DOI: 10.5061/dryad.mpg4f4qzb
title: The underground life of homeodomain-leucine zipper transcription factors

description: Roots are the anchorage organs of plants, responsible for water and
 nutrient uptake, exhibiting high plasticity. Root architecture is driven
 by the interactions of biomolecules, including transcription factors (TFs)
 and hormones that are crucial players regulating root plasticity. Multiple
 TF families are involved in root development; some, such as ARFs and LBDs,
 have been well characterized, whereas others remain less investigated. In
 this review, we synthesize the current knowledge about the involvement of
 the large family of homeodomain-leucine zipper (HD-Zip) TFs in root
 development. This family is divided into four subfamilies (I to IV),
 mainly according to structural features, such as additional motifs aside
 from HD-Zip, as well as their size, gene structure, and expression
 patterns. We explored and analyzed public databases and the scientific
 literature regarding HD-Zip TFs in Arabidopsis and other species. Most
 members of the four HD-Zip subfamilies are expressed in specific cell
 types and several ones from each group have assigned functions in root
 development. Notably, a high proportion of the studied proteins are part
 of intricate regulation pathways involved in primary and lateral root
 growth and development.
description: <strong>Methods used for the analysis of data from the
 repositories BAR and Root Atlas</strong>
 <strong>Analysis of tissue-specific BAR
 data</strong><br> Raw normalized expression levels for the
 HD-Zip genes and selected marker genes were downloaded from the BAR
 repository of the University of Toronto (http://bar.utoronto.ca/; Brady et
 al., 2007; Dinneny et al., 2008; Kilian et al., 2007) (Supplementary Table
 S1). As markers, we selected genes with known expression in a given tissue
 or cell type of the root. They were analyzed in the tissue for which they
 were chosen (image below). Their expression levels were averaged between
 samples (Supplementary Table S1). Expression was available for tissues in
 13 regions. For clearness, we simplified this by collapsing them, i.e.
 averaging replicates and samples together as follows: regions 1 to 7 of
 the root tip were considered zone 1; 8 to 10, zone 2; and 11 to 13 zone 3.
 The inspection of marker distribution means expression in the
 corresponding tissues, which was dispersed, allowing us to define a
 conservative expression threshold of 50, for which most of the markers had
 at least one mean expression value above it. We then applied this
 threshold to HD-Zip genes mean expression, obtained by an analogous
 procedure, to classify TFs as expressed, or not, in each tissue or cell
 type (Supplementary Table S2). Mean expression of marker genes in the
 corresponding tissue in which they have reported as expressed. Horizontal
 lines show expression levels of 10, 50, and 100, of which 50 was chosen as
 a threshold. <strong>Analysis of abiotic stress
 data from the BAR repository</strong><br> Raw expression data
 for Abiotic Stress was downloaded from the BAR repository and averaged
 between replicates. Responses to different stress factors were the
 collapsed into Early responses, 15 and 30 min, 1, 3 and 4 hours after
 treatment; and Late responses, 6, 12 and 24 hours aftertreatment. To
 recover the expression peak in each time window, the maximum expression
 level, and not the average, was kept among mean expression values of all
 considered time points. We then applied the same threshold, defined for
 tissues (50), which was capable of identifying many HD-Zip I TFs known for
 their response to several of the abiotic stresses evaluated. Using this
 threshold, we classified all HD-Zip TFs as expressed, or not, in early or
 late stress responses (Supplementary Table S3).
 <strong>Analysis data from the Root Atlas
 repository</strong><br> Raw LogNormalized expression data for
 all HD-Zip genes and 7695 cells were downloaded from the Root Atlas
 repository (Zhang et al., 2019; http://wanglab.sippe.ac.cn/rootatlas;
 Supplementary Table S1). Provided cluster assignment for each cell allowed
 us to analyze cell expression distribution for each HD Zip gene in each of
 the 24 clusters. In this case, we defined a threshold based on the
 expression of HZ-Zip III TFs in roots according to the literature. We then
 defined a gene to be expressed in a cluster if a proportion of 0.03 of the
 cells in the cluster presented an expression level above 0.5. Based on
 this criterion, we classified HD-Zip genes as expressed, or not expressed,
 in each cluster (Supplementary Table S4).