotton protoplasts ( Figure 1G,  Supplementary data 3). Transactivation of the GhMYB25 promoter, fused to the luciferase reporter, did occur in the presence of GhMYB25Like, but only when co-expressed with the HDzip factor GhHD-1, recently shown to be involved in fiber initiation (Walford et al., 2012), so the two may be involved in a complex to activate GhMYB25.

CONCLUSION
The MYBMIXTA-like subgroup appears to have expanded in cotton compared to non-fiber plants, probably following a cotton specific triplication as reported in Paterson et al. (2012), thus contributing to the evolution of seed fiber. The spatiotemporal expression study of the different GhMML genes reported here distinguishes them according to the plant organs and/or ovule tissues in which they are expressed and the timing of their expression during the early stages of seed fiber development. The newly described GhMML2 is one of the more highly expressed MML genes in the OI along with GhMML7/GhMYB25 and GhMML3/GhMYB25Like which have already been shown to be involved in seed fiber initiation (Machado et al., 2009;Walford et al., 2011), but GhMML2 shows a different pattern of expression with highest transcript accumulation before the fiber initiation step. GhMML2, together with GhMML6, transcripts are increased rather than decreased at 0 and +2 DPA in GhMYB25Like RNAi plants, suggesting a molecular function different to GhMML7, 8, and 9 that are down-regulated in this transgenic background. The differences in GhMML expression in OI in the two MMLs silenced plants highlights a possible hierarchical network between MMLs; with MML3/GhMYB25-like activating the transcription of MML7/GhMYB25, MML8, and MML9 and perhaps being involved in the repression of MML2 and MML6 at 0 and +2 DPA as the fibers begin to expand and elongate. MML7/GhMYB25 might be involved in a feedback loop to transcriptionally activate MML3, 4, 8, and 9 at 0 DPA. We were able to show that MML3/GhMYB25Like is able to transactivate the MML7/MYB25 promoter, but only when associated with GhHD-1. Such results suggest the presence of a transcriptional complexes, involving MML(s) and GhHD-1, necessary for fiber development that are similar to the sorts of complexes seen in Arabidopsis trichome development (Ishida et al., 2008), although clearly involving different factors. Here, we identified some potential new players in fiber development that belong to the same phylogenetic group of MYBs and suggest a very subtle networking involving both protein-protein and protein-DNA interactions. The next steps involve the functional characterization of these novel cotton MML genes by silencing and over-expression in transgenic cotton and the study of their interactions in both cotton protoplasts and yeast to confirm their roles in generating the fibers of commercially important cotton species.