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The stay-green trait is recognized as a key drought adaptation mechanism in cereals worldwide. Stay-green sorghum plants exhibit delayed senescence of leaves and stems, leading to prolonged growth, a reduced risk of lodging, and higher grain yield under end-of-season drought stress. More than 45 quantitative trait loci (QTL) associated with stay-green have been identified, including two major QTL (Stg1 and Stg2). However, the contributing genes that regulate functional stay-green are not known. Here we show that the PIN FORMED family of auxin efflux carrier genes induce some of the causal mechanisms driving the stay-green phenotype in sorghum, with SbPIN4 and SbPIN2 located in Stg1 and Stg2, respectively. We found that nine of 11 sorghum PIN genes aligned with known stay-green QTL. In transgenic studies, we demonstrated that PIN genes located within the Stg1 (SbPIN4), Stg2 (SbPIN2), and Stg3b (SbPIN1) QTL regions acted pleiotropically to modulate canopy development, root architecture, and panicle growth in sorghum, with SbPIN1, SbPIN2, and SbPIN4 differentially expressed in various organs relative to the non-stay-green control. The emergent consequence of such modifications in canopy and root architecture is a stay-green phenotype. Crop simulation modelling shows that the SbPIN2 phenotype can increase grain yield under drought.

Raman and infrared (IR) are two complementary vibrational spectroscopy techniques that enable label-free, noninvasive, and nondestructive structural characterization of analyzed specimens. IR spect...

The stems of bioenergy sorghum hybrids at harvest are > 4 m long, contain > 40 internodes and account for ~ 80% of harvested biomass. In this study, bioenergy sorghum hybrids were grown at four planting densities (~ 20,000 to 132,000 plants/ha) under field conditions for 60 days to investigate the impact shading has on stem growth and biomass accumulation. Increased planting density induced a > 2-fold increase in sorghum internode length and a ~ 22% decrease in stem diameter, a typical shade avoidance response. Shade-induced internode elongation was due to an increase in cell length and number of cells spanning the length of internodes. SbGA3ox2 (Sobic.003G045900), a gene encoding the last step in GA biosynthesis, was expressed ~ 20-fold higher in leaf collar tissue of developing phytomers in plants grown at high vs. low density. Application of GA3 to bioenergy sorghum increased plant height, stem internode length, cell length and the number of cells spanning internodes. Prior research showed that sorghum plants lacking phytochrome B, a key photoreceptor involved in shade signaling, accumulated more GA1 and displayed shade avoidance phenotypes. These results are consistent with the hypothesis that increasing planting density induces expression of GA3-oxidase in leaf collar tissue, increasing synthesis of GA that stimulates internode elongation.

Main conclusion Developmental and organ-specific expression of genes in dhurrin biosynthesis, bio-activation, and recycling offers dynamic metabolic responses optimizing growth and defence responses in Sorghum. Abstract Plant defence models evaluate the costs and benefits of resource investments at different stages in the life cycle. Poor understanding of the molecular regulation of defence deployment and remobilization hampers accuracy of the predictions. Cyanogenic glucosides, such as dhurrin are phytoanticipins that release hydrogen cyanide upon bio-activation. In this study, RNA-seq was used to investigate the expression of genes involved in the biosynthesis, bio-activation and recycling of dhurrin in Sorghum bicolor. Genes involved in dhurrin biosynthesis were highly expressed in all young developing vegetative tissues (leaves, leaf sheath, roots, stems), tiller buds and imbibing seeds and showed gene specific peaks of expression in leaves during diel cycles. Genes involved in dhurrin bio-activation were expressed early in organ development with organ-specific expression patterns. Genes involved in recycling were expressed at similar levels in the different organ during development, although post-floral initiation when nutrients are remobilized for grain filling, expression of GSTL1 decreased > tenfold in leaves and NITB2 increased > tenfold in stems. Results are consistent with the establishment of a pre-emptive defence in young tissues and regulated recycling related to organ senescence and increased demand for nitrogen during grain filling. This detailed characterization of the transcriptional regulation of dhurrin biosynthesis, bioactivation and remobilization genes during organ and plant development will aid elucidation of gene regulatory networks and signalling pathways that modulate gene expression and dhurrin levels. In-depth knowledge of dhurrin metabolism could improve the yield, nitrogen use efficiency and stress resilience of Sorghum.

SUMMARY Stems of bioenergy sorghum (Sorghum bicolor L. Moench.), a drought‐tolerant C4 grass, contain up to 50 nodes and internodes of varying length that span 4–5 m and account for approximately 84% of harvested biomass. Stem internode growth impacts plant height and biomass accumulation and is regulated by brassinosteroid signaling, auxin transport, and gibberellin biosynthesis. In addition, an AGCVIII kinase (Dw2) regulates sorghum stem internode growth, but the underlying mechanism and signaling network are unknown. Here we provide evidence that mutation of Dw2 reduces cell proliferation in internode intercalary meristems, inhibits endocytosis, and alters the distribution of heteroxylan and mixed linkage glucan in cell walls. Phosphoproteomic analysis showed that Dw2 signaling influences the phosphorylation of proteins involved in lipid signaling (PLDδ), endomembrane trafficking, hormone, light, and receptor signaling, and photosynthesis. Together, our results show that Dw2 modulates endomembrane function and cell division during sorghum internode growth, providing insight into the regulation of monocot stem development., Significance Statement Bioenergy sorghum (Sorghum bicolor L. Moench.) is a promising drought‐tolerant high‐biomass crop that produces 4–5 m stems that account for approximately 80% of harvested biomass. In this study, Dw2, a signaling kinase shown to regulate stem growth, altered the phosphorylation of proteins involved in lipid signaling and endomembrane function and modified endocytosis, internode cell proliferation, vascular bundle anatomy, and the localization of heteroxylan and mixed linkage glucan in cell walls.

The genetic engineering of value-added traits such as the accumulation of bioproducts in high biomass C4 grass stems is one promising strategy to make plant-derived biofuels more economical for industrial use. A first step toward achieving this goal is to identify stem-specific promoters that can drive the expression of genes of interest with good temporal and spatial specificity. However, a comprehensive characterization of the spatial-temporal regulatory elements of stem tissue-specific promoters for C4 grasses has not been reported. Therefore, we performed anin-silicoanalysis onSorghum bicolor cv BTx623transcriptomes from multiple tissues over development to identify stem-expressed genes. The analysis identified 10 genes that are “Always-On-Stem-Specific,” 59 genes that are “Temporally-Stem-Specific during early development,” and 21 genes that are “Temporally-Stem-Specific during late development.” Promoter analysis revealed common and/or unique cis-regulatory elements in promoters of genes within each of the three categories. Subsequent gene regulatory network (GRN) analysis revealed that different transcriptional regulatory programs are responsible for the temporal activation of the stem-expressed genes. The analysis of temporal stem GRNs between sweet (cv Della) and grain (cv BTx623) sorghum varieties revealed genetic variation that could influence the regulatory landscape. This study provides new insights about sorghum stem biology, and information for future genetic engineering efforts to fine-tune the spatial-temporal expression of transgenes in C4 grass stems.

Stem internodes of bioenergy sorghum inbred R.07020 are longer at high plant density (shade) than at low plant density (control). Initially, the youngest newly‐formed subapical stem internodes of shade‐treated and control plants are comparable in length. However, full‐length internodes of shade‐treated plants are three times longer than the internodes of the control plants. To identify the early molecular events associated with internode elongation in response to shade, we analyzed the transcriptome of the newly‐formed internodes of shade‐treated and control plants sampled between 4 and 6 hr after the start of the light period (14 hr light/10 hr dark). Sorghum genes homologous to the Arabidopsis shade marker genes ATHB2 and PIL1 were not differentially expressed. The results indicate that shade signals promote internode elongation indirectly because sorghum internodes are not illuminated and grow while enclosed with leaf sheaths. Sorghum genes homologous to the Arabidopsis morning‐phased circadian clock genes LHY, RVE, and LNK were downregulated and evening‐phased genes such as TOC1, PRR5, and GI were upregulated in young internodes in response to shade. We hypothesize that a change in the function or patterns of expression of the circadian clock genes is the earliest molecular event associated with internode elongation in response to shade in bioenergy sorghum. Increased expression of CycD1, which promotes cell division, and decreased expression of cell wall‐loosening and MBF1‐like genes, which promote cell expansion, suggest that shade signals promote internode elongation in bioenergy sorghum in part through increasing cell number by delaying transition from cell division to cell expansion.

Sorghum offers enormous potential as a feedstock for the production of fuels and chemicals from both water-extractable sugars and the cell wall biopolymers, while its within-plant structural and co ...

A significant increase in agricultural productivity will be required by 2050 to meet the needs of an expanding and rapidly developing world population, without allocating more land and water resources to agriculture, and despite slowing rates of grain yield improvement. This review examines the proposition that high-biomass C4 grasses could help fill the yield gap. High-biomass C4 grasses exhibit high yield due to C4 photosynthesis, long growth duration, and efficient capture and utilization of light, water, and nutrients. These C4 grasses exhibit high levels of drought tolerance during their long vegetative growth phase ideal for crops grown in water-limited regions of agricultural production. The stems of some high-biomass C4 grasses can accumulate high levels of non-structural carbohydrates that could be engineered to enhance biomass yield and utility as feedstocks for animals and biofuels production. The regulatory pathway that delays flowering of high-biomass C4 grasses in long days has been elucidated enabling production and deployment of hybrids. Crop and landscape-scale modeling predict that utilization of high-biomass C4 grass crops on land and in regions where water resources limit grain crop yield could increase agricultural productivity.

Phytochrome B (phyB) enables plants to modify shoot branching or tillering in response to varying light intensities and ratios of red and far-red light caused by shading and neighbor proximity. Tillering is inhibited in sorghum genotypes that lack phytochrome B (58M, phyB-1) until after floral initiation. The growth of tiller buds in the first leaf axil of wild-type (100M, PHYB) and phyB-1 sorghum genotypes is similar until 6 d after planting when buds of phyB-1 arrest growth, while wild-type buds continue growing and develop into tillers. Transcriptome analysis at this early stage of bud development identified numerous genes that were up to 50-fold differentially expressed in wild-type/phyB-1 buds. Up-regulation of terminal flower1, GA2oxidase, and TPPI could protect axillary meristems in phyB-1 from precocious floral induction and decrease bud sensitivity to sugar signals. After bud growth arrest in phyB-1, expression of dormancy-associated genes such as DRM1, GT1, AF1, and CKX1 increased and ENOD93, ACCoxidase, ARR3/6/9, CGA1, and SHY2 decreased. Continued bud outgrowth in wild-type was correlated with increased expression of genes encoding a SWEET transporter and cell wall invertases. The SWEET transporter may facilitate Suc unloading from the phloem to the apoplast where cell wall invertases generate monosaccharides for uptake and utilization to sustain bud outgrowth. Elevated expression of these genes was correlated with higher levels of cytokinin/sugar signaling in growing buds of wild-type plants.

The development of a robust method to non-invasively visualize root morphology in natural soils has been hampered by the opaque, physical, and structural properties of soils. In this work we describe a novel technology, low field magnetic resonance imaging (LF-MRI), for imaging energy sorghum (Sorghum bicolor (L.) Moench) root morphology and architecture in intact soils. The use of magnetic fields much weaker than those used with traditional MRI experiments reduces the distortion due to magnetic material naturally present in agricultural soils. A laboratory based LF-MRI operating at 47 mT magnetic field strength was evaluated using two sets of soil cores: 1) soil/root cores of Weswood silt loam (Udifluventic Haplustept) and a Belk clay (Entic Hapluderts) from a conventionally tilled field, and 2) soil/root cores from rhizotrons filled with either a Houston Black (Udic Haplusterts) clay or a sandy loam purchased from a turf company. The maximum soil water nuclear magnetic resonance (NMR) relaxation time T2 (4 ms) and the typical root water relaxation time T2 (100 ms) are far enough apart to provide a unique contrast mechanism such that the soil water signal has decayed to the point of no longer being detectable during the data collection time period. 2-D MRI projection images were produced of roots with a diameter range of 1.5–2.0 mm using an image acquisition time of 15 min with a pixel resolution of 1.74 mm in four soil types. Additionally, we demonstrate the use of a data-driven machine learning reconstruction approach, Automated Transform by Manifold Approximation (AUTOMAP) to reconstruct raw data and improve the quality of the final images. The application of AUTOMAP showed a SNR (Signal to Noise Ratio) improvement of two fold on average. The use of low field MRI presented here demonstrates the possibility of applying low field MRI through intact soils to root phenotyping and agronomy to aid in understanding of root morphology and the spatial arrangement of roots in situ.

Sorghum (Sorghum bicolor L. Moench) offers substantial potential as a feedstock for the production of sugar-derived biofuels and biochemical products from cell wall polysaccharides (i. e., cellulose and hemicelluloses) and water-extractable sugars (i.e., glucose, fructose, sucrose, and starch). A number of preprocessing schemes can be envisioned that involve processes such as sugar extraction, pretreatment, and densification that could be employed in decentralized, regional-scale biomass processing depots. In this work, an energy sorghum exhibiting a combination of high biomass productivity and high sugar accumulation was evaluated for its potential for integration into several potential biomass preprocessing schemes. This included counter-current extraction of water-soluble sugars followed by mild NaOH or liquid hot water pretreatment of the extracted bagasse. A novel processing scheme was investigated that could integrate with current diffuser-type extraction systems for sugar extraction. In this approach, mild NaOH pretreatment (i.e., 8%) at high metabolic yields without detoxification using a Saccharomyces cerevisiae strain metabolically engineered and evolved to ferment xylose.

The cuticle covers external surfaces of plants, protecting them from biotic and abiotic stress factors. Epicuticular wax on the outer surface of the cuticle modifies reflectance and water loss from plant surfaces and has direct and indirect effects on photosynthesis. Variation in epicuticular wax accumulation, composition, and nanoscale structural organization impacts its biological function. Atomic force microscope infrared spectroscopy (AFM-IR) was utilized to investigate the internal and external surfaces of the cuticle of Sorghum bicolor, an important drought-tolerant cereal, forage, and high-biomass crop. AFM-IR revealed striking heterogeneity in chemical composition within and between the surfaces of the cuticle. The wax aggregate crystallinity and distribution of chemical functional groups across the surfaces was also probed and compared. These results, along with the noninvasive nondestructive nature of the method, suggest that AFM-IR can be used to investigate mechanisms of wax deposition and transport of charged molecules through the plant cuticle.

Sorghum bicolor is a drought‐resilient C4 grass used for production of grain, forage, sugar, and biomass. Sorghum genotypes capable of accumulating high levels of stem sucrose have solid stems that contain low levels of aerenchyma. The D‐locus on SBI06 modulates the extent of aerenchyma formation in sorghum stems and leaf midribs. A QTL aligned with this locus was identified and fine‐mapped in populations derived from BTx623*IS320c, BTx623*R07007, and BTx623*Standard broomcorn. Analysis of coding polymorphisms in the fine‐mapped D‐locus showed that genotypes that accumulate low levels of aerenchyma encode a truncated NAC transcription factor (Sobic.006G147400, SbNAC_d1), whereas parental lines that accumulate higher levels of stem aerenchyma encode full‐length NAC TFs (SbNAC‐D). During vegetative stem development, aerenchyma levels are low in nonelongated stem internodes, internode growing zones, and nodes. Aerenchyma levels increase in recently elongated internodes starting at the top of the internode near the center of the stem. SbNAC_D was expressed at low levels in nonelongated internodes and internode growing zones and at higher levels in regions of stem internodes that form aerenchyma. SbXCP1, a gene encoding a cysteine protease involved in programmed cell death, was induced in SbNAC_D genotypes in parallel with aerenchyma formation in sorghum stems but not in SbNAC_d1 genotypes. Several sweet sorghum genotypes encode the recessive SbNAC_d1 allele and have low levels of stem aerenchyma. Based on these results, we propose that SbNAC_D is the D‐gene identified by Hilton (1916) and that allelic variation in SbNAC_D modulates the extent of aerenchyma formation in sorghum stems.

Sweet sorghums were identified that accumulate up to ~9% of their total stem dry weight as starch. Starch accumulated preferentially in stem pith parenchyma in close proximity to vascular bundles. Stem starch accumulated slowly between floral initiation and anthesis and more rapidly between anthesis and 43 days post‐anthesis before declining in parallel with tiller outgrowth. Genes involved in stem starch metabolism were identified through phylogenetic approaches and RNA‐seq analysis of Della stem gene expression during the starch accumulation phase of development. Genes differentially expressed in stems were identified that are involved in starch biosynthesis (i.e., AGPase SS/LS, starch synthases, starch‐branching enzymes), degradation (i.e., glucan‐water dikinase, β‐amylase, disproportionating enzyme, alpha‐glucan phosphorylase) and amyloplast sugar transport (glucose‐6‐P translocator). Transcripts encoding AGPase SS and LS subunits with plastid localization were differentially induced during stem starch accumulation indicating that ADP‐glucose for starch biosynthesis is primarily generated in stem plastids. Cytosolic heteroglucan metabolism may play a role in stem sucrose/starch accumulation because genes encoding cytosolic forms of the disproportionating enzyme and alpha‐glucan phosphorylase were induced in parallel with stem sucrose/starch accumulation. Information on the stem starch pathway obtained in this study will be useful for engineering sorghum stems with elevated starch thereby improving forage quality and the efficiency of biomass conversion to biofuels and bio‐products.

Shoot branches or tillers develop from axillary buds. The dormancy versus outgrowth fates of buds depends on genetic, environmental and hormonal signals. Defoliation inhibits bud outgrowth indicating the role of leaf-derived metabolic factors such as sucrose in bud outgrowth. In this study, the sensitivity of bud outgrowth to selective defoliation was investigated. At 6 d after planting (6 DAP), the first two leaves of sorghum were fully expanded and the third was partially emerged. Therefore, the leaves were selectively defoliated at 6 DAP and the length of the bud in the first leaf axil was measured at 8 DAP. Bud outgrowth was inhibited by defoliation of only 2 cm from the tip of the second leaf blade. The expression of dormancy and sucrose-starvation marker genes was up-regulated and cell cycle and sucrose-inducible genes was down-regulated during the first 24 h post-defoliation of the second leaf. At 48 h, the expression of these genes was similar to controls as the defoliated plant recovers. Our results demonstrate that small changes in photosynthetic leaf area affect the propensity of tiller buds for outgrowth. Therefore, variation in leaf area and photosynthetic activity should be included when integrating sucrose into models of shoot branching.

Recent advances in variant calling made available in the Genome Analysis Toolkit (GATK) enable the use of validated single-nucleotide polymorphisms and indels to improve variant calling. However, large collections of variants for this purpose often are unavailable to research communities. We introduce a workflow to generate reliable collections of single-nucleotide polymorphisms and indels by leveraging available genomic resources to inform variant calling using the GATK. The workflow is demonstrated for the crop plant Sorghum bicolor by (i) generating an initial set of variants using reduced representation sequence data from an experimental cross and association panels, (ii) using the initial variants to inform variant calling from whole-genome sequence data of resequenced individuals, and (iii) using variants identified from whole-genome sequence data for recalibration of the reduced representation sequence data. The reliability of variants called with the workflow is verified by comparison with genetically mappable variants from an independent sorghum experimental cross. Comparison with a recent sorghum resequencing study shows that the workflow identifies an additional 1.62 million high-confidence variants from the same sequence data. Finally, the workflow’s performance is validated using Arabidopsis sequence data, yielding variant call sets with 95% sensitivity and 99% positive predictive value. The Recalibration and Interrelation of genomic sequence data with the GATK (RIG) workflow enables the GATK to accurately identify genetic variation in organisms lacking validated variant resources.

This study was conducted to document the extent and basis of compositional variation of shoot biomass of the energy Sorghum bicolor hybrid TX08001 during development under field conditions. TX08001 is capable of accumulating ~40 Mg/ha of dry biomass under good growing conditions and this genotype allocates ~80% of its shoot biomass to stems. After 150 days of growth TX08001 stems had a fresh/dry weight ratio of ~3:1 and soluble biomass accounted for ~30% of stem biomass. A panel of diverse energy sorghum genotypes varied ~6-fold in the ratio of stem structural to soluble biomass after 150 days of growth. Near-infrared spectroscopic analysis (NIRS) showed that TX08001 leaves accumulated higher levels of protein, water extractives and ash compared to stems, which have higher sugar, cellulose, and lignin contents. TX08001 stem sucrose content varied during development, whereas the composition of TX08001 stem cell walls, which consisted of ~45-49% cellulose, ~27-30% xylan, and ~15-18% lignin, remained constant after 90 days post emergence until the end of the growing season (180 days). TX08001 and Della stem syringyl (S)/guaiacyl (G) (0.53-0.58) and ferulic acid (FA)/para-coumaric acid (pCA) ratios were similar whereas ratios of pCA/(S+G) differed between these genotypes. Additionally, an analysis of irrigated versus non-irrigated TX08001 revealed that non-irrigated hybrids exhibited a 50% reduction in total cell wall biomass, an ~2-fold increase in stem sugars, and an ~25% increase in water extractives relative to irrigated hybrids. This study provides a baseline of information to help guide further optimization of energy sorghum composition for various end-uses.

International audience; This paper presents a 3D plant segmentation method with an emphasis on segmentation of the leaves. This method is part of a 3D plant phenotyping project with a main objective that deals with the development of the leaf area over time. First, a 3D point cloud of a plant is obtained with Structure from Motion technique and the cloud is then segmented into the main components of a plant: the stem and the leaves. As the main objective is to measure leaf area over time, an emphasis was placed on accurate segmentation and the labelling of the leaves. This article presents an original approach which starts by finding the stem in a 3D point cloud and then the leaves. Moreover, this method relies on the model of a plant as well as the agronomic rules to affect a unique label that do not change over time. This method is evaluated using two morphologically distinct plants, sunflower and sorghum.

Plant waxes and staygreen are distinct phenotypic traits that have been independently implicated in heat and drought tolerance among grasses. The association between these two traits has not been fully explored, which makes the exploitation of synergy between them difficult. This study assessed the association between QTL regulating the staygreen (Stg) trait in sorghum and those regulating epicuticular wax load (WL) and associated canopy temperature depression (TD). Using a recombinant inbred line (RIL) population derived from Tx642 and Tx7000, phenotypic data were collected in three replicated field trials and one greenhouse trial. High absolute TD generally corresponded to high WL. The r2 of TD against WL was highest under non-stress conditions in the greenhouse while it was much larger in the cooler and irrigated field conditions compared to hotter, drier field trials. The genetic correlations between the two traits also followed this pattern. Composite interval mapping identified a total of 28 QTL, 15 of which had significant overlaps between different traits. Most of the wax QTL were associated with pre-anthesis drought tolerant Tx7000. However, one QTL for WL overlapped with a QTL for staygreen (Stg2) and was represented by a single, isolated marker near the centromeric region on the short arm of SBI-01. The marker is identified by a Cis-acting regulatory module and is part of a 2-kb multifunctional motif-rich region which includes core promoter and enhancer regions and transcription elements, including a drought-responsive MYB binding site. We suggest that this QTL may be pleiotropic for important stress tolerance mechanisms regulating both staygreen and leaf wax in sorghum.

Background Bioenergy sorghum accumulates 75% of shoot biomass in stem internodes. Grass stem internodes are formed during vegetative growth and elongate in response to developmental and environmental signals. To identify genes and molecular mechanisms that modulate the extent of internode growth, we conducted microscopic and transcriptomic analyses of four successive sub-apical vegetative internodes representing different stages of internode development of the bioenergy sorghum genotype R.07020. Results Stem internodes of sorghum genotype R.07020 are formed during the vegetative phase and their length is enhanced by environmental signals such as shade and floral induction in short days. During vegetative growth, the first visible and youngest sub-apical internode was ~0.7 cm in length, whereas the fourth fully expanded internode was ~5 cm in length. Microscopic analyses revealed that all internode tissue types including pith parenchyma and vascular bundles are present in the four successive internodes. Growth in the first two sub-apical internodes occurred primarily through an increase in cell number consistent with expression of genes involved in the cell cycle and DNA replication. Growth of the 3rd internode was associated with an increase in cell length and growth cessation in the 4th internode was associated with up-regulation of genes involved in secondary cell wall deposition. The expression of genes involved in hormone metabolism and signaling indicates that GA, BR, and CK activity decreased while ethylene, ABA, and JA increased in the 3rd/4th internodes. While the level of auxin appears to be increasing as indicated by the up-regulation of ARFs, down-regulation of TIR during development indicates that auxin signaling is also modified. The expression patterns of transcription factors are closely associated with their role during the development of the vegetative internodes. Conclusions Microscopic and transcriptome analyses of four successive sub-apical internodes characterized the developmental progression of vegetative stem internodes from initiation through full elongation in the sorghum genotype R.07020. Transcriptome profiling indicates that dynamic variation in the levels and action of GA, CK, IAA, BR, ethylene, ABA, and JA modulate gene expression and growth during internode growth and development. This study provides detailed microscopic and transcriptomic data useful for identifying genes and molecular pathways regulating internode elongation in response to various developmental and environmental signals. Electronic supplementary material The online version of this article (doi:10.1186/s13068-017-0848-3) contains supplementary material, which is available to authorized users.

Sorghum bicolor is a drought tolerant C4 grass used for the production of grain, forage, sugar, and lignocellulosic biomass and a genetic model for C4 grasses due to its relatively small genome (approximately 800 Mbp), diploid genetics, diverse germplasm, and colinearity with other C4 grass genomes. In this study, deep sequencing, genetic linkage analysis, and transcriptome data were used to produce and annotate a high-quality reference genome sequence. Reference genome sequence order was improved, 29.6 Mbp of additional sequence was incorporated, the number of genes annotated increased 24% to 34 211, average gene length and N50 increased, and error frequency was reduced 10-fold to 1 per 100 kbp. Subtelomeric repeats with characteristics of Tandem Repeats in Miniature (TRIM) elements were identified at the termini of most chromosomes. Nucleosome occupancy predictions identified nucleosomes positioned immediately downstream of transcription start sites and at different densities across chromosomes. Alignment of more than 50 resequenced genomes from diverse sorghum genotypes to the reference genome identified approximately 7.4 M single nucleotide polymorphisms (SNPs) and 1.9 M indels. Large-scale variant features in euchromatin were identified with periodicities of approximately 25 kbp. A transcriptome atlas of gene expression was constructed from 47 RNA-seq profiles of growing and developed tissues of the major plant organs (roots, leaves, stems, panicles, and seed) collected during the juvenile, vegetative and reproductive phases. Analysis of the transcriptome data indicated that tissue type and protein kinase expression had large influences on transcriptional profile clustering. The updated assembly, annotation, and transcriptome data represent a resource for C4 grass research and crop improvement.

Bioenergy sorghum is targeted for production in water-limited annual cropland therefore traits that improve plant water capture, water use efficiency, and resilience to water deficit are necessary to maximize productivity. A crop modeling framework, APSIM, was adapted to predict the growth and biomass yield of energy sorghum and to identify potentially useful traits for crop improvement. APSIM simulations of energy sorghum development and biomass accumulation replicated results from field experiments across multiple years, patterns of rainfall, and irrigation schemes. Modeling showed that energy sorghum's long duration of vegetative growth increased water capture and biomass yield by ~30% compared to short season crops in a water-limited production region. Additionally, APSIM was extended to enable modeling of VPD-limited transpiration traits that reduce crop water use under high vapor pressure deficits (VPDs). The response of transpiration rate to increasing VPD was modeled as a linear response until a VPD threshold was reached, at which the slope of the response decreases, representing a range of responses to VPD observed in sorghum germplasm. Simulation results indicated that the VPD-limited transpiration trait is most beneficial in hot and dry regions of production where crops are exposed to extended periods without rainfall during the season or to a terminal drought. In these environments, slower but more efficient transpiration increases biomass yield and prevents or delays the exhaustion of soil water and onset of leaf senescence. The VPD-limited transpiration responses observed in sorghum germplasm increased biomass accumulation by 20% in years with lower summer rainfall, and the ability to drastically reduce transpiration under high VPD conditions could increase biomass by 6% on average across all years. This work indicates that the productivity and resilience of bioenergy sorghum grown in water-limited environments could be further enhanced by development of genotypes with optimized VPD-limited transpiration traits and deployment of these crops in water limited growing environments. The energy sorghum model and VPD-limited transpiration trait implementation are made available to simulate performance in other target environments.

2ABSTRACTSorghum bicoloris a drought tolerant C4 grass used for production of grain, forage, sugar, and lignocellulosic biomass and a genetic model for C4 grasses due to its relatively small genome (~800 Mbp), diploid genetics, diverse germplasm, and colinearity with other C4 grass genomes. In this study, deep sequencing, genetic linkage analysis, and transcriptome data were used to produce and annotate a high quality reference genome sequence. Reference genome sequence order was improved, 29.6 Mbp of additional sequence was incorporated, the number of genes annotated increased 24% to 34,211, average gene length and N50 increased, and error frequency was reduced 10-fold to 1 per 100 kbp. Sub-telomeric repeats with characteristics of Tandem Repeats In Miniature (TRIM) elements were identified at the termini of most chromosomes. Nucleosome occupancy predictions identified nucleosomes positioned immediately downstream of transcription start sites and at different densities across chromosomes. Alignment of the reference genome sequence to 56 resequenced genomes from diverse sorghum genotypes identified ~7.4M SNPs and 1.8M indels. Large scale variant features in euchromatin were identified with periodicities of ~25 kbp. An RNA transcriptome atlas of gene expression was constructed from 47 samples derived from growing and developed tissues of the major plant organs (roots, leaves, stems, panicles, seed) collected during the juvenile, vegetative and reproductive phases. Analysis of the transcriptome data indicated that tissue type and protein kinase expression had large influences on transcriptional profile clustering. The updated assembly, annotation, and transcriptome data represent a resource for C4 grass research and crop improvement.

A significant increase in agricultural productivity will be required by 2050 to meet the needs of an expanding and rapidly developing world population, without allocating more land and water resources to agriculture, and despite slowing rates of grain yield improvement. This review examines the proposition that high-biomass C

Recombinant inbred populations of many plant species exhibit more heterozygosity than expected under the Mendelian model of segregation. This segregation distortion causes the overestimation of recombination frequencies and consequent genetic map expansion. Here we build upon existing genetic models of differential zygotic viability to model a heterozygote fitness term and calculate expected genotypic proportions in recombinant inbred populations propagated by selfing. We implement this model using the existing open-source genetic map construction code base for R/qtl to estimate recombination fractions. Finally, we show that accounting for excess heterozygosity in a sorghum recombinant inbred mapping population shrinks the genetic map by 213 cM (a 13% decrease corresponding to 4.26 fewer recombinations per meiosis). More accurate estimates of linkage benefit linkage-based analyses used in the identification and utilization of causal genetic variation.

Sorghum is emerging as an excellent genetic model for the design of C4 grass bioenergy crops. Annual energy Sorghum hybrids also serve as a source of biomass for bioenergy production. Elucidation of Sorghum's flowering time gene regulatory network, and identification of complementary alleles for photoperiod sensitivity, enabled large-scale generation of energy Sorghum hybrids for testing and commercial use. Energy Sorghum hybrids with long vegetative growth phases were found to accumulate more than twice as much biomass as grain Sorghum, owing to extended growing seasons, greater light interception, and higher radiation use efficiency. High biomass yield, efficient nitrogen recycling, and preferential accumulation of stem biomass with low nitrogen content contributed to energy Sorghum's elevated nitrogen use efficiency. Sorghum's integrated genetics-genomics-breeding platform, diverse germplasm, and the opportunity for annual testing of new genetic designs in controlled environments and in multiple field locations is aiding fundamental discovery, and accelerating the improvement of biomass yield and optimization of composition for biofuels production. Recent advances in wide hybridization between Sorghum and other C4 grasses could allow the deployment of improved genetic designs of annual energy Sorghums in the form of wide-hybrid perennial crops. The current trajectory of energy Sorghum genetic improvement indicates that it will be possible to sustainably produce biofuels from C4 grass bioenergy crops that are cost competitive with petroleum-based transportation fuels.

* Stay-green is an integrated drought adaptation trait characterized by a distinct green leaf phenotype during grain filling under terminal drought. We used sorghum (Sorghum bicolor), a repository of drought adaptation mechanisms, to elucidate the physiological and genetic mechanisms underpinning stay-green. * Near-isogenic sorghum lines (cv RTx7000) were characterized in a series of field and managed-environment trials (seven experiments and 14 environments) to determine the influence of four individual stay-green (Stg1–4) quantitative trait loci (QTLs) on canopy development, water use and grain yield under post-anthesis drought. * The Stg QTL decreased tillering and the size of upper leaves, which reduced canopy size at anthesis. This reduction in transpirational leaf area conserved soil water before anthesis for use during grain filling. Increased water uptake during grain filling of Stg near-isogenic lines (NILs) relative to RTx7000 resulted in higher post-anthesis biomass production, grain number and yield. Importantly, there was no consistent yield penalty associated with the Stg QTL in the irrigated control. * These results establish a link between the role of the Stg QTL in modifying canopy development and the subsequent impact on crop water use patterns and grain yield under terminal drought.

Sorghum bicolor is a drought-resilient facultative short-day C4 grass that is grown for grain, forage, and biomass. Adaptation of sorghum for grain production in temperate regions resulted in the selection of mutations in Maturity loci (Ma1 – Ma6) that reduced photoperiod sensitivity and resulted in earlier flowering in long days. Prior studies identified the genes associated with Ma1 (PRR37), Ma3 (PHYB), Ma5 (PHYC) and Ma6 (GHD7) and characterized their role in the flowering time regulatory pathway. The current study focused on understanding the function and identity of Ma2. Ma2 delayed flowering in long days by selectively enhancing the expression of SbPRR37 (Ma1) and SbCO, genes that co-repress the expression of SbCN12, a source of florigen. Genetic analysis identified epistatic interactions between Ma2 and Ma4 and located QTL corresponding to Ma2 on SBI02 and Ma4 on SBI10. Positional cloning and whole genome sequencing identified a candidate gene for Ma2, Sobic.002G302700, which encodes a SET and MYND (SYMD) domain lysine methyltransferase. Nine sorghum genotypes previously identified as recessive for Ma2 contained the mutated version of Sobic.002G302700 present in 80M (ma2).

The nitrogen use efficiency (NUE) of high biomass energy sorghum hybrid plants increased during 180 days of growth to a maximum of 370 g DW g−1 N−1. Shoot N uptake was biphasic and continued for 120 days. Leaf N accumulation was rapid until day 60. Specific leaf nitrogen (SLN) varied from 0.9 to 1.7 g N m−2 green leaf area, a typical range for C4 grass canopies. Stem N increased to a maximum at day 120. NUE increased during development in parallel with increasing stem to leaf biomass ratio and as stems decreased from 0.7% to 0.2% N. At the end of the season, green leaves were ∼1% N, represented 17% of total shoot biomass and accounted for 50% of N present in shoots (above ground biomass) while stems were ∼0.2% N, comprised 83% of shoot biomass and accounted for 50% of shoot N. High NUE was due in part to N-remobilization from lower leaves and stem nodes/internodes to upper portions of the canopy. Up to 70% of dry weight and 90% of N was remobilized during senescence of lower leaves and 70% of N was remobilized from lower stem nodes/internodes. The NUE of energy sorghum was similar to Saccharum officinarum and Miscanthus x giganteus, and higher than grain Sorghum bicolor, Zea mays, and Panicum virgatum. High NUE of energy S. bicolor is due to long duration of vegetative growth, high stem to leaf biomass ratio, and very efficient N-remobilization from lower leaves and stem internodes during development.

Dissecting the genetic basis of complex traits is aided by frequent and non-destructive measurements. Advances in range imaging technologies enable the rapid acquisition of three-dimensional (3D) data from an imaged scene. A depth camera was used to acquire images of Sorghum bicolor, an important grain, forage, and bioenergy crop, at multiple developmental timepoints from a greenhouse-grown recombinant inbred line population. A semi-automated software pipeline was developed and used to generate segmented, 3D plant reconstructions from the images. Automated measurements made from 3D plant reconstructions identified quantitative trait loci (QTL) for standard measures of shoot architecture such as shoot height, leaf angle and leaf length, and for novel composite traits such as shoot compactness. The phenotypic variability associated with some of the QTL displayed differences in temporal prevalence; for example, alleles closely linked with the sorghum Dwarf3 gene, an auxin transporter and pleiotropic regulator of both leaf inclination angle and shoot height, influence leaf angle prior to an effect on shoot height. Furthermore, variability in composite phenotypes that measure overall shoot architecture, such as shoot compactness, is regulated by loci underlying component phenotypes like leaf angle. As such, depth imaging is an economical and rapid method to acquire shoot architecture phenotypes in agriculturally important plants like sorghum to study the genetic basis of complex traits.

Summary Biomass accumulated preferentially in leaves of the sweet sorghum Della until floral initiation, then stems until anthesis, followed by panicles until grain maturity, and apical tillers. Sorghum stem RNA-seq transcriptome profiles and composition data were collected for approximately 100 days of development beginning at floral initiation. The analysis identified >200 differentially expressed genes involved in stem growth, cell wall biology, and sucrose accumulation. Genes encoding expansins and xyloglucan endotransglucosylase/hydrolases were differentially expressed in growing stem internodes. Genes encoding enzymes involved in the synthesis of cellulose, lignin, and glucuronoarabinoxylan were expressed at elevated levels in stems until approximately 7 days before anthesis and then down-regulated. CESA genes involved in primary and secondary cell wall synthesis showed different temporal patterns of expression. Following floral initiation, the level of sucrose and other non-structural carbohydrates increased to approximately 50% of the stem's dry weight. Stem sucrose accumulation was inversely correlated with >100-fold down-regulation of SbVIN1, a gene encoding a vacuolar invertase. Accumulation of stem sucrose was also correlated with cessation of leaf and stem growth at anthesis, decreased expression of genes involved in stem cell wall synthesis, and approximately 10-fold lower expression of SbSUS4, a gene encoding sucrose synthase that generates UDP-glucose from sucrose for cell wall biosynthesis. Genes for mixed linkage glucan synthesis (CSLF) and turnover were expressed at high levels in stems throughout development. Overall, the stem transcription profile resource and the genes and regulatory dynamics identified in this study will be useful for engineering sorghum stem composition for improved conversion to biofuels and bio-products.

A first-generation energy sorghum hybrid with enhanced photoperiod sensitivity and long growth duration accumulated more than twice as much biomass as grain sorghum. The energy sorghum produced more leaves (~45 vs 17–20), longer stems (~4 vs 1.5 meters) and had a higher stem-to-leaf biomass ratio than grain sorghum. At the end of the season, energy sorghum stems represented 83% of the plant's shoot biomass. The greater biomass accumulation was due to longer growth duration, a higher leaf area index, greater radiation interception, and higher radiation use efficiency. When grown under dryland or limited irrigation conditions, the rate of biomass accumulation by the energy sorghum hybrid was reduced in mid-season. This decrease was most apparent in August due to summer water deficit; however plants recovered when rain occurred in September. Crop growth modeling and biomass accumulation rates measured under optimal field conditions show that energy sorghum, like other C4 energy grasses, has excellent biomass yield potential. The greenhouse gas offset values of energy sorghum hybrids, grown in large and small field plots, and under fully irrigated and dryland conditions, ranged from 63–78% for cellulosic ethanol production and 88–95% for power generation. This study shows that drought-tolerant, annual energy sorghum hybrids have the genetic yield potential to contribute significantly to bioenergy production. Sorghum is a genetically tractable, diverse species with a good genomics platform, making energy sorghum a promising genetic model for the design of C4 grass energy crops. © 2012 Society of Chemical Industry and John Wiley & Sons, Ltd

Optimal flowering time is critical to the success of modern agriculture. Sorghum is a short-day tropical species that exhibits substantial photoperiod sensitivity and delayed flowering in long days. Genotypes with reduced photoperiod sensitivity enabled sorghum's utilization as a grain crop in temperate zones worldwide. In the present study, Ma 1 , the major repressor of sorghum flowering in long days, was identified as the pseudoresponse regulator protein 37 (PRR37) through positional cloning and analysis of SbPRR37 alleles that modulate flowering time in grain and energy sorghum. Several allelic variants of SbPRR37 were identified in early flowering grain sorghum germplasm that contain unique loss-of-function mutations. We show that in long days SbPRR37 activates expression of the floral inhibitor CONSTANS and represses expression of the floral activators Early Heading Date 1 , FLOWERING LOCUS T , Zea mays CENTRORADIALIS 8 , and floral induction. Expression of SbPRR37 is light dependent and regulated by the circadian clock, with peaks of RNA abundance in the morning and evening in long days. In short days, the evening-phase expression of SbPRR37 does not occur due to darkness, allowing sorghum to flower in this photoperiod. This study provides insight into an external coincidence mechanism of photoperiodic regulation of flowering time mediated by PRR37 in the short-day grass sorghum and identifies important alleles of SbPRR37 that are critical for the utilization of this tropical grass in temperate zone grain and bioenergy production.

Digestion and fermentation of lignocellulosic biomass (i.e., structural carbohydrates) are predicted to deliver higher yields of energy per hectare than sugar and starch (nonstructural carbohydrates), yet little research on genetic variation in crop feedstock biomass traits has been conducted. We investigated the genetic basis of leaf and stem biomass yield and composition in a population derived from a high-biomass sweet sorghum, ‘Rio’, and a grain sorghum inbred line, ‘BTx623’, and compared these results with those from analyses of grain and stem sugar traits that we reported previously. Thirty-one traits were evaluated and a total of 110 quantitative trait loci (QTL) were identifi ed across three locations. Many QTL for structural and nonstructural carbohydrate yields colocalized with loci for height, fl owering time, and stand density–tillering. Quantitative trait loci for composition had little colocalization across tissues and environments. Separate genetic control for leaf and stem structural carbohydrate composition was identifi ed, as well as separate genetic control of protein accumulation in leaf, stem, and grain. To maximize energy yields from grain and dedicated biomass sorghums, results suggest yield traits should be targeted for improvement before composition traits.

Genetic improvement of sorghum [Sorghum bicolor (L.) Moench] has traditionally focused on a single nonstructural carbohydrate, either grain starch or stem sugar. Sorghum starch and sugar may both be used as feedstocks for biofuel production. To investigate genetic tradeoffs between grain and stem sugar, a population derived from sweet sorghum cultivar Rio and grain sorghum ‘BTx623’ was evaluated for 27 traits related to grain and stem sugar yield and composition. Across three environments, a total of 129 quantitative trait loci (QTL) were identifi ed. Tradeoffs identifi ed between grain and stem sugar yield QTL colocalized with height and fl owering time QTL. Most importantly, QTL were identifi ed that increased yield and altered the composition of stem sugar and grain without pleiotropic effects. For example, a QTL on chromosome 3 that explained 25% of the genetic variance for stem sugar concentration did not colocalize with any grain QTL. These results suggest that total nonstructural carbohydrate yield could be increased by selecting for major QTL from both grain and sweet sorghum types. We conclude that altering grain and stem sugar genetic potential for yield traits should lead to greater feedstock improvement than altering composition traits.

Apomixis, asexual reproduction through seed, is widespread among angiosperm families. Gametophytic apomixis in Pennisetum squamulatum and Cenchrus ciliaris is controlled by the apospory-specific genomic region (ASGR), which is highly conserved and macrosyntenic between these species. Thirty-two ASGR bacterial artificial chromosomes (BACs) isolated from both species and one ASGR-recombining BAC from P. squamulatum, which together cover approximately 2.7 Mb of DNA, were used to investigate the genomic structure of this region. Phrap assembly of 4,521 high-quality reads generated 1,341 contiguous sequences (contigs; 730 from the ASGR and 30 from the ASGR-recombining BAC in P. squamulatum, plus 580 from the C. ciliaris ASGR). Contigs containing putative protein-coding regions unrelated to transposable elements were identified based on protein similarity after Basic Local Alignment Search Tool X analysis. These putative coding regions were further analyzed in silico with reference to the rice (Oryza sativa) and sorghum (Sorghum bicolor) genomes using the resources at Gramene (www.gramene.org) and Phytozome (www.phytozome.net) and by hybridization against sorghum BAC filters. The ASGR sequences reveal that the ASGR (1) contains both gene-rich and gene-poor segments, (2) contains several genes that may play a role in apomictic development, (3) has many classes of transposable elements, and (4) does not exhibit large-scale synteny with either rice or sorghum genomes but does contain multiple regions of microsynteny with these species.