biostudies-arrayexpressPriscilla GlennSorghum bicolorhttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15090Bioenergy sorghum’s large and deep nodal root system and associated microbiome enables uptake of water and nutrients from and deposition of soil organic carbon into soil profiles, key contributors to the crop’s resilience and sustainability. The goal of this study was to increase our understanding of bioenergy sorghum nodal root bud development. Sorghum nodal root bud initiation was first observed on the stem node of the 7th phytomer below the shoot apex. Buds were initiated near the upper end of the stem node pulvinus on the side of the stem opposite the tiller bud, then additional buds were added over the next 6-8 days forming a ring of 10-15 nascent nodal root buds around the stem. Later in plant development, a second ring of nodal root buds began forming on the 17th stem node immediately above the first ring of buds. Overall, nodal root bud development can take ~40 days from initiation to onset of nodal root outgrowth. Nodal root buds were initiated in close association with vascular bundles in the rind of the pulvinus. Stem tissue forming nascent nodal root buds expressed sorghum homologs of genes associated with root initiation (WOX4), auxin transport (LAX2, PIN4), meristem activation (NGAL2), and genes involved in cell proliferation. Expression of WOX11 and WOX5, genes involved in root stem niche formation, increased early in nodal root bud development followed by genes encoding PLTs, LBDs (LBD29), LRP1, SMB, RGF1 and root cap LEAs later in development. A nodal root bud gene regulatory network module expressed during nodal root bud initiation predicted connections linking PFA5, SPL9 and WOX4 to genes involved in hormone signaling, meristem activation, and cell proliferation. A network module expressed later in development predicted connections among SOMBRERO, a gene involved in root cap formation, and GATA19, BBM, LBD29 and RITF1/RGF1 signaling. Overall, this study provides a detailed description of bioenergy sorghum nodal root bud development and transcriptome information useful for understanding the regulation of sorghum nodal root bud formation and development.biostudies-arrayexpressSample Collection - At 120 days after emergence (DAE), five TX08001 field-grown plants were harvested for morphological analysis and RNA-seq. Leaf blades were removed for transport, and in the laboratory, leaf sheaths were dissected using a scalpel to expose the stem tissue. The relative position, number, and size of nodal root buds (NRBs) on each stem node were measured. NRB tissue samples were collected from stem nodes of phytomers 7-21 by excising a 3-4 mm deep wedge of pulvinus rind tissue containing the NRBs. Three replicate plants were sampled, and mid-internode stem rind samples from phytomers 6, 8, 9, 20, and 21, where NRBs do not form, were collected as controls. All tissue samples were flash-frozen in liquid nitrogen and stored in Whirl-Pak® bags for further analysis. In addition, plants containing 7 phytomers with fully elongated internodes were harvested at 40 DAE to investigate the number of NRB rings on the nodes of plants containing less than 17 developed phytomers.Growth Protocol - The bioenergy sorghum hybrid TX08001 was planted at the Texas A&M University Farm in Burleson County, TX on 4/29/2021; plant emergence occurred 6-days after planting on 5/5/2021. Plot size was thirty-two 30 m long rows at a row spacing of 76 cm. At the time of planting, a solution of liquid ammonium polyphosphate (11-37-0), UAN 32%, and zinc sulfate was applied at a depth of 5 cm to the side of the seed for a total fertilizer yield of 45-63-0 + 5 Zn kg/ha. The soil consisted of Weswood Silt Loam (Staff, 2022). The plot was sown with enough seeds to allow thinning to 15 cm plant spacing within rows at 21 days after emergence (DAE). Seeds, before planting, were treated with Concept III®, a herbicide protectant, Nugro® a systemic insecticide and Apron X®, a systemic fungicide.Nucleic Acid Extraction - 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 c 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.Sequencing - 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 et al., 2015).Library Construction - Plate-based RNA sample prep was performed on the PerkinElmer Sciclone NGS robotic liquid handling system using Illumina's TruSeq Stranded mRNA HT sample prep kit utilizing poly-A selection of mRNA following the protocol outlined by Illumina in their user guide: https://support.illumina.com/sequencing/sequencing_kits/truseq-stranded-mrna.html, and with the following conditions: total RNA starting material was 1000 ng per sample and 8 cycles of PCR was used for library amplification. The prepared libraries were quantified using KAPA Biosystems' next-generation sequencing library qPCR kit and run on a Roche LightCycler 480 real-time PCR instrument. Sequencing of the flowcell was performed on the Illumina NovaSeq sequencer using NovaSeq XP V1.5 reagent kits, S4 flowcell, following a 2x151 indexed run recipe.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - The transcriptome assembly and TPM normalization were conducted using StringTie version 1.3 (Pertea et al., 2015). The script prepDE.py https://github.com/gpertea/stringtie/blob/master/prepDE.py and https://ccb.jhu.edu/software/stringtie/index.shtml?t=manual was used to convert nucleotide coverage data from StringTie into read counts that were readable by differential expression statistical packages using the formula: reads_per_transcript = coverage * transcript_length/read_length. Read length was 151 bp per read. Functional annotations of the transcripts were obtained from the Sorghum bicolor V3.1 genome which is available from Phytozome 13 (McCormick et al., 2018).UnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq XBioenergy sorghum's large and deep nodal root system and associated microbiome enables uptake of water and nutrients from and deposition of soil organic carbon into soil profiles, key contributors to the crop's resilience and sustainability. The goal of this study was to increase our understanding of bioenergy sorghum nodal root bud development. Sorghum nodal root bud initiation was first observed on the stem node of the 7th phytomer below the shoot apex. Buds were initiated near the upper end of the stem node pulvinus on the side of the stem opposite the tiller bud, then additional buds were added over the next 6-8 days forming a ring of 10-15 nascent nodal root buds around the stem. Later in plant development, a second ring of nodal root buds began forming on the 17th stem node immediately above the first ring of buds. Overall, nodal root bud development can take ~40 days from initiation to onset of nodal root outgrowth. Nodal root buds were initiated in close association with vascular bundles in the rind of the pulvinus. Stem tissue forming nascent nodal root buds expressed sorghum homologs of genes associated with root initiation (WOX4), auxin transport (LAX2, PIN4), meristem activation (NGAL2), and genes involved in cell proliferation. Expression of WOX11 and WOX5, genes involved in root stem niche formation, increased early in nodal root bud development followed by genes encoding PLTs, LBDs (LBD29), LRP1, SMB, RGF1 and root cap LEAs later in development. A nodal root bud gene regulatory network module expressed during nodal root bud initiation predicted connections linking PFA5, SPL9 and WOX4 to genes involved in hormone signaling, meristem activation, and cell proliferation. A network module expressed later in development predicted connections among SOMBRERO, a gene involved in root cap formation, and GATA19, BBM, LBD29 and RITF1/RGF1 signaling. Overall, this study provides a detailed description of bioenergy sorghum nodal root bud development and transcriptome information useful for understanding the regulation of sorghum nodal root bud formation and development.RNA-seq of coding RNASorghum bicolorBioenergy sorghum nodal root bud development: morphometric, transcriptomic and gene regulatory network analysisBrian McKinleyAustin Lamb, Evan Kurtz, Priscilla Glenn, Brian A. McKinley, John MulletJohn MulletPriscilla GlennfalseNodal Root Bud Development - Transcriptome AnalysisBioenergy sorghum’s large and deep nodal root system and associated microbiome enables uptake of water and nutrients from and deposition of soil organic carbon into soil profiles, key contributors to the crop’s resilience and sustainability. The goal of this study was to increase our understanding of bioenergy sorghum nodal root bud development. Sorghum nodal root bud initiation was first observed on the stem node of the 7th phytomer below the shoot apex. Buds were initiated near the upper end of the stem node pulvinus on the side of the stem opposite the tiller bud, then additional buds were added over the next 6-8 days forming a ring of 10-15 nascent nodal root buds around the stem. Later in plant development, a second ring of nodal root buds began forming on the 17th stem node immediately above the first ring of buds. Overall, nodal root bud development can take ~40 days from initiation to onset of nodal root outgrowth. Nodal root buds were initiated in close association with vascular bundles in the rind of the pulvinus. Stem tissue forming nascent nodal root buds expressed sorghum homologs of genes associated with root initiation (WOX4), auxin transport (LAX2, PIN4), meristem activation (NGAL2), and genes involved in cell proliferation. Expression of WOX11 and WOX5, genes involved in root stem niche formation, increased early in nodal root bud development followed by genes encoding PLTs, LBDs (LBD29), LRP1, SMB, RGF1 and root cap LEAs later in development. A nodal root bud gene regulatory network module expressed during nodal root bud initiation predicted connections linking PFA5, SPL9 and WOX4 to genes involved in hormone signaling, meristem activation, and cell proliferation. A network module expressed later in development predicted connections among SOMBRERO, a gene involved in root cap formation, and GATA19, BBM, LBD29 and RITF1/RGF1 signaling. Overall, this study provides a detailed description of bioenergy sorghum nodal root bud development and transcriptome information useful for understanding the regulation of sorghum nodal root bud formation and development.2025-05-10T00:00:00Z2025-04-24T16:59:07.286Z2025-04-24T16:59:07.286ZE-MTAB-1509039498396ERP171954EFO_0002944EFO_0004170EFO_0003789EFO_0005518EFO_0003816EFO_0003738EFO_000418410.3389/fpls.2024.1456627