DOI: 10.5061/dryad.tdz08kq5k
title: Data from: A eudicot MIXTA family ancestor likely functioned in both conical cells and trichomes

description: The MIXTA family of MYB transcription factors modulate the development of
 diverse epidermal features in land plants. This study investigates the
 evolutionary history and function of the MIXTA gene family in the
 early-diverging eudicot model lineage Thalictrum (Ranunculaceae), with
 R2R3 SBG9-A MYB transcription factors representative of the pre
 core-eudicot duplication and thus hereby referred to as “paleoMIXTA”
 (PMX). Cloning and phylogenetic analysis of Thalictrum paleoMIXTA (ThPMX)
 orthologs across 23 species reveals a genus-wide duplication coincident
 with a Whole Genome Duplication. Expression analysis by qPCR confirmed the
 highest expression is found in carpels, while newly revealing high
 expression in leaves and nuanced differences between paralogs in
 representative polyploid species. The single copy ortholog from the
 diploid species T. thalictroides (TthPMX, previously TtMYBML2), which has
 petaloid sepals with conical papillate cells and trichomes on leaves, was
 functionally characterized by virus induced gene silencing (VIGS) and its
 role in leaves was also assessed from heterologous over-expression in
 tobacco. Another ortholog from a species with conical papillate cells on
 stamen filaments, TclPMX, was also targeted for silencing. Overexpression
 assays in tobacco provide further evidence that the paleoMIXTA lineage has
 the potential for leaf trichome function in a core eudicot. Transcriptome
 analysis by RNA-Seq on leaves of VIGS-treated plants suggests that TthPMX
 modulates leaf trichome development and morphogenesis through
 microtubule-associated mechanisms and that this may be a conserved pathway
 for eudicots. These experiments provide evidence for a combined role for
 paleoMIXTA orthologs in (leaf) trichomes and (floral) conical papillate
 cells that, together with data from other systems, makes the functional
 reconstruction of a eudicot ancestor most likely as also having a combined
 function.
description: This dataset contains qPCR data, trichome measurements in wild
 type and transgenic Thalictrum and tobacco plants, top 30 DEGs for RNAseq
 experiments, a (trimmed) coding sequence alignment and a phylogeny of the
 MIXTA gene family in <em>Thalictrum</em> and outgroups (see
 associated publication for details).
description: # A eudicot MIXTA family ancestor likely functioned in both conical cells
 and trichomes
 [https://doi.org/10.5061/dryad.tdz08kq5k](https://doi.org/10.5061/dryad.tdz08kq5k) This README file was generated on 2024-02-29 by Simra Zahid. GENERAL INFORMATION 1\. Title of Dataset: A eudicot MIXTA family ancestor likely functioned in both conical cells and trichomes 2\. Link to dataset: [https://doi.org/10.5061/dryad.tdz08kq5k](https://doi.org/10.5061/dryad.tdz08kq5k) 3\. Date of data collection: 2020-2021 This dataset contains qPCR data, trichome measurements in wild type and transgenic Thalictrum and tobacco plants, top 30 DEGs for RNAseq experiments, a (trimmed) coding sequence alignment (Zahid_Schulfer_and_Di_Stilio_FPS_2023_MIXTA_alignment) and a phylogeny (Zahid_Schulfer_and_Di_Stilio_FPS_2023_MIXTA_phylogeny) of the MIXTA gene family in Thalictrum and outgroups (see associated publication for details). Refer to the associated publication for detailed methods, background and results. DATA & FILE OVERVIEW 1\. Sheets in the Excel workbook A) qPCR VIGS TthPMX B) qPCR VIGS PDS C) qPCR Tth Tissue expression D) qPCR Tcl, Tth sepal VIGS E) qPCR Tcl, Tth dissected F) Tth VIGS stomatal density G) Tth VIGS trichome height H) Tobacco Trichome density I) Tth VIGS RNA-seq validation J) RNA-seq Significant DEGs 2\. Relationship between files, if important: None \######################################################################### DATA-SPECIFIC INFORMATION FOR: qPCR VIGS TthPMX 1\. Variables: 10 2\. Number of cases/rows: 22 3\. Variable List: \* Treatment: All Thalictrum thalictroides plants treated with TRV-TthPMX-PDS, or TRV-TthPMX or Mock Empty vector control (EV) or untreated Wild type (WT) \* Average CT TthPMX: Average cycle threshold (CT) value of gene TthPMX of 3 technical replicates \* Relative Expression to Actin: Relative expression of experimental gene TthPMX to housekeeping gene Actin \* Relative Epression to EEF1a: Relative expression of experimental gene TthPMX to housekeeping gene EEF1-alpha \* 2-ΔΔCT Actin: Ct value of target gene TthPMX to internal control gene ACTIN (using the formula: 2ΔΔCT) \* 2-ΔΔ CT EEF1a: Ct value of target gene TthPMX to internal control gene EEF1-alpha (using the formula: 2ΔΔCT) \* Geomean: Normalization of 2-ΔΔCT Actin and 2-ΔΔ CT EEF1a values \* Bio-replicate average (green): Green tabs correspond to average of three bio-replicates for corresponding treatment TRV-TthPMX-PDS, or TRV-TthPMX or Mock Empty vector control (EV) or untreated Wild type (WT) \* Expression normalized to EV: Exxpression of treated bio-replicates normalized to control group Empty Vector (EV) \* Graph: Expression of samples used for graphing purposes 4\. Missing data codes: n/a 5\. Specialized formats or other abbreviations used: \* avg: average \* WT: Wild Type \* EV: Empty Vector \######################################################################### DATA-SPECIFIC INFORMATION FOR: qPCR VIGS PDS 1\. Variables: 10 2\. Number of cases/rows: 18 3\. Variable List: \* Treatment: All Thalictrum thalictroides plants treated with TRV-TthPMX-PDS, or Mock Empty vector control (EV) or untreated Wild type (WT) \* Average CT TthPMX: Average cycle threshold (CT) value of gene TthPMX of 3 technical replicates \* Relative Expression to Actin: Relative expression of experimental gene TthPMX to housekeeping gene Actin \* Relative Epression to EEF1a: Relative expression of experimental gene TthPMX to housekeeping gene EEF1-alpha \* 2-ΔΔCT Actin: Ct value of target gene TthPMX to internal control gene ACTIN (using the formula: 2ΔΔCT) \* 2-ΔΔ CT EEF1a: Ct value of target gene TthPMX to internal control gene EEF1-alpha (using the formula: 2ΔΔCT) \* Geomean: Normalization of 2-ΔΔCT Actin and 2-ΔΔ CT EEF1a values \* Bio-replicate average (green): Green tabs correspond to average of three bio-replicates for corresponding treatment TRV-TthPMX-PDS, or Mock Empty vector control (EV) or untreated Wild type (WT) \* Expression normalized to EV: Exxpression of treated bio-replicates normalized to control group Empty Vector (EV) \* Graph: Expression of samples used for graphing purposes 4\. Missing data codes: n/a 5\. Specialized formats or other abbreviations used: \* avg: average \* WT: Wild Type \* EV: Empty Vector \######################################################################### DATA-SPECIFIC INFORMATION FOR: qPCR Tth Tissue expression 1\. Variables: 12 2\. Variable List: \* Gene name: Target gene TthPMX or TdiPMX1a or TdiPMX1b or TdaPMXb.1 or TdaPMX1a or TdaPMX2b or TdaPMX1b.2 \* Sample type: Sample taken from LEAF or SEPAL or CARPEL or STAMEN \* Average CT TthPMX: Average cycle threshold (CT) value of gene TthPMX of 3 technical replicates \* Relative Expression to ACTIN: Relative expression of experimental gene TthPMX to housekeeping gene ACTIN \* Relative Epression to EEF1a: Relative expression of experimental gene TthPMX to housekeeping gene EEF1-alpha \* 2-ΔΔ CT Actin: Ct value of target gene TthPMX to internal control gene ACTIN (using the formula: 2ΔΔCT) \* 2-ΔΔ CT EEF1a: Ct value of target gene TthPMX to internal control gene EEF1-alpha (using the formula: 2ΔΔCT) \* GeoMean: Normalization of 2-ΔΔCT Actin and 2-ΔΔ CT EEF1a values \* Avg of bio-reps: Average of Three biological replicates \* GeoMean: Normalization of 2-ΔΔCT Actin and 2-ΔΔ CT EEF1a values \* Expression relative to highest paralog: Average expression of 3 bio-replicates to highest epressing paralog for T. thalictroides or T. dioicum or T. dasycarpum \* Relative expression of biorep avg to highest organ (carpel): Average Expression of the bio-replicates to highest expressing organ i.e, carpels 4\. Missing data codes: n/a 5\. Specialized formats or other abbreviations used: SEP=sepals, STA=stamens, CA=carpels FL=female leaf, FSEP= female sepals, ML=male leaf, MSEP=male sepals Avg= average, ct= cycle threshold \######################################################################### DATA-SPECIFIC INFORMATION FOR: qPCR Tcl, Tth sepal VIGS 1\. Variables: 3 2\. Variable List: \* Thalictrum thalictroides or Thalictrum clavatum Expression (2 -ΔΔ CT) relative to EV (TRV-EV): Relative expression of experimental gene (TthPMX or TclPMX) in empty vector treated samples to Empty vector controls \* Thalictrum thalictroides or Thalictrum clavatum Expression (2 -ΔΔ CT) relative to EV (Untreated): Relative expression of experimental gene (TthPMX or TclPMX) in Untreated samples to Empty vector controls \* Thalictrum thalictroides or Thalictrum clavatum Expression (2 -ΔΔ CT) relative to EV (Treated): Relative expression of experimental gene (TthPMX or TclPMX) in VIGS samples to Empty vector controls 4\. Missing data codes: n/a 5\. Specialized formats or other abbreviations used: EV= Empty vector, Bioreps= biological replicates, CT= cycle threshold \######################################################################### DATA-SPECIFIC INFORMATION FOR: qPCR Tcl, Tth dissected 1\. Variables: 3 2\. Variable List: \* Thalictrum thalictroides or Thalictrum clavatum Expression (2 -ΔΔ CT) in sepal relative to highest expressing organ (carpel) \* Thalictrum thalictroides or Thalictrum clavatum Expression (2 -ΔΔ CT) in stamen relative to highest expressing organ (carpel) \* Thalictrum thalictroides or Thalictrum clavatum Expression (2 -ΔΔ CT) in carpel relative to highest expressing organ (carpel) 4\. Missing data codes: n/a 5\. Specialized formats or other abbreviations used: AVG= average, Bioreps= biological replicates, CT= cycle threshold \######################################################################### DATA-SPECIFIC INFORMATION FOR: Tth VIGS stomatal density 1\. Variables: 5 2\. Variable List: \* Quadrant: sample taken from Top or mid or base of leaf section in 3 bio-replicates \* Stomatal density 1mmx1mm: The number of stomates counted on 1mm x 1mm area \* Density Avg: Average stomatal density of 3 technical replicates \* Treatment VIGS OR EV: treated samples with target gene silencing (VIGS) or empty vector control (EV) \* Average (TOP, MID, BASE): Average stomatal density i.e top, base, or mid of leaf section 4\. Missing data codes: n/a 5\. Specialized formats or other abbreviations used: mid= middle, VIGS= VIRUS INDUCED GENE SILENCING, EV= empty vector, AVG= average \######################################################################### DATA-SPECIFIC INFORMATION FOR: Tth VIGS trichome height 1\. Variables: 2 \* Sample: Sample taken from Empty vector, Target gene silenced plants TRV-PDS-PMX (PHOTOBLEACHED OR GREEN SECTORS) or TRV-PMX \* Trichome Length (µm): Trichome length in micro meters in leaf samples taken from Empty vector, Target gene silenced plants TRV-PDS-PMX (PHOTOBLEACHED OR GREEN SECTORS) or TRV-PMX 4\. Missing data codes: none 5\. Specialized formats or other abbreviations used: PB= Photobleached, µm= micrometers, PDS= PHYTODESATURASE GENE (SILENCED) \######################################################################### DATA-SPECIFIC INFORMATION FOR: Tobacco Trichome density 1\. Variables: 2 \* Treatment on Adaxial 5mm^2 or Abaxial 5mm^2: Trichome density of adaxial or abaxial leaf surface (5mm ^2) in Target gene TthPMX overexpressors (OE) or wild type plants \* Trichome type: Type of trichome counted (T1, T2, T3, T4, T5) and with normal or abnormal appearance 4\. Missing data codes: none 5\. Specialized formats or other abbreviations used: OE= over expressor, WT= wild type, T1= type 1, T2= type 2, T3= type 3, T4= type 4, T5= type 5 \######################################################################### DATA-SPECIFIC INFORMATION FOR: Tth VIGS RNA-seq validation 1\. Variables: 5 \* Gene name= name of gene i.e. TthPMX or EEF1A or Actin \* Empty Vector TPM: Transcript per million (expression) from mock treated plants i.e. TthEV11, TthEV2, TthEV14 \* Treated TPM: Transcript per million (expression) from VIGS plants i.e. TthVIGS39, TthVIGS25, TthVIGS9 \* AVG of biorep: Average expression of gene (TthPMX, ACTIN, EEF1a) from 3 bio replicates in Empty vector treated or VIGS treated plants \* Relative expression to Mock: Average expression of target gene (TthPMX) relative to Empty vector control 4\. Missing data codes: none 5\. Specialized formats or other abbreviations used: TPM= transcript per million, VIGS= virus induced gene silencing, biorep= biological replicates, AVG= average \######################################################################### DATA-SPECIFIC INFORMATION FOR: RNA-seq Significant DEGs 1\. Variables: 10 2\. Variables list: \* ID Annotation: Unique identifier for Gene in RNA sequencing database \* log2FoldChange: change in transcripts expression between target and control group \* pvalue: chance data could occur given no difference actually exists with a cut off value of 0.05 means \* padj: p-values adjusted for multiple testing with the Benjamini-Hochberg procedure \* EV11: Expression of selected gene in control sample EV11 \* EV2: Expression of selected gene in control sample EV2 \* EV14: Expression of selected gene in control sample EV14 \* VIGS39: Expression of selected gene in comparison sample VIGS39 \* VIGS25: Expression of selected gene in comparison sample VIGS25 \* VIGS9: Expression of selected gene in comparison sample VIGS9 4\. Missing data codes: none 5\. Specialized formats or other abbreviations used: ID= Identifier, VIGS= virus induced gene silencing, padj= p value adjusted, EV= empty vector