ABSTRACT
Small signaling peptides (SSPs) are important regulators of plant growth, development, and responses to biotic and abiotic stress, yet their role in the C4 grass Sorghum bicolor is largely uncharacterized. To help fill this knowledge gap, 219 sorghum genes that encode SSPs were identified based on SSP sequences previously identified in Arabidopsis thaliana , Zea mays , Oryza sativa , Triticum aestivum , and Brachypodium distachyon . The 219 sorghum SSP‐encoding genes were assigned to 19 gene families, analyzed for the presence of motifs, and aligned with genes that encode SSPs in other plants using phylogenetic analysis. Sorghum genes in 12 of the 19 SSP gene families had not been previously characterized. Expression of the 219 SSP‐encoding genes in sorghum organs, during stem development, and in stem tissues and cell types revealed distinct spatial, temporal, and developmental patterns of expression. Genes associated with the SbCEP and SbRGF families were preferentially expressed in roots, whereas SbEPF genes were expressed in stem epidermal and pith parenchyma cells and panicles. The expression of genes during bioenergy sorghum stem growth and development was investigated because stems account for ~80% of harvested biomass and serve as conduits for water and nutrient transport between leaves and roots. During stem development, 28 SSP genes in several families (CLE, EPF, CEP, GASS, PSY, ES, PSK, CAPE, POE) were expressed at higher levels in zones of cell proliferation. For example, the TDIF homologs SbCLE41 and SbCLE42 were expressed at high levels in nascent stem nodes where they may regulate vascular bundle cambial activity and cell differentiation. A different set of 15 genes in the CIF, POE, CAPE, PSY, CEP, RALF, and CLE families were expressed at higher levels in zones of stem tissue differentiation highlighted by elevated expression of five SbRALFRs in the stem nodal plexus. Cell type–specific expression of many sorghum genes that encode SSPs was observed in fully elongated internodes indicating gene expression is regulated with high spatial resolution. Overall, the results provide a foundation of information for analysis of SSP function in sorghum that can be integrated with knowledge of sorghum gene regulatory networks to modulate traits important for production of sorghum crops.
Keywords: bioenergy sorghum, growth regulation, phylogenetics, small signaling peptides, stem development, transcriptome
1. Introduction
Small signaling peptides (SSPs) are hormone‐like regulators of plant growth and development, mediating intercellular communication and modulation of meristem activity, vascular patterning, cell elongation, and stress responses. Large gene families encode related peptides such as CLAVATA3/Embryo Surrounding Region (CLE) peptides, C‐terminally encoded peptides (CEP), PHYTOSULFOKINE (PSK), and RAPID ALKALINIZATION FACTOR (RALF) (Lu and Xiao 2024; Xiao et al. 2025). SSPs are encoded as prepropeptide precursors that have three functional domains: an N‐terminal secretion signal peptide (~20 aa) that routes the precursor through the ER and Golgi apparatus into the secretory pathway, a central variable region of 50–100 aa, and a C‐terminal domain of 5 to ~100 aa containing the mature bioactive peptide sequence (Fletcher 2020; Datta et al. 2024; Zhou, Zheng, et al. 2024). During secretory pathway transit, many precursors undergo post‐translational modifications (PTMs) including tyrosine sulfation, proline hydroxylation, and hydroxyproline arabinosylation, which alter peptide charge, hydrophilicity, and conformation to regulate receptor binding affinity (Stührwohldt et al. 2021; Royek et al. 2022).
Subtilisin‐like serine proteases (SBTs) recognize conserved cleavage motifs within SSP precursors and process these proteins in secretory compartments such as the Golgi/trans‐Golgi network or after secretion into the apoplast depending on the SSP family and subtilase involved (Schardon et al. 2016; Stührwohldt et al. 2020; Deng et al. 2025). Following PTM, SSPs move via the symplast or apoplast, locally or over long distances through the plant vasculature. SSPs interact with plasma membrane‐localized leucine‐rich repeat receptor‐like kinases (LRR‐RLKs) by binding to their extracellular domains, with co‐receptors often conferring signaling specificity (Narasimhan and Simon 2022; Datta et al. 2024). Ligand binding activates the LRR‐RLK's intracellular kinase domain, initiating downstream signaling cascades involving MAPK modules, calcium signaling, and ROS, which converge on transcription factors that regulate genes governing cell proliferation, differentiation, organ development, or stress adaptation, ultimately producing changes at the cell, tissue, organ, or whole‐plant level (Fletcher 2020; Zhou et al. 2022; Xiao et al. 2025).
Bioenergy sorghum ( Sorghum bicolor ) is a highly productive drought tolerant C4 grass that is well adapted to annual US cropland designated for bioenergy crops (Rooney et al. 2007; Mullet et al. 2014; Langholtz et al. 2024). Sorghum's C4 photosynthesis enhances its productivity, reduces nitrogen requirements and water use (Byrt et al. 2011) while its deep and extensive root system contributes to nutrient uptake and deposition of soil organic carbon (Lamb et al. 2022). Bioenergy sorghum's productivity, low carbon intensity (Olson et al. 2012; Gautam et al. 2020), low input requirements (Olson et al. 2013), and resilience make it a promising bioenergy crop for annual bioenergy cropland (Rooney et al. 2007; Maw et al. 2017).
Bioenergy sorghum hybrids are very photoperiod sensitive, which delays flowering in long days, increasing the duration of vegetative phase growth and biomass yield. At the end of the growing season, bioenergy sorghum stems are often 4–5 m long and account for ~80% of harvested biomass (Olson et al. 2012). Stem biomass is composed of cell walls composed primarily of cellulose, glucuronoarabinoxylan (GAX), and lignin and nonstructural carbohydrates such as sucrose, glucose, fructose, starch, and mixed‐linkage glucans (MLGs) (McKinley et al. 2016; McKinley, Olson, et al. 2018). Sugars derived from cellulose and nonstructural carbohydrates can be readily converted into biofuels such as ethanol, butanol, and sustainable aviation fuel (SAF) and other bioproducts.
Grass stems are composed of nodes and internodes. Stem nodes span the pulvinus, a tissue that produces tiller buds (Kebrom et al. 2017) and nodal root buds (Lamb et al. 2024), and the nodal plexus, a stem tissue located below the pulvinus where the leaf sheath joins the stem. Stem internode tissue develops between nodes from an intercalary meristem (IM) located at the base of the internode just above the pulvinus (Serrano‐Mislata and Sablowski 2018; McKim 2019; Yu et al. 2021). During the vegetative phase, a new stem node is formed below the shoot apical meristem approximately every 3–4 days. Therefore, at the end of bioenergy sorghum's long vegetative growing season, plants contain > 30 node–internode stem segments (Olson et al. 2012). The formation, growth, and development of stem node–internode segments is a key determinant of bioenergy sorghum biomass accumulation since longer, thicker, and more dense stems contribute to greater stem biomass accumulation (Kebrom et al. 2017; McKinley, Olson, et al. 2018; Dos Santos et al. 2020).
Stem internode growth is regulated by genetic, hormonal, and environmental factors. Sorghum dwarfing loci that are used to reduce grain sorghum stem length provided early insights into the regulation of stem internode growth. Sorghum Dw3 encodes an ABCB19 auxin (IAA) export transporter (Multani et al. 2003), Dw1 encodes a protein involved in brassinosteroid (BR) signaling (Hilley et al. 2016; Yamaguchi et al. 2016), and Dw2 encodes an AGCVIII kinase (Hilley et al. 2017) that modulates endomembrane trafficking (Oliver et al. 2021). Shade avoidance responses mediated by PhyB‐signaling increase the length of sorghum internodes in part by increasing expression of GA3ox2 and through gibberellin (GA)‐enhanced stem internode elongation (Beall et al. 1991; Yu et al. 2021). In addition, BR (Mantilla Perez et al. 2014), cytokinins (CK) (Amzallag et al. 1992), and ethylene (Finlayson et al. 1999) also modulate stem growth.
Studies of sorghum stem development provided further insight into the molecular basis of stem growth regulation. Stem node–internode segments formed during the juvenile phase contain minimal internode tissue. The crown root system grows out from this initial stack of below ground stem nodes. In contrast, aboveground stem nodes produced during the adult vegetative phase are separated from each other by internodes of varying length. The early stage of internode tissue development is characterized by active cell proliferation and elongation. This is followed by secondary cell wall formation, cell wall lignification, and inactivation of the IM (Kebrom et al. 2017). Genes involved in auxin (IAA), GA, BR, and CK hormone metabolism and signaling are differentially regulated during internode development. Analysis of gene expression in the stem internode IM during sorghum internode development enabled the identification of a gene regulatory network that modulates the expression of genes involved in cell proliferation in response to changes in IAA, GA, BR, and CK (Yu et al. 2022).
Sorghum homologs of GA responsive AtGASA4 and AtGASA14 genes are present in a gene regulatory network that modulates stem IM activity (Yu et al. 2022). GASA SSPs, characterized by their conserved cysteine‐rich motifs, are regulated by gibberellins and influence cell proliferation, elongation, and stress adaptation (Herzog et al. 1995; Bouteraa et al. 2023). They mediate hormonal crosstalk between GA, BRs, and ABA (Chen et al. 2021), with roles in stem elongation (AtGASA2; Bouteraa et al. 2023), grain size regulation (OsGASR9; Li et al. 2019), and stress tolerance (i.e., AtGASA11 in ROS scavenging) (Chen et al. 2021). Additional functions include zinc homeostasis (OsGASR10; Nanda et al. 2017), pathogen defense (OsGASR7; Boonpa et al. 2018), and reproductive development (AtGASA13; Fan et al. 2017).
The potential importance of SSPs in bioenergy sorghum growth and development and their role in plant resilience encouraged us to conduct further analysis of sorghum genes that encode SSPs. Previous studies have identified sorghum SSPs like CEP (Roberts et al. 2013; Ogilvie et al. 2014; Aggarwal et al. 2020), CLE (Goad et al. 2017), EPF (Takata et al. 2013; Li et al. 2014; Caine et al. 2016), GASA (Muhammad et al. 2019), POE (Jiménez‐López et al. 2011), RALF (Sharma et al. 2016; Campbell and Turner 2017; Abarca et al. 2021), and RGF (Fang et al. 2021). However, sorghum genes encoding other SSP families such as CAPE (Chien et al. 2015), CIF (Okuda et al. 2020; Fujita 2021), DVL (Narita et al. 2004; Wen et al. 2004), ECL (Sprunck et al. 2012), ES (Woriedh et al. 2015), IDA (Vie et al. 2015), MEG (Xiong et al. 2014), PIP (Hou et al. 2014; Zhou et al. 2022), PNP (Turek and Gehring 2016), PSK (Matsubayashi et al. 2006; Stührwohldt et al. 2021), PSY (Tost et al. 2021), and TPD (Huang et al. 2016) had not been identified and analyzed in sorghum. The goal of the current study is to identify genes that encode SSPs in sorghum, determine their relationship to SSPs in other plant species and analyze their expression in different organs, stages of stem development, tissues, and cell types. A more complete characterization of SSPs gene expression and function in sorghum may elucidate ways to modify SSP‐based regulation to enhance bioenergy sorghum performance and sustainability.
2. Materials and Methods
2.1. Identification of Sorghum Genes That Encode SSPs
SSPs, and the genes that encode them, have been identified in Arabidopsis thaliana , Zea mays , Oryza sativa , Sorghum bicolor , Triticum aestivum , and Brachypodium distachyon . Prior studies identified sorghum genes that encode SSPs named CEP (Roberts et al. 2013, Ogilvie et al. 2014, Aggarwal et al. 2020), CLE (Goad et al. 2017), EPF (Takata et al. 2013, Li et al. 2014, Caine et al. 2016), GASA (Muhammad et al. 2019), POE (Jiménez‐López et al. 2011), RALF (Sharma et al. 2016, Campbell and Turner 2017, Abarca et al. 2021), and RGF (Fang et al. 2021). Other SSP gene families including CAPE (Chien et al. 2015), CIF (Okuda et al. 2020, Fujita 2021), DVL (Narita et al. 2004, Wen et al. 2004), ECL (Sprunck et al. 2012), ES (Woriedh et al. 2015), IDA (Vie et al. 2015), MEG (Xiong et al. 2014), PIP (Hou et al. 2014, Zhou et al. 2022), PNP (Turek and Gehring 2016), PSK (Matsubayashi et al. 2006, Stührwohldt et al. 2021), PSY (Tost et al. 2021), and TPD (Huang et al. 2016) were identified in Arabidopsis thaliana , Zea mays , or Triticum aestivum and those sequences were utilized to identify sorghum SSP genes. Additionally, Triticum aestivum and Zea mays sequences for these SSP families were extracted from a large‐scale analysis (Li et al. 2014; Tian et al. 2022).
The published SSP sequences were used to identify sorghum genes in the BTx623 reference genome v3.1.1 (Phytozome v13) using BLASTP analysis. The searches utilized published SSP sequences from Arabidopsis thaliana TAIR10, Zea mays RefGen_V4, Oryza sativa v7.0, Sorghum bicolor v3.1.1, Triticum aestivum v2.2, and/or Brachypodium distachyon v3.2 as queries. To validate this method for identifying previously unannotated SSP in sorghum, well‐annotated SSPs like CLE, GASA, RALF, and RGF families were analyzed and the results compared to prior analyses. The resulting Sorghum bicolor SSP‐encoding genes are reported for CLE (Table S2), GASA (Table S3), RALF (Table S4), and RGF (Table S5). Among these four families, only the RALF family contained SSPs not previously reported in Sorghum bicolor . The seven putative RALF encoding sorghum genes exhibited some regions of sequence similarity (E‐value < 1 × 10−4) to RALF SSPs identified in other species, but further confirmation will be required to determine if these genes encode functional RALFs. Identifying genes that encode signaling SSPs can be challenging due to their short open reading frames and highly variable central regions (Hu et al. 2021; Liang et al. 2021; Ren et al. 2024).
Some SSP families contained conserved domain annotations, which were used to further validate the identity of genes encoding these SSPs. The domain annotations included: RALF (PF05498), EPF (PTHR33109), PSK (PF06404), GASA (PF02704), POE (PF01190), and DVL (PF08137).
To identify conserved motifs, SSP sequences from each family were analyzed using MEME Suite Version 5.5.7 in classic mode. The motif distribution was set to “Zero or One Occurrence Per Sequence” (ZOOPS), and the minimum motif width was fixed at six amino acids for all families. For the CLE and RGF families, the maximum motif width was set to 50 amino acids, while for the GASA and RALF families, it was increased to 60 amino acids to accommodate their longer open reading frames and extended SSP lengths. The number of motifs to be identified was set to three for the CLE and RGF families and four for the GASA and RALF families, reflecting the increased sequence complexity of the latter. All input sequences were retained in the MEME output, and the resulting motif images were arranged in tabular format according to phylogenetic order to facilitate visual comparison of motif patterns.
2.2. Phylogenetic Analysis
Full preprocessed SSP sequences and their variants encoded by each sorghum gene described above were collected from BioMart on Phytozome v13. Phylogenetic analysis was conducted separately for each SSP family. Multiple sequence alignments were performed using MUSCLE, and the best fit protein substitution model was determined using ModelTest‐NG v0.1.7 based on the lowest AICc value. All SSP families followed a gamma‐distributed model; however, the CAPE, ES, PIP, and PSY families additionally required invariant sites. The WAG model was selected for CAPE, ES, PIP, and PNP; variable‐rate models for CEP, CIF, GASA, MEG, POE, and RGF; PMB for CLE, EPF, and RALF; and JTT for DVL, ECL, IDA, PSK, PSY, and TPD. Maximum‐likelihood phylogenetic trees were inferred using RAxML v8.2.12 with 1000 bootstrap replicates, running on the Terra and Grace supercomputers at the Texas A&M High Performance Research Computing (HPRC) facility. Trees were visualized and annotated using iTOL v7 (Letunic and Bork 2024) and Dendroscope3, enabling interactive exploration and comparative analysis. The best homolog for a sorghum gene encoding a SSP was extracted from the phylogenetic tree based on proximity of a gene encoding a SSP in Zea mays , Oryza sativa , and Arabidopsis thaliana in the phylogenetic tree topology (Table S6–S9). Phylogenetic trees were also generated for Sorghum bicolor SSPs independently to determine their relative order and assign gene names accordingly.
2.3. Collection of Stem Tissues From TX08001 Internodes for Transcriptome Analysis
Sorghum genotype TX08001 was grown in the Automated Precision Phenotyping Greenhouse at Texas A&M University during the fall under long‐day conditions until 52 days after emergence (DAE) in the vegetative stage. Plants were cultivated in rhizotrons filled with a mixture of 30% clay and 70% sand. Fertilization was performed using two tablespoons of Osmocote 14–14–14, and watering was provided every 2–3 days. Shoots were harvested in biological quadruplicates, and tissues were collected from Phytomers 1–4 and 6–7. Full stem node–internode samples were taken from Phytomers 1–4, the youngest phytomers below the shoot apical meristem. For Phytomers 6 and 7, which had partially elongated stem internodes, internode tissue samples were collected from the IM, located at the base of the internode, to the top of the internode adjacent to the nodal plexus. The pulvinus and nodal plexus were excised separately as 5–10‐mm sections, while the IM was sampled as a 5‐mm section. Additionally, 10‐mm sections above the IM were collected to capture internode tissues at various stages of elongation and maturation. The upper and oldest portion of each stem internode was divided into 20‐mm sections for analysis of later stages of internode maturation. Diagrams showing the pattern of stem dissection are included in Figures 4b and 5b.
FIGURE 4.
Expression of genes encoding SSPs that are highly expressed in sorghum stem–growing zones. (a) Heat map of peptide gene expression in stems of sorghum genotype TX08001. Stem tissues with active cell proliferation (highlighted in yellow) were identified using SbCDKB2 as a cell proliferation biomarker. Nascent nodes of Phytomers 1–4 (Phy1–4) and the internode intercalary meristem (IM) and adjacent zone of elongation (ZoE) of phytomers (Phy) 6 and 7 were identified as regions of cell proliferation. (b) Stem tissue identification based on the analysis of Yu et al. (2022). Stem tissues collected for RNA‐seq analysis included apical tissue spanning the SAM (shoot apical meristem), IM (intercalary meristem), and ZoE (zone of elongation) of the internode (Int). Nascent nodes were collected from Phy1–4. Stem Internode Sections 6‐1 and 7‐1 were 5 mm long, whereas 6‐2 and 7‐2 were 10‐mm sections.
FIGURE 5.
Expression of genes that encode SSPs in sorghum stem–differentiating zones. (a) Heat map of peptides encoding genes that show elevated expression during stem tissue differentiation of sorghum genotype TX08001. Regions of cell differentiation were identified using SbCESA4 as a secondary cell wall formation biomarker. The orange highlight was used to denote stem nodal plexus (NP) tissue whereas the green highlight was used to denote stem pulvinus tissue. The purple highlight corresponds to tissues within the internode (Int) zone of maturation (ZoM) that have completed the elongation process and expressing genes involved in secondary cell wall synthesis. (b) Stem tissue identification based on the analysis of Yu et al. (2022). The sections corresponding to 6‐3 and 7‐3 were 5 mm long, 7‐4 and 7‐5 were 10 mm long, and Section 7‐6 was 20 mm in length.
2.4. K‐Means Clustering of TX08001 SSP Transcriptome Data
Transcriptome data on sorghum genes that encode SSPs shown in Figures 4a and 5b were clustered to identify cohorts with distinct expression patterns. The optimum number of cohorts was identified using the “wss” (within sum of square) and/or “silhouette” methods from the “factoextra” package in R (Lamb et al. 2024). K‐means clustering was performed using the “cluster” package to identify the genes within each cohort. For each cohort extracted, the median expression of the cluster was calculated and plotted using “ggplot” package in R.
2.5. RNA Purification, Sequencing, and RNA‐Seq Analysis
Methods used for RNA purification and RNA‐seq data collection and analysis were the same as described in previous publications (Lamb et al. 2024; Yu et al. 2025). 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 MiniPrep kit. Purity and the concentration of the RNA were analyzed using a Thermo Scientific NanoDrop One Microvolume UV–Vis Spectrophotometer. The integrity of sample RNA was assessed with 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 the HISAT2 aligner (Kim et al. 2015). 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. Sequence read length was 151 bp. Functional annotations of the transcripts were obtained from the Sorghum bicolor V3.1 genome, which is available from Phytozome 13 (McCormick et al. 2018). RNA‐seq data were obtained from three biological replicates of all tissues analyzed. Differential gene expression analysis was conducted using the “limma” package in R. TPM normalized transcript data were log2‐transformed. A linear model was fitted to the data using the log2‐transformed expression values. An empirical Bayes method was applied to the model to assess the standard errors of the estimated log‐fold changes. Differentially expressed genes were identified, and the results were adjusted for multiple testing using the false discovery rate (FDR) method. Genes with an adjusted p‐value < 0.05 were used in the analysis. Fold changes were converted from log2 scale to linear scale for ease of interpretation. TAU analysis was conducted on portions of the data set to provide a gauge of organ and cell‐type specificity (Yanai et al. 2005). Some figures contain results of analyses of genes expressed at five TPM or more in at least one sample of the selected dataset to provide a simplified survey of genes with relatively high expression. Data on all genes were included in Supporting Information tables. Some tables and figures excluded genes that showed differential expression of less than fivefold to show patterns of expression focused on genes with higher variation in expression during development. Expression data on SSP‐genes with max TPM < 5 is included in the Supporting Information tables.
2.6. Analysis of SSP‐Encoding Gene Expression in Plant Organs
Some RNA‐seq data used in this study were obtained from prior analyses of leaves, stems, roots, and panicles of several sorghum genotypes at different stages of development (McKinley et al. 2016; Kebrom et al. 2017; McCormick et al. 2018; McKinley, Casto, et al. 2018; McKinley, Olson, et al. 2018; Kebrom et al. 2020; Lamb et al. 2022; Koleva et al. 2025). This dataset was analyzed to estimate relative organ‐level expression of genes that encode SSPs by averaging the expression of genes in tissues that comprise each organ (average of TPM values). The tissues analyzed for each organ were as follows. Leaf organ expression was based on RNA‐seq data from the whole leaf, leaf blade, leaf sheath, and leaf whorl. Stem organ expression was based on data from the juvenile stem, stem apex, apical dome, full internode, nodal plexus, pulvinus, and IM. Root organ expression was based on RNA‐seq analysis of mature roots, elongating roots, meristematic roots, and roots furthest from the stem and deeper in the soil profile. Reproductive stage organ expression was based on RNA‐seq data from the peduncle, upper and lower panicle, and seeds.
2.7. Expression of SSP‐Encoding Genes in Developing Stem Internodes
RNA‐seq data from a prior study of R07020 internode development (Kebrom et al. 2017) were used in the current study to characterize the expression of SSP‐encoding genes in internodes that were beginning to elongate (Internode 1), in the process of elongation (Internodes 2 and 3), and a fully elongated internode (Internode 4).
2.8. Stem Cell–Type Expression of SSP‐Encoding Genes
Laser capture microdissection (LCM) RNA‐seq data from a prior study (Fu et al. 2024) was utilized to characterize the expression of genes that encode SSPs in different stem internode cell types of Phytomer 8 from the Wray genotype grown to 74 DAE. The selectivity of the expression of genes that encode SSPs in stem cell types was characterized using TAU analysis (Yanai et al. 2005).
3. Results
3.1. Identification of Sorghum Genes That Encode SSPs
Sorghum genes that encode SSPs were identified from prior studies (see Section 2.1), from information in Phytozome, and by BLASTP analysis using SSP sequences identified in other plants. Preprocessed SSP sequences were obtained from the genomes of Arabidopsis thaliana, Brachypodium distachyon , Sorghum bicolor , Triticum aestivum , Zea mays , and Oryza sativa and used to identify 219 sorghum genes that encode SSPs through homology searches on Phytozome v13 ( Sorghum bicolor , v3.1.1). Previously annotated sorghum genes encoding 42 CLEs, 13 GASSs, 23 RALFs, 13 RGFs, 9 CEPs, 11 EPFs, and 30 POEs were identified (Table S1). Among the 23 RALFs were genes encoding 7 RALF‐related proteins not reported in prior studies. Sorghum genes that encode 9 CAPEs, 4 CIFs, 26 DVLs, 2 ECLs, 3 ESs, 4 IDAs, 7 MEGs, 4 PIPs, 3 PNPs, 6 PSKs, 6 PSYs, and 4 TPDs were identified in sorghum (Table S1). Phylogenetic analysis of SSP families was carried out utilizing sequence information from sorghum and the species listed above (Figures S1–S19). SSP genes that encode peptides from the species used for the phylogenetic analysis are listed in Table S14. In addition, phylogenetic analysis using only sorghum sequences was carried out and the sorghum genes/SSPs in each family were named (numbered) based on their phylogenetic order (Figures S20–S37).
3.2. Sorghum Gene Families Encoding RGF, CLE, GASS, and RALF SSPs
The identification of genes that encode SSPs is often challenging because the conserved sequences that encode the SSPs are short. Therefore, additional information about the SSPs encoded by the sorghum genes predicted to encode RGF, CLE, GASS, and RALF SSPs was collected to help validate the methods used for gene identification.
3.2.1. Sorghum RGF/GLV/CLEL Gene Family
The 13 sorghum genes that encode SbRGF SSPs identified in the current study were annotated in Phytozome and encode proteins that exhibit sequence similarity (E‐value < 1 × 10−16) to previously published monocot RGFs (Fang et al. 2021). RGFs play a role in the maintenance and regulation of root meristem cell proliferation capacity (Keerthana et al. 2025). RGFs are typically 13–18 amino acids long and post‐translationally modified. RGF SSPs exhibit a conserved DY motif at the N‐terminus and an H/(H/N) residue at the C‐terminus (Fang et al. 2021; Shinohara 2021). Motif analysis confirmed the presence of a conserved C‐terminal RGF motif and an N‐terminal secretion signal peptide sequence in all 13 sorghum RGF SSPs. Sequence analysis of mature RGF SSP sequences confirmed that each sorghum RGF contained a conserved N‐terminal DY motif and a C‐terminal HH/HN motif (Table S5). Sobic.006G11200 (SbRGF3) is highly related to maize Zm00001d025645, with a bootstrap support of 91 (Table S9).
A study of sorghum nodal root buds that form on the stem pulvinus identified several genes encoding RGF SSPs (Lamb et al. 2024). The RGF genes were part of a gene regulatory network that modulates root tissue formation prior to outgrowth (Lamb et al. 2024). RGF SSPs, also known as GOLVEN (GLV), regulate root meristem maintenance, differentiation, and hormonal signaling through interactions with RGFR receptor kinases (Stührwohldt et al. 2020). They modulate auxin distribution, root gravitropism, and stem cell proliferation (AtRGF9 and AtRGF4) (Matsuzaki et al. 2010; Whitford et al. 2012) and influence lateral root formation (AtRGF7; Matsuzaki et al. 2010, Zhong et al. 2020). Crosstalk with auxin and cytokinin further integrates RGFs into root patterning and stress responses (i.e., AtRGF6) (Matsuzaki et al. 2010; Stührwohldt et al. 2020).
3.2.2. Sorghum GASS SSP Gene Family
Genes encoding GA‐Stimulated in Sorghum (SbGASS) SSPs, originally reported by (Muhammad et al. 2019), were all annotated in Phytozome and showed sequence similarity to GA‐stimulated SSPs in other monocots (E‐value < 1 × 10−34) (Muhammad et al. 2019). In the SbGASS gene family (Table S7), Sobic.009G119800 (SbGASS5) shows a high sequence similarity to the well‐characterized maize gene Zm00001d38056 (ZmGSL1), with a bootstrap support of 96 (Zhang et al. 2018). GASA SSPs are characterized by a cysteine‐rich motif that stabilizes the SSP's helix–turn–helix structure. Variation resulting in the loss of cysteine residues impacts structural integrity (Muhammad et al. 2019; Bouteraa et al. 2023; Shang et al. 2023). Each of the SbGASS SSPs contained two C‐terminal cysteine‐rich motif regions and a predicted N‐terminal signal peptide. Sequence analysis confirmed that each SbGASS SSP contained a strongly conserved cysteine rich region containing 10 well‐conserved cysteine residues (Table S3).
3.2.3. Sorghum CLE SSP Gene Family
Forty two Sorghum bicolor genes were previously reported to encode CLE (SbCLE) SSPs (Goad et al. 2017) although only 28 SbCLE genes are annotated in Phytozome 13. All but one of the 42 SbCLEs exhibited sequence similarity to monocot CLE‐encoding genes (E‐value < 1 × 10−5) as previously reported (Goad et al. 2017; Tian et al. 2022). A homolog for SbCLE17 (Sobic.002G361800) was not identified in maize or rice, suggesting that this sorghum gene is highly diverged and possibly nonfunctional. Sobic.004G041700 (SbCLE32) is closely related to maize ZmCLE18 (Zm00001d015282), with a bootstrap support of 92 (Je et al. 2016) (Table S6).
Mature CLE/CLV SSPs are less than 20 amino acids in length and contain numerous PTMs (Keerthana et al. 2025). Preprocessed CLE proteins contain an N‐terminal secretion signal peptide and a C‐terminal signaling peptide that is cleaved off for biological activity. All SbCLE proteins except SbCLE29 possessed a predicted N‐terminal secretion signal peptide sequence (Table S2). Biologically active CLE SSPs typically contain a dibasic KR motif, an N‐terminal H/N and central prolines or glycines (Fiers et al. 2005; Ito et al. 2006; Ohyama et al. 2009; Katsir et al. 2011). Motif analysis confirmed that each SbCLE SSP harbored a single conserved C‐terminal CLE motif whereas SbCLE37, SbCLE38, and SbCLE40 contained multiple CLE motifs within their preprocessed sequences. Sequence analysis showed that each mature CLE SSP contained an N‐terminal R/K except for SbCLE16, 39, 41, and 32. The SSPs with multiple CLE motifs were not analyzed further since it is not clear which sequence represents the mature peptide sequence. All mature SbCLE SSPs contained a conserved P–X–G–P–X–P central motif or related sequence. All SSPs also contained the C‐terminal H/N on the mature peptide except for SbCLE16 (Table S2).
3.2.4. Sorghum RALF SSP Gene Family
Sixteen of the 23 sorghum genes that encode RALFs identified in this study were previously identified by (Campbell and Turner 2017), and 18 were annotated in Phytozome. Seven of these RALFs were not identified in previous studies. Of these, Sobic.003G415500 (SbRALF6) shares a high sequence similarity with the maize gene Zm00001d011921 (ZmRALF10) (Table S8), with a bootstrap support of 98 (Zhou, Wang, et al. 2024). SbRALF genes that were not annotated in Phytozome exhibited some sequence similarity to previously published monocot RALF SSPs that lacked or contained modified RALF motifs (Sharma et al. 2016; Tian et al. 2022). These SSPs were labeled RALF‐related (RALFR#). All of the SbRALFs exhibited homology (E‐value < 10−6) to previously identified monocot RALFs (Sharma et al. 2016; Campbell and Turner 2017).
Preprocessed RALF SSPs possess the conserved YISY motif and a dibasic RR cleavage site near its C‐terminus. Sequence variation is mainly observed in the YISY motif and cleavage site, which are conserved between monocots and dicots (Matos et al. 2008; Srivastava et al. 2009; Sharma et al. 2016; Abarca et al. 2021). Motif and sequence analysis revealed that SbRALFR1, SbRALFR2, SbRALF3, 5, 6, 7, 8, 9, 11, 12, 13, 14, SbRALFR17, SbRALFR18, and SbRALF21 each had an identifiable RRXL cleavage site whereas the other RALF or RALFR SSPs did not contain an easily identifiable cleavage site. SbRALFR2, SbRALF3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, RALFR17, SbRALFR20, and SbRALFR23 all contained a YISY motif or similar sequence (i.e., SIGY) (Table S4).
SbRALFR1, 2, and 17–23 displayed divergent motifs compared to canonical RALFs. SbRALFR22 showed sequence similarity to SbRALF20; however, SbRALFR22 lacked recognizable RALF motifs and is not homologous to any previously identified RALF, suggesting it may represent a different class of SSP. Our results are consistent with a prior study that identified a divergent class of clade IV RALF SSPs that lack the YISY motif and RRXL cleavage site; however, these retain some sequence similarity to other RALF SSPs within their cystine rich region (Campbell and Turner 2017). Functional characterization will be required to determine if these SSPs function as RALFs (Table S4).
3.3. Expression of SSP‐Encoding Genes in Sorghum Organs
Expression of the sorghum genes that encode SSPs in leaves, stems, roots, and panicles was examined using RNA‐seq data derived from those organs/tissues (Table S10). The average expression of each gene in the four organs was used to calculate TAU values, a gauge of tissue specificity, with values closer to 1 indicating high tissue specificity (Yanai et al. 2005). Forty‐three of the 219 sorghum genes that encode SSPs were expressed in a highly organ‐specific manner (τ > 0.95).The relative expression of sorghum genes that encode SbCLE, SbGASS, and SbRALF genes in leaves, stems, roots, and panicles is shown in Figure 1a. A max TPM > 5 across the multiple tissue samples was used for average calculation across an organ and TAU calculation on organ averages to identify SSPs with relatively high expression in each organ type and determine their organ specificity. One hundred fifty‐eight SSP‐encoding genes had max TPM > 5 (Figure S38). The other 61 SSPs with max TPM < 5 in each tissue sample is also included in (Table S10). While these gene families are expressed in all four organs, some genes show relatively high organ‐specific expression whereas others are expressed in more than one organ. For example, SbCLE1, 3, 6, 7, 9, 10, 11, 12, 15, 24, 25, and 26 exhibited relatively high expression in roots whereas SbCLE31 displayed relatively high expression in leaves and SbCLE23 showed relatively high expression in the stem. SbCLE37 and 38 were specifically expressed in the panicle, while SbCLE18, 20, 23, 27, 28, 41, and 42 showed relatively high expression in the stem. SbCLE18, 20, 24, 25, 26, and 27 also had relatively high expression in the panicle. Among the sorghum genes encoding “GA‐stimulated in sorghum” SSPs (SbGASS), SbGASS3, 4, 5, 6, 7, 8, 9, and 13 exhibited relatively high expression in stems and SbGASS2, 4, and 6 were expressed in leaves. SbGASS2 was also expressed in roots and SbGASS1, 2, 3, 4, 5, 7, 9, 10, 11, 12, and 13 were expressed in the panicle. SbRALFR1, 2, 17, 19, 22 and 23 and SbRALF5, 7, 9, 11, and 12 were expressed in the stem, while SbRALFR2, 21, and 23 and SbRALF9 were expressed in the root. Several RALF genes, including SbRALFR1, 20, and 22 were expressed in the leaf, whereas SbRALF6, 10, 13, 14, 15, and 16 exhibited relatively high expression in the panicle. Expression of SbRALF5, 7, 9 11, and 12 and SbRALFR1, 2, 17, 19, 22, and 23 appeared to be the most stem specific, while SbRALF21 was root specific (τ = 0.92).
FIGURE 1.
Expression of genes that encode SSPs in sorghum leaf, stem, root, and panicle tissues. Genes were ordered within families based on phylogenetic analysis. Gene encoding SSPs shown were expressed with an average of > 5 TPM in at least one organ. Expression values are reported in TPM, with blue indicating high expression and green indicating low expression. (a) Expression of genes encoding CLE, GASS, and RALF peptide families. (b) Expression of genes encoding CEP, EPF, and RGF peptide families that show elevated organ‐specific expression. The Max column is the highest TPM expression of the four organs. The τ column is the gauge of organ specificity.
The RGF and CEP gene families were enriched in genes expressed in roots consistent with prior studies of RGF and CEP SSP function (Keerthana et al. 2025). For example, SbRGF1, 3, and 5 were more highly expressed in the roots than leaves, stems, and panicles; however, SbRGF7 was expressed in stems and leaves (Figure 1b). Similarly, eight of the nine SbCEP genes were highly expressed in roots compared to leaves, stems, and panicles. SbCEP6 was unique as it was expressed specifically in stem (τ = 0.94) (Figure 1b). In contrast, the SbEPF genes were highly expressed in stems, and some family members were also expressed at somewhat lower levels in panicles, possibly because panicles contain stem‐like structures (i.e., rachis) (Figure 1b).
3.4. Stem Cell–Type Expression of Genes That Encode SSPs
Expression of sorghum genes in stem internode cell types was characterized using transcriptome data generated previously from cells isolated by laser capture microdissection (Fu et al. 2024). The stem tissue/cell types targeted for transcriptome profiling included the epidermis, pith parenchyma cells, xylem fibers, vascular parenchyma, and phloem (Figure 2). Analysis of all SSP‐encoding genes expressed at > 5 TPM in fully expanded stem internode tissue showed that two genes encoding IDA peptides were differentially expressed in the epidermis and five genes encoding EPFs were highly expressed in the epidermis, pith parenchyma or both the epidermis and pith (Figure 2a). Two SbPSY genes were highly expressed in vascular parenchyma (Figure 2a). In contrast, the six members of the SbPSK gene family were expressed selectively in the epidermis, pith parenchyma, vascular parenchyma, or phloem (Figure 2a). Several SbCLE genes were expressed in the epidermis and other members of that gene family were expressed in the pith parenchyma, vascular parenchyma, and phloem. For example, SbCLE42, a homolog of TDIF, was highly expressed in pith parenchyma. SbCLE3 was highly expressed in vascular parenchyma, with additional expression in the phloem and xylem fibers. SbCLE19 was most highly expressed in the phloem, with additional expression in vascular parenchyma and xylem fibers (Figure 2a). Many SbRALFR genes such as SbRALFR19, SbRALFR2, and SbRALFR22 with SbRALFR20 and SbRALFR21 were expressed in vascular parenchyma and/or phloem cells. SbRALFR23 shows selective expression in the epidermis and phloem whereas SbRALFR17 is expressed in epidermis and pith parenchyma. The other SbRALFs and SbRALFRs show expression in multiple cell types (i.e., SbRALF11, SbRALF12, SbRALF5, SbRALF9, and SbRALFR1) (Figure 2a). Two SbCIF genes were more highly expressed in cells of vascular bundles (xylem fibers, vascular parenchyma, phloem) (Figure 2a).
FIGURE 2.
Expression of genes that encode SSPs in cell types of a fully elongated internode of sorghum genotype Wray. (a) Expression of all SSP‐encoding genes expressed at > 5 TPM in at least one stem internode cell type, sorted by peptide family. Genes that encompass a family are color‐coded. (b) Peptide encoding genes that exhibit high cell type–specific expression (τ > 0.89).
SSP‐encoding genes expressed with high cell‐type specificity were identified by Tau analysis (> 0.89) and sorted by cell type (Figure 2b). Tau analysis identified numerous genes with highly specific expression in the epidermis, pith parenchyma, vascular parenchyma, or phloem (Figure 2b). Specific CLE and PSK gene family members were expressed at high specificity in each of the four cell types (Figure 2b). In contrast, two IDA genes were expressed at high levels only in the epidermis, while SbEPF1 was highly expressed in the epidermis and SbEPF4 was highly expressed in stem pith parenchyma cells (Figure 2b). Three SbRALF genes were expressed with high cell‐type specificity in stem vascular parenchyma; three SbPOE genes were expressed highly in the epidermis along with two SbGASS peptides (Figure 2b). SbPOE2 was selectively expressed in the vascular parenchyma whereas SbGASS6 was expressed in the pith parenchyma, and SbGASS2.2 was expressed in xylem fiber cells (Figure 2b). The SbDVL family has two genes expressed in vascular parenchyma, along with another gene, SbDVL26 with specific expression in the pith parenchyma (Figure 2b). SbPSY2 is specifically expressed in vascular parenchyma whereas SbPNP3 is specifically expressed in the phloem cells (Figure 2b). Among the other genes encoding SSPs (Table S13), SbGASS8 showed relatively high expression levels in the epidermis and pith parenchyma cells, and SbPOE11 is expressed in all cell types with its highest expression in the stem epidermis whereas SbDVL22 is expressed in stem core pith/parenchyma (Table S13). SbCIF1.3, SbPOE14, SbPOE1, and SbPOE2 have relatively high expression in the stem vascular parenchyma along with some expression in xylem fibers and phloem (Table S13). SbMEG5 showed high expression in the stem phloem cells; SbPOE3 is expressed in xylem fibers, stem vascular parenchyma, and highest expression in stem phloem. SbDVL25 and SbPOE21 are expressed in each of the cell types analyzed (Table S13).
3.5. Expression of SSP‐Encoding Genes in Developing Stem Internodes
The potential involvement of SSPs in stem growth regulation was investigated by analyzing the expression of SSP‐encoding genes during stem internode development. In a previous study, transcriptome profiles were collected from four stages of R07020 stem internode growth and development (Kebrom et al. 2017). Genes involved in cell proliferation (i.e., SbCDKB2) were highly expressed in the youngest internode (INT1) and genes involved in lignin synthesis and secondary cell wall formation (i.e., SbCESA4) were highly expressed in a fully elongated internode (INT4) (Kebrom et al. 2017). In the current study, expression of genes that encode SSPs in INT1–INT4 was investigated to identify genes that are differentially expressed during this phase of internode growth and development. The analysis identified genes that encode SSPs that were highly expressed early in internode development (INT1) that showed decreasing expression during development and in fully elongated internodes (INT4) (INT1 > INT4), whereas other genes were expressed at low levels in INT1 and higher levels during development and in INT4 (INT4 > INT1) suggesting they may play roles in internode differentiation following cessation of growth (Figure 3). For example, SbGASS8, SbGASS9, SbGASS3, SbGASS13, and SbGASS2 showed a INT1 > INT4 pattern of expression during internode development. Similarly, SbRALF7, SbCAPE9, SbDVL24, SbEPF7/8, SbES1, SbPOE19, and SbPSY3 exhibited an INT1 > INT4 expression pattern, suggesting a potential role in regulating growth processes during early internode development. Conversely, several members of the RALF SSP family, including SbRALF5, SbRALFR2, SbRALFR20, SbRALFR22, and SbRALFR19 displayed an INT4 > INT1 pattern of expression highlighting their possible involvement in processes associated with internode differentiation and secondary cell wall formation. Expression of SbCIF1, SbPIP2, SbPOE2/14/27/28/29, and SbPSK3/5 also increased during internode development (INT4 > INT1) (Figure 3). SSPs that expressed > 5 TPMs in at least one internode but did not have an FC > 5 are included in Table S11 along with SSPs that expressed < 5 TPMs.
FIGURE 3.
Expression of genes that encode SSPs during development of internodes of sorghum genotype R07020. The youngest internodes (INT1/INT2) showed elevated expression of SbCDK2, a marker gene for cell proliferation. Older internodes (INT3/INT4) showed elevated expression of SbCESA4. Genes were sorted based on their differential expression in young (INT1) versus older internodes (INT1 > INT4 or INT4 > INT1). Genes included in this figure had a fold change (FC) > 5 (adjusted p‐value < 0.05).
3.6. Expression of SSP‐Encoding Genes During TX08001 Stem Development
Sorghum stems are composed of node–internode segments that are part of phytomers initially formed below the stem apical meristem approximately every 3–4 days during vegetative growth. The node–internode segments of each phytomer are composed of the nodal plexus, where the leaf sheath joins the stem, internode tissue produced by an IM, and the pulvinus where the formation of tiller buds and nodal root buds takes place. An IM zone of cell division (ZoD) is located at the base of a growing internode just above the pulvinus, with an internode zone of cell elongation (ZoE) located immediately above the IM and a zone of cell maturation (ZoM) above the ZoE (Yu et al. 2022) (Figures 4b and 5b). To better understand the potential role of SSPs in stem growth and development, we investigated the expression of genes that encode SSPs in stem tissues during stem growth and development. Nascent stem nodal tissue was collected from Phytomers 1–4 (Phy1–4) without further dissection. Stem node–internode segments of Phytomers 6 and 7 were dissected into the nodal plexus, internode, IM, and pulvinus for RNA‐seq analysis. Stem internode tissue was divided into 5‐, 10‐, and 20‐mm sections starting from the base of the internode (5 mm, IM) to the nodal plexus (20 mm) for transcriptome analysis. The expression of the 219 sorghum genes that encode SSPs in this set of stem tissues was investigated to identify genes that are expressed during stem development. The subset of genes with expression of > 5 TPM in at least one stem sample was identified and further sorted into those with higher expression in regions of cell proliferation/growth (Figure 4a) and those with higher relative expression in nongrowing tissues (Figure 5a). Genes with these patterns of expression but at < 5 TPM were included in Table S12. Expression of SbCDK2 (Sobic.010G215300), a gene associated with high rates of cell proliferation, and SbCESA4 (Sobic.003G296400), a gene involved in secondary cell wall formation, was used as biomarkers for tissue/cells that are proliferating or differentiating, respectively (Yu et al. 2022). SbCDK2 was highly expressed in stem tissue associated with the youngest nascent nodes of Phytomers 1–4 (Figure 4a) and the IM, whereas SbCESA4 expression was highest in the nodal plexus of Phytomer 6 and nodes–internodes of Phytomer 7 (Figure 5a).
Genes encoding SSPs expressed in nascent nodal stem tissue from the youngest phytomers (1–4) but at low levels in stem tissue of older phytomers (6–7) indicate they play a role in early stages of stem node development (Figure 4a). Examples of such SSPs include SbCLE18, SbCLE41, and SbCLE42. Expression of SbGASS8, SbGASS3, SbGASS9, and SbGASS4 was elevated in apical and IM tissues that are characterized by high rates of cell proliferation (Yu et al. 2022). SSP‐encoding genes such as SbGASS7, SbGASS6, and SbGASS13 were also highly expressed in the stem apical phytomers and showed lower expression in stem tissues from Phytomer 7.
SbCLE26 and SbGASS2 genes showed an intriguing expression pattern, with elevated expression in a 10‐mm section of the internode just above the IM of Phytomer 6. At this stage of internode tissue development, rates of cell proliferation decrease, coinciding with increased cell elongation followed by cessation of growth and synthesis of secondary cell walls. Expression of SbCESA4, a biomarker associated with secondary cell wall formation (McKinley et al. 2016), was highest in the upper regions of internodes that had completed elongation, suggesting the 10‐mm section of Phytomer 6 above the IM represents a transitional zone where cell elongation increases. This correlation suggests SbCLE26 and SbGASS2 may regulate the transition from cell proliferation to cell elongation.
SbCLE24 was highly expressed in internodes undergoing secondary cell wall formation, while SbCLE10 and SbCLE6 were predominantly expressed in the pulvinus and nodal plexus of older phytomers (Figure 5a). SbGASS12 was uniquely expressed in the internode and pulvinus of Phytomer 6 (Table S12). Members of the SbRALF gene family, including SbRALFR23, SbRALFR2, SbRALFR19, SbRALFR20, and SbRALFR23, were expressed more highly in the nodal plexus and upper internodes of Phytomers 6 and 7, suggesting a role in nodal plexus development (Figure 5a).
The other genes that encode SSPs like SbDVL24, SbDVL23, SbEPF7, and SbEPF9 exhibited elevated expression in young stem tissue of Phytomers 1–4 (Figure 4a). SbEPF8, SbEPF11, SbEPF6, SbES1, SbPOE19, SbPSK2, and SbPSY3 were expressed in meristematic tissues of the internode. SbCAPE9, one variant of SbCIF1.3, SbDVL25, SbDVL1, SbEPF4, SbPOE11, and SbPSK1 were expressed at higher levels during development in all the internode tissues. SbCAPE5, a variant of SbCIF1.2, SbEPF1, SbIDA1, SbPOE3, SbPOE26, SbPOE6, SbPOE14, SbPOE28, SbPOE29, and SbPSY5, shows a pattern of increased expression during stem development, with interesting patterns in the nodal plexus tissue. SbCEP6, SbCIF4, SbDVL22, SbDVL26, SbDVL21, SbPOE23, SbPOE2, SbPOE21, SbPSK3, SbPSK5, SbPSK6, and SbPSY2 showed elevated expression in many of the mature tissues (Figures 4a and 5a and Table S12).
3.7. Cluster Analysis of SSP Gene Expression in TX08001 Stems During Development
The transcriptome data from stem tissues at various stages of development that were used to construct Figures 4a and 5a were subjected to clustering analysis to identify cohorts of SSP genes that have similar patterns of expression during stem development (Figure 6a–c). Transcriptome clustering analysis identified three groups of genes with distinct patterns of expression in nascent nodes and developing regions of elongating internodes (Figure 6a). Cluster 1 included 17 SSP genes that showed peak expression in nascent nodes but lower expression in the growing zone tissue of Phytomer 6. These genes were co‐expressed with the cell division marker SbCDKB2, indicating association with tissues with high rates of cell proliferation. Cluster 2 comprised three SSP genes with relatively lower expression in nascent nodes and higher expression in elongating stem tissues. Cluster 3 included eight SSP genes that showed high expression across nascent nodes, the zone of elongation (ZoE), and the IM, suggesting roles in actively growing internode tissues.
FIGURE 6.
Clustering analysis of SSP genes across stem growth and differentiation zones. (a) Line plots showing the median expression profiles of SSP genes within each cluster derived from the dataset encompassing nascent nodes and elongating internode regions. Genes assigned to each cluster are indicated. (b) Schematic of stem tissues used for clustering analysis, including nascent nodes, nodal tissues, and internode regions. P refers to the pulvinus. (c) Line plots showing the median expression profiles of SSP genes within each cluster derived from the dataset encompassing nodal tissues and the zone of maturation. Genes assigned to each cluster are indicated.
Analysis of the transcriptome data from nongrowing stem tissues shown in Figure 5a identified two distinct groups of SSP genes that are expressed in stem nodal tissues (nodal plexus and pulvinus) and the maturing zone of the internode (Figure 6c). Cluster 1 consisted of 11 SSP genes preferentially expressed in the pulvinus and nodal plexus. Cluster 2 was composed of four SSP genes with elevated expression in the stem internode zone of maturation that were co‐expressed with the secondary cell wall marker SbCESA4, indicating association with tissues undergoing secondary cell wall formation.
4. Discussion
SSPs regulate plant growth, differentiation, adaptation to abiotic stress, and responses to plant pathogens and pests (Czyzewicz et al. 2013; Matsubayashi 2014; Xie et al. 2022; Xiao et al. 2025). The action of SSPs is complementary to and integrated with regulation of similar processes by the plant hormones ABA, IAA, CK, GA, JA, and ethylene. While the role of plant hormones in sorghum stem growth regulation has been investigated (i.e., Multani et al. 2003, Hilley et al. 2016, Kebrom et al. 2017, Yu et al. 2021), the function of SSPs is largely unexplored. Therefore, the goal of this study was to update the identification of sorghum genes that encode SSPs and analyze the expression of these genes in sorghum organs, in stem cell types, and during stem growth and development. The current study identified 219 sorghum genes that encode SSPs that were members of 19 SSP families. Some of the sorghum genes were identified previously in a study of rice SSP‐encoding genes (Muhammad et al. 2019) based on sorghum genome sequence V1 (Paterson et al. 2009). The current study annotated sorghum genes that encode SSPs encoded in the BTx623 reference genome sequence V3.1.1 (McCormick et al. 2018) based on homology to SSP‐encoding genes identified in other plant species, motif identification, phylogenetic analysis, and expression profiling. Twelve of the gene families that encode CAPE, CIF, DVL, ECL ES, IDA, MEG, PIP, PNP, PSK, PSY, and TPD SSPs were previously unannotated in sorghum.
4.1. Organ‐ and Cell‐Specific Expression
Sorghum genes that encode SSPs were expressed in leaves, stems, roots, and panicles. Several gene families were mostly expressed in one organ. For example, genes encoding SbCEP and SbRGF SSPs were largely expressed in roots, whereas genes encoding EPF SSPs were primarily expressed in stems and panicles. Specific genes encoding members of other SSP families (i.e., SbCLE, SbGASS, and SbRALF genes) were expressed in different organs or, in some cases, more than one organ. Expression of sorghum genes that encode SSPs in stem cell types was examined using LCM‐derived cell specific transcriptome data from a fully expanded stem internode (Fu et al. 2024). Cell‐type transcriptome analysis showed that many of the sorghum SSP‐encoding genes are expressed in a cell type–specific manner (τ > 0.89). One gene family was selectively expressed in cells of the stem epidermis (i.e., IDAs) while genes encoding EPF SSPs were expressed in the epidermis and pith parenchyma. Specific members of other gene families (i.e., CLE and PSK) were expressed in each of the stem cell types analyzed (epidermis, pith, vascular parenchyma, phloem). Overall, the analysis showed that expression of many of the SSP‐encoding genes in sorghum occurs in a highly organ‐ and cell type–specific manner that would enable targeted spatial regulation of cell‐type growth and differentiation.
4.2. Expression of SSP Genes During Stem Development
Bioenergy sorghum stems account for ~80% of harvested biomass at the end of the growing season (Olson et al. 2012). Sorghum stem architecture has an impact on leaf distribution in the canopy; stems provide a conduit for water, sugars, and nutrient exchange between roots and leaves; and stem anatomy and composition influence stalk lodging and conversion of biomass to bioproducts. Sorghum stem growth and development is highly regulated and influenced by genotype (Multani et al. 2003; Hilley et al. 2016; Hilley et al. 2017), environmental factors (i.e., shading) (Yu et al. 2021), and mechanical stress (Zargar et al. 2022).
The current study found that numerous sorghum SSP‐encoding genes are differentially expressed in stem zones of cell proliferation (apical, IM) or at later stages of stem tissue development. For example, 13 genes were differentially expressed early in internode development during the cell proliferation stage, and a different set of 14 genes were induced later in internode development when cells are differentiating. Analysis of stem development starting with nascent nodes located in stem apex (Phytomers 1–4) and extending through stem tissues in Phytomers 6 and 7 revealed additional complexity of SSP gene expression. For example, several SbCLE‐genes were expressed at their highest levels in nascent nodes of Phytomer 1 (SbCLE18, 20, 41, 42) followed by decreased in expression in stem tissue of older phytomers. Other genes were highly expressed in the apical nascent nodes and in the internode IM and adjacent ZoE (i.e., SbGASS3 and SbPSY3), indicating a more general role in the regulation of cell proliferation and enlargement consistent with prior studies (Yu et al. 2022; Zhang et al. 2025). Other genes were expressed at low levels in nascent nodes and at higher levels in the nodal plexus and fully elongated portions of internodes in older phytomers (i.e., SbPOE14, 28, 29, SbCAPE5, SbRALFR2, 19, 20, 22, and 23). This high‐resolution analysis enabled the classification of SSP genes into clusters defined by distinct expression patterns across stem developmental stages, including nascent nodes, elongating regions, nodal tissues, and maturing internode segments undergoing secondary cell wall formation. The clustering analysis grouped SSP genes into cohorts associated with early stem development, growing zones, stem nodes, and nongrowing internode tissue that was undergoing secondary cell wall deposition.
4.2.1. SbGASS Genes
Homologs of SSP‐encoding GASA (GA‐Stimulated in Arabidopsis) genes are found in many plant species including sorghum (Muhammad et al. 2019). GASA homologs are involved in growth regulation (i.e., Sun et al. 2013, Han et al. 2017, Lee et al. 2017), salt tolerance (Lee et al. 2015), regulation of ROS levels (Rubinovich and Weiss 2010) and regulation of flowering and seed development (Roxrud et al. 2007). Three sorghum GASS genes were previously found to be differentially expressed in the sorghum stem IM (Yu et al. 2022) consistent with GA regulation of cell proliferation in stem internodes (Yu et al. 2021). The current study showed that six SbGASS genes (SbGASS2, 3, 4, 8, 9, 13) were expressed in nascent stem nodes of Phytomers 1–4 and/or in the internode IM consistent with a role in GA‐modulated cell proliferation. SbGASS3 and SbGASS13 were also expressed in the epidermis of a fully elongated section of the internode of Phytomer 7.
4.2.2. SbCLE Genes
In the current study, SbCLE18, 20, 41, and 42 were highly expressed in nascent stem nodes; SbCLE26 was expressed in the internode zone of cell elongation, and SbCLE6, 10, and 24 were expressed in the pulvinus or internode tissue later in stem development. SbCLE41 and SbCLE42 are homologs of TDIFs that promote expression of WOX4 and vascular cambium activity and inhibit xylem differentiation (Qiang et al. 2013). SbCLE42 is a homolog of ZmCLE2‐1, which interacts with WUS2 to regulate meristem activity (Dong et al. 2023). Further analysis of the SbCLE genes that are expressed in apical nascent stem nodes may provide insight into the early stages of sorghum stem growth and vascular bundle cell development. Analysis of stem internode cell‐type expression showed that SbCLE42 was selectively expressed in pith parenchyma of a fully elongated portion of the internode associated with Phytomer 7. Continued expression of SbCLE42 in pith cells that lack secondary cell walls is consistent with TDIF's additional role in repressing xylem cell differentiation and secondary cell wall formation (Qiang et al. 2013).
4.2.3. SbRALF Genes
RALF SSPs have been found to regulate plant growth, root hair elongation, pollen development, and plant responses to adverse environmental conditions (Murphy and De Smet 2014; Blackburn et al. 2020; Liu et al. 2024; Zhang et al. 2025). RALF SSPs are essential for cell wall integrity, receptor activation, and stress adaptation. They interact with FERONIA receptor kinases to modulate calcium signaling, pH homeostasis, and endocytosis (Yu et al. 2018; Zhu et al. 2020). They regulate pollen tube reception (AtRALF4; Mecchia et al. 2017, Feng et al. 2019), root elongation (AtRALF1; Yu et al. 2018, Zhu et al. 2020), and cell wall modification via pectin interactions (AtRALF4 and AtRALF22; Moussu et al. 2023, Schoenaers et al. 2024). Stress responses are mediated through FER receptor internalization (AtRALF1; Liu et al. 2024), linking RALFs to environmental adaptation.
Seven previously unidentified sorghum genes that encode SbRALFs were found in this current study. Analysis of the expression of the RALF gene family shows SbRALF5, S bRALFR2, SbRALFR 19, SbRALFR 20, SbRALFR 22, and SbRALFR23 were expressed during stem development at low levels in regions of cell proliferation/elongation and at higher levels in tissues that were undergoing differentiation. Elevated expression of these SbRALF genes in the nodal plexus and pulvinus of Phytomer 6/7 was noteworthy. In addition, SbRALFR2, 19, and 22 were expressed in internode vascular parenchyma cells in fully elongated internode tissue of Phytomer 7. The function of the SbRALF/R genes listed above in stem tissue differentiation is currently unknown.
5. Conclusion, Next Steps, and Study Limitations
This study characterized the expression of 219 sorghum genes that encode SSPs in sorghum organs, stem cell types, and stages of stem development. Since biomass production is largely dependent on stem growth, we hypothesize that SSPs expressed during stem growth could be used to further improve stem morphometrics and possibly biomass yield. The specific functions of sorghum SSPs will require the generation of overexpression or knockout lines to validate or discover SSP roles in stem growth and development. Given their small size and bioactive properties, synthetic SSPs could also be applied exogenously to test their effects on stem growth, cell spacing, and cell differentiation. Furthermore, since many SSPs exhibit cell type–specific expression, integrating developmental time‐course data from single cell RNA‐seq studies could refine our understanding of their roles. This study expands knowledge of SSPs in sorghum, a drought tolerant C4 grass used to produce grain, forage, and biomass for bioenergy/biofuels. By integrating phylogenetics, transcriptomics, and expression profiling during stem development, this research provides a useful dataset for understanding the roles of SSPs in stem development.
Author Contributions
E.K. and J.M. conceived the original research plan. E.K. performed the experiments. E.K., J.M., and B.M. analyzed the data. E.K. and J.M. wrote the manuscript.
Funding
This work was funded by the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science Grant/Award: DE‐SC0018409), Texas AgriLife Research, and the Perry Adkisson Chair in Agricultural Biology (JEM).
Conflicts of Interest
The authors declare no conflicts of interest.
Supporting information
Table S1: List of SSP‐encoding genes in sorghum with their gene names based on phylogenetic order in their respective sorghum phylogenetic analysis.
Table S2: CLE SSPs in sorghum identified from homology and literature sources. This table also includes any annotations provided by Phytozome v13 for a given peptide. Provided is the best E‐value to a respective published homolog query from another species to sorghum. Each preprocessed peptide length is provided along with the peptide structure visual provided from the MEME analysis. Previously published conserved sequences for the respective mature peptide sequences were analyzed, and their presence or absence is provided in tabular format. The results for the motif consensus are also provided as an image along with a color‐coded key to show which sequence is present in the peptide of interest. The settings used for the MEME search can also be located here.
Table S3: GASS SSPs in sorghum identified from homology and literature sources. This table also includes any annotations provided by Phytozome v13 for a given peptide. Provided is the best E‐value to a respective published homolog query from another species to sorghum. Each preprocessed peptide length is provided along with the peptide structure visual provided from the MEME analysis. Previously published conserved sequences for the respective mature peptide sequences were analyzed, and their presence or absence is provided in tabular format. The results for the motif consensus are also provided as an image along with a color‐coded key to show which sequence is present in the peptide of interest. The settings used for the MEME search can also be located here.
Table S4: RALF and RALFR SSPs in sorghum identified from homology and literature sources. This table also includes any annotations provided by Phytozome v13 for a given peptide. Provided is the best E‐value to a respective published homolog query from another species to sorghum. Each preprocessed peptide length is provided along with the peptide structure visual provided from the MEME analysis. Previously published conserved sequences for the respective mature peptide sequences were analyzed, and their presence or absence is provided in tabular format. The results for the motif consensus are also provided as an image along with a color‐coded key to show which sequence is present in the peptide of interest. The settings used for the MEME search can also be located here.
Table S5: RGF SSPs in sorghum identified from homology and literature sources. This table also includes any annotations provided by Phytozome v13 for a given peptide. Provided is the best E‐value to a respective published homolog query from another species to sorghum. Each preprocessed peptide length is provided along with the peptide structure visual provided from the MEME analysis. Previously published conserved sequences for the respective mature peptide sequences were analyzed, and their presence or absence is provided in tabular format. The results for the motif consensus are also provided as an image along with a color‐coded key to show which sequence is present in the peptide of interest. The settings used for the MEME search can also be located here.
Table S6: The best homolog of a Sorghum bicolor gene encoding peptide from the maximum‐likelihood analysis of CLE SSPs. The best homolog was determined by topological proximity. The best homolog for a given Sorghum bicolor is provided for Zea mays , Oryza sativa , and Arabidopsis thaliana along with their bootstrap values.
Table S7: The best homolog of a Sorghum bicolor gene encoding peptide from the maximum‐likelihood analysis of GASS SSPs. The best homolog was determined by topological proximity. The best homolog for a given Sorghum bicolor is provided for Zea mays , Oryza sativa , and Arabidopsis thaliana along with their bootstrap values.
Table S8: The best homolog of a Sorghum bicolor gene encoding peptide from the maximum‐likelihood analysis of RALF SSPs. The best homolog was determined by topological proximity. The best homolog for a given Sorghum bicolor is provided for Zea mays , Oryza sativa , and Arabidopsis thaliana along with their bootstrap values.
Table S9: The best hits of a Sorghum bicolor gene for homology from the maximum‐likelihood analysis of RGF SSPs determined by topological proximity. The best homolog for a given Sorghum bicolor is provided for Zea mays , Oryza sativa , and Arabidopsis thaliana along with their bootstrap values.
Table S10: All 219 SSPs in Sorghum bicolor expression throughout different organ samples. All SSPs from this analysis are included along with their respective max TPM across the dataset and TAU values.
Table S11: Genes encoding SSPs expressed in each internode, 1–4. This table includes genes that were not differentially expressed more than five FC and the rest that had max expression across the four tissues analyzed less than five from the analysis in Figure 2.
Table S12: Expression of genes encoding SSPs that increase in expression during stem differentiation or throughout stem differentiation. Genes included are expressed throughout P1–P6 stem development from Figures 4a and 5a. SSPs that had TPM max < 5 were also included.
Table S13: Expression of genes encoding 219 SSPs in the LCM analysis from Figure 2. Genes encoding SSPs that are expressed at > 5 TPM across the five cell types shown in Figure 2 are included along with their TAU values. In addition, all genes encoding SSPs expressed at < 5 TPM in the data set and also included in Genes that encompass a family are color‐coded.
Table S14: List of SSP‐encoding genes in Sorghum bicolor, Arabidopsis thaliana, Oryza sativa, Zea mays, Brachypodium distachyon, and Triticum aestivum.
Figure S1: Maximum‐likelihood phylogenetic analysis of CLE SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S20. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S2: Maximum‐likelihood phylogenetic analysis of GASA SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S21. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S3: Maximum‐likelihood phylogenetic analysis of RALF SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S22. Genes encoding SSPs in other species are named based on published nomenclature. The tree was rooted to AtRALF18. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S4: Maximum‐likelihood phylogenetic analysis of RGF SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S23. Genes encoding SSPS in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S5: Maximum‐likelihood phylogenetic analysis of CAPE SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S24. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S6: Maximum‐likelihood phylogenetic analysis of CEP SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S25. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S7: Maximum‐likelihood phylogenetic analysis of CIF SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S26. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S8: Maximum‐likelihood phylogenetic analysis of DVL SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S27. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S9: Maximum‐likelihood phylogenetic analysis of ECL SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S10: Maximum‐likelihood phylogenetic analysis of EPF SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S28. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S11: Maximum‐likelihood phylogenetic analysis of ES SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S29. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S12: Maximum‐likelihood phylogenetic analysis of IDA SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S30. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S13: Maximum‐likelihood phylogenetic analysis of MEG SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S31. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S14: Maximum‐likelihood phylogenetic analysis of PIP SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S32. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S15: Maximum‐likelihood phylogenetic analysis of PNP SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S33. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S16: Maximum‐likelihood phylogenetic analysis of POE SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S34. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S17: Maximum‐likelihood phylogenetic analysis of PSK SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S35. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S18: Maximum‐likelihood phylogenetic analysis of PSY SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S36. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S19: Maximum‐likelihood phylogenetic analysis of TPD SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S37. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S20: Maximum‐likelihood phylogenetic analysis of CLE SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S21: Maximum‐likelihood phylogenetic analysis of GASS SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S22: Maximum‐likelihood phylogenetic analysis of RALF SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. Tree was annotated and visualized in iTOL v7.0.
Figure S23: Maximum‐likelihood phylogenetic analysis of RGF SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S24: Maximum‐likelihood phylogenetic analysis of CAPE SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S25: Maximum‐likelihood phylogenetic analysis of CEP SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S26: Maximum‐likelihood phylogenetic analysis of CIF SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S27: Maximum‐likelihood phylogenetic analysis of DVL SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S28: Maximum‐likelihood phylogenetic analysis of EPF SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S29: Maximum‐likelihood phylogenetic analysis of ES SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S30: Maximum‐likelihood phylogenetic analysis of IDA SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S31: Maximum‐likelihood phylogenetic analysis of MEG SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. Tree was annotated and visualized in iTOL v7.0.
Figure S32: Maximum‐likelihood phylogenetic analysis of PIP SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S33: Maximum‐likelihood phylogenetic analysis of PNP SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S34: Maximum‐likelihood phylogenetic analysis of POE SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S35: Maximum‐likelihood phylogenetic analysis of PSK SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S36: Maximum‐likelihood phylogenetic analysis of PSY SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S37: Maximum‐likelihood phylogenetic analysis of TPD SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S38: Expression of 158 genes encoding small signaling SSPs in 19 small signaling peptide families in leaf, stem, root, and panicle tissues. SSPs were sorted based on phylogenetic order and 158 of the 219 were selected using a > 5 TPM threshold across the averaged expression values within each organ. SbECL2 has a max TPM of 3 but was included to not remove the family. Expression values are reported in TPM, with blue indicating high expression and green indicating low expression.
Data S1: Supporting information.
Acknowledgments
The authors thank Kerrie Barry and the Joint Genome Institute for contributing to RNA‐seq analysis. The work (proposal: 10.46936/10.25585/60001026 and 10.46936/10.25585/60000987) conducted by the US Department of Energy Joint Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science User Facility, is supported by the Office of Science of the US Department of Energy operated under contract no. DE‐AC02‐05CH11231. The authors acknowledge Brock D. Weers for developing methods for building rhizotrons for bioenergy sorghum.
Data Availability Statement
The data supporting this study are available from multiple repositories. RNA‐seq data from developing stem internodes (Internodes 1–4) of R.07020, generated by Kebrom et al. 2017, are available at the NCBI under accession number GSE98817. Organ specificity data used for Tau analysis in the BTx623 Atlas dataset are accessible via the JGI Genome Portal under project IDs 1051037, 1051406, and 1053825. Laser capture microdissection (LCM) RNA‐seq data from Fu et al. 2024, are available in the NCBI Sequence Read Archive under accession numbers SRA558272, SRA558514, and SRA558539. Additionally, data from the Sorghum bicolor TX08001 Compendium Set2 Gene Expression Profiling project can be accessed through the JGI Genome Portal (https://genome.jgi.doe.gov/portal) under JGI Project ID 1454838. The phylogenetic trees can be analyzed directly in iTOL via link https://itol.embl.de/shared/1gT26PPKrO3dq or can be accessed with a phylogenetic viewer of choice by downloading the .tree files within the Supporting Information.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Table S1: List of SSP‐encoding genes in sorghum with their gene names based on phylogenetic order in their respective sorghum phylogenetic analysis.
Table S2: CLE SSPs in sorghum identified from homology and literature sources. This table also includes any annotations provided by Phytozome v13 for a given peptide. Provided is the best E‐value to a respective published homolog query from another species to sorghum. Each preprocessed peptide length is provided along with the peptide structure visual provided from the MEME analysis. Previously published conserved sequences for the respective mature peptide sequences were analyzed, and their presence or absence is provided in tabular format. The results for the motif consensus are also provided as an image along with a color‐coded key to show which sequence is present in the peptide of interest. The settings used for the MEME search can also be located here.
Table S3: GASS SSPs in sorghum identified from homology and literature sources. This table also includes any annotations provided by Phytozome v13 for a given peptide. Provided is the best E‐value to a respective published homolog query from another species to sorghum. Each preprocessed peptide length is provided along with the peptide structure visual provided from the MEME analysis. Previously published conserved sequences for the respective mature peptide sequences were analyzed, and their presence or absence is provided in tabular format. The results for the motif consensus are also provided as an image along with a color‐coded key to show which sequence is present in the peptide of interest. The settings used for the MEME search can also be located here.
Table S4: RALF and RALFR SSPs in sorghum identified from homology and literature sources. This table also includes any annotations provided by Phytozome v13 for a given peptide. Provided is the best E‐value to a respective published homolog query from another species to sorghum. Each preprocessed peptide length is provided along with the peptide structure visual provided from the MEME analysis. Previously published conserved sequences for the respective mature peptide sequences were analyzed, and their presence or absence is provided in tabular format. The results for the motif consensus are also provided as an image along with a color‐coded key to show which sequence is present in the peptide of interest. The settings used for the MEME search can also be located here.
Table S5: RGF SSPs in sorghum identified from homology and literature sources. This table also includes any annotations provided by Phytozome v13 for a given peptide. Provided is the best E‐value to a respective published homolog query from another species to sorghum. Each preprocessed peptide length is provided along with the peptide structure visual provided from the MEME analysis. Previously published conserved sequences for the respective mature peptide sequences were analyzed, and their presence or absence is provided in tabular format. The results for the motif consensus are also provided as an image along with a color‐coded key to show which sequence is present in the peptide of interest. The settings used for the MEME search can also be located here.
Table S6: The best homolog of a Sorghum bicolor gene encoding peptide from the maximum‐likelihood analysis of CLE SSPs. The best homolog was determined by topological proximity. The best homolog for a given Sorghum bicolor is provided for Zea mays , Oryza sativa , and Arabidopsis thaliana along with their bootstrap values.
Table S7: The best homolog of a Sorghum bicolor gene encoding peptide from the maximum‐likelihood analysis of GASS SSPs. The best homolog was determined by topological proximity. The best homolog for a given Sorghum bicolor is provided for Zea mays , Oryza sativa , and Arabidopsis thaliana along with their bootstrap values.
Table S8: The best homolog of a Sorghum bicolor gene encoding peptide from the maximum‐likelihood analysis of RALF SSPs. The best homolog was determined by topological proximity. The best homolog for a given Sorghum bicolor is provided for Zea mays , Oryza sativa , and Arabidopsis thaliana along with their bootstrap values.
Table S9: The best hits of a Sorghum bicolor gene for homology from the maximum‐likelihood analysis of RGF SSPs determined by topological proximity. The best homolog for a given Sorghum bicolor is provided for Zea mays , Oryza sativa , and Arabidopsis thaliana along with their bootstrap values.
Table S10: All 219 SSPs in Sorghum bicolor expression throughout different organ samples. All SSPs from this analysis are included along with their respective max TPM across the dataset and TAU values.
Table S11: Genes encoding SSPs expressed in each internode, 1–4. This table includes genes that were not differentially expressed more than five FC and the rest that had max expression across the four tissues analyzed less than five from the analysis in Figure 2.
Table S12: Expression of genes encoding SSPs that increase in expression during stem differentiation or throughout stem differentiation. Genes included are expressed throughout P1–P6 stem development from Figures 4a and 5a. SSPs that had TPM max < 5 were also included.
Table S13: Expression of genes encoding 219 SSPs in the LCM analysis from Figure 2. Genes encoding SSPs that are expressed at > 5 TPM across the five cell types shown in Figure 2 are included along with their TAU values. In addition, all genes encoding SSPs expressed at < 5 TPM in the data set and also included in Genes that encompass a family are color‐coded.
Table S14: List of SSP‐encoding genes in Sorghum bicolor, Arabidopsis thaliana, Oryza sativa, Zea mays, Brachypodium distachyon, and Triticum aestivum.
Figure S1: Maximum‐likelihood phylogenetic analysis of CLE SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S20. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S2: Maximum‐likelihood phylogenetic analysis of GASA SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S21. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S3: Maximum‐likelihood phylogenetic analysis of RALF SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S22. Genes encoding SSPs in other species are named based on published nomenclature. The tree was rooted to AtRALF18. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S4: Maximum‐likelihood phylogenetic analysis of RGF SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S23. Genes encoding SSPS in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S5: Maximum‐likelihood phylogenetic analysis of CAPE SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S24. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S6: Maximum‐likelihood phylogenetic analysis of CEP SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S25. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S7: Maximum‐likelihood phylogenetic analysis of CIF SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S26. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S8: Maximum‐likelihood phylogenetic analysis of DVL SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S27. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S9: Maximum‐likelihood phylogenetic analysis of ECL SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S10: Maximum‐likelihood phylogenetic analysis of EPF SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S28. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S11: Maximum‐likelihood phylogenetic analysis of ES SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S29. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S12: Maximum‐likelihood phylogenetic analysis of IDA SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S30. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S13: Maximum‐likelihood phylogenetic analysis of MEG SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S31. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S14: Maximum‐likelihood phylogenetic analysis of PIP SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S32. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S15: Maximum‐likelihood phylogenetic analysis of PNP SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S33. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S16: Maximum‐likelihood phylogenetic analysis of POE SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S34. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S17: Maximum‐likelihood phylogenetic analysis of PSK SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S35. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S18: Maximum‐likelihood phylogenetic analysis of PSY SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S36. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S19: Maximum‐likelihood phylogenetic analysis of TPD SSP sequences in multiple plant species. The phylogeny includes sequences from Arabidopsis thaliana TAIR10 (At), Oryza sativa v7.0 (Os), Brachypodium distachyon v3.2 (Bd), Triticum aestivum v2.2 (Ta), Zea mays RefGen_V4 (Zm), and Sorghum bicolor v3.1.1 (Sb). Sorghum genes encoding SSPs are highlighted in blue. Genes encoding sorghum SSPs are named based on their phylogenetic order in Figure S37. Genes encoding SSPs in other species are named based on published nomenclature. The tree was midpoint rooted. The scale bar represents the average number of amino acid substitutions per site. Trees were annotated in Dendroscope3 and visualized using iTOL v7.0.
Figure S20: Maximum‐likelihood phylogenetic analysis of CLE SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S21: Maximum‐likelihood phylogenetic analysis of GASS SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S22: Maximum‐likelihood phylogenetic analysis of RALF SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. Tree was annotated and visualized in iTOL v7.0.
Figure S23: Maximum‐likelihood phylogenetic analysis of RGF SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S24: Maximum‐likelihood phylogenetic analysis of CAPE SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S25: Maximum‐likelihood phylogenetic analysis of CEP SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S26: Maximum‐likelihood phylogenetic analysis of CIF SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S27: Maximum‐likelihood phylogenetic analysis of DVL SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S28: Maximum‐likelihood phylogenetic analysis of EPF SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S29: Maximum‐likelihood phylogenetic analysis of ES SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S30: Maximum‐likelihood phylogenetic analysis of IDA SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S31: Maximum‐likelihood phylogenetic analysis of MEG SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. Tree was annotated and visualized in iTOL v7.0.
Figure S32: Maximum‐likelihood phylogenetic analysis of PIP SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S33: Maximum‐likelihood phylogenetic analysis of PNP SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S34: Maximum‐likelihood phylogenetic analysis of POE SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S35: Maximum‐likelihood phylogenetic analysis of PSK SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S36: Maximum‐likelihood phylogenetic analysis of PSY SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S37: Maximum‐likelihood phylogenetic analysis of TPD SSP sequences for Sorghum bicolor . The scale bar represents the average number of amino acid substitutions per site. The tree was annotated and visualized in iTOL v7.0.
Figure S38: Expression of 158 genes encoding small signaling SSPs in 19 small signaling peptide families in leaf, stem, root, and panicle tissues. SSPs were sorted based on phylogenetic order and 158 of the 219 were selected using a > 5 TPM threshold across the averaged expression values within each organ. SbECL2 has a max TPM of 3 but was included to not remove the family. Expression values are reported in TPM, with blue indicating high expression and green indicating low expression.
Data S1: Supporting information.
Data Availability Statement
The data supporting this study are available from multiple repositories. RNA‐seq data from developing stem internodes (Internodes 1–4) of R.07020, generated by Kebrom et al. 2017, are available at the NCBI under accession number GSE98817. Organ specificity data used for Tau analysis in the BTx623 Atlas dataset are accessible via the JGI Genome Portal under project IDs 1051037, 1051406, and 1053825. Laser capture microdissection (LCM) RNA‐seq data from Fu et al. 2024, are available in the NCBI Sequence Read Archive under accession numbers SRA558272, SRA558514, and SRA558539. Additionally, data from the Sorghum bicolor TX08001 Compendium Set2 Gene Expression Profiling project can be accessed through the JGI Genome Portal (https://genome.jgi.doe.gov/portal) under JGI Project ID 1454838. The phylogenetic trees can be analyzed directly in iTOL via link https://itol.embl.de/shared/1gT26PPKrO3dq or can be accessed with a phylogenetic viewer of choice by downloading the .tree files within the Supporting Information.