DOI: 10.5061/dryad.rbnzs7hhn
title: Data from: Sorghum bicolor TX08001 nodal root tissue development gene expression profiling

description: Bioenergy sorghum’s large nodal root system enables deposition of soil
 organic carbon deep in soil profiles aiding production of low carbon
 intensity biofuels from this crop. During bioenergy sorghum’s long growing
 season, plants produce ~175 nodal roots In review bearing lateral roots
 that take up water and nutrients from &gt;2 m deep in soil profiles,
 and aerial roots that support a complex phyllosphere. In the current
 study, nodal root bud development, a slow process spanning ~40 days, was
 characterized using microscopy and transcriptome analysis. A first ring of
 10-15 nodal root buds was initiated in the stem pulvinus of phytomer 7
 near sub-epidermal vascular bundles. A second ring of buds formed above
 the first ring much later in phytomer development. Nascent nodal root buds
 from phytomer 7 exhibited relatively high expression of pericycle marker
 genes (PFA) and genes involved in auxin transport (ABCB19, PIN4, LAX2),
 cytokinin signaling (TSO, MYB3R1), and cell proliferation (CYCB2;4,
 CDKB2;1, REM1). Following initiation, expression of genes involved in cell
 proliferation and cytokinin-signaling decreased while expression of genes
 involved in proliferative arrest, ABA-signaling, dormancy and stress
 tolerance increased. Further bud development was correlated with increased
 expression of WOX11 and PLT5 followed by PLT2, PLT4 and genes encoding RGF
 peptides that regulate PLT-expression and bud development. Expression of
 the ARF7-regulated LBD29, a gene required for nodal root formation,
 increased in parallel with increasing bud size to a maximum late in NRB
 development. Appearance of the nodal root bud cap late in development
 coincided with expression of SMB and FEZ, whereas genes such as WOX5 and
 two MYB36 family members were expressed at higher levels in outgrowing
 aerial roots. Genes involved in gibberellin, brassinosteroid,
 strigolactone, ethylene, jasmonate, salicyclic acid, and eATP signaling
 showed complex patterns of expression during nodal root bud formation.
 Overall, this study provides a detailed description of bioenergy sorghum
 nodal root bud development and transcriptome information useful for
 molecular analysis of networks that regulate nodal root development.
description: NRB samples for RNA-Seq were harvested from TX08001 field grown
 plants at 120 DAE. Prior to NRB tissue collection, leaf sheaths were
 removed using a scalpel exposing NRBs on stem nodes. A sterile razor was
 used to excise a wedge of stem pulvinus rind tissue approximately 3-4 mm
 deep that contained the nodal root buds. Three biological replicates were
 taken from node 7 through node 21 for a total of 40 samples. Node 17 was
 sampled but the resulting data was not included in the analysis. After
 excision, the tissue samples were placed in Whirl-Pak® sample bags and
 flash-frozen in liquid nitrogen. Tissues were ground to a fine powder with
 a heat sterilized mortar and pestle filled with liquid nitrogen then
 transferred into liquid- nitrogen chilled sterile 1.5 mL centrifuge tubes.
 RNA was extracted using the Zymo RNA Mini- Prep kit. Purity and
 concentration of the RNA was analyzed using a Thermo ScientificTM NanoDrop
 One Microvolume UV-Vis Spectrophotometer before being sent for
 fragmentation analysis on an Agilent 5300 Fragment Analyzer using software
 version 3.1.0.12. RNA that passed QC was sent to the Joint Genome
 Institute for sequencing to a depth of 30–50 million reads. Sequenced
 reads were aligned to the Sorghum bicolor V3.1 genome using HISAT2 aligner
 (Kim, Langmead et al. 2015, McCormick, Truong et al. 2018). The
 transcriptome assembly and TPM normalization were conducted using
 StringTie version 1.3 In review (Pertea, Pertea et al. 2015). The script
 prepDE.py was used to convert nucleotide coverage data from StringTie into
 read counts that were readable by differential expression statistical
 packages. Functional annotations of the transcripts were obtained from the
 Sorghum bicolor V3.1 genome which is available from Phytozome 13
 (McCormick, Truong et al. 2018). Aerial nodal root sample 1 (AR1) extended
 from the root tip to 4-5 mm into the growing zone and aerial nodal root
 sample 2 (AR2) was a 10 mm section of the root adjacent to AR1 that
 extended into the more developed portion of the root.
 Visualization of the relationships between biological replicates
 of NRB samples and the aerial nodal root samples was conducted using UMAP
 (McInnes, Healy et al. 2018). Figures were generated using GIMP 2.10.30
 (Team 2019).