Data from: Sorghum bicolor TX08001 nodal root tissue development gene expression profiling
Data files
Jul 31, 2023 version files 27.69 MB
Abstract
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 >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.
Methods
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).