The AGCVIII kinase Dw2 modulates cell proliferation, endomembrane trafficking, and MLG/xylan cell wall localization in elongating stem internodes of Sorghum bicolor.

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.

Ruggedized, field-ready snapshot light-guide-based imaging spectrometer for environmental and remote sensing applications.

A field-ready, fiber-based high spatial sampling snapshot imaging spectrometer was developed for applications such as environmental monitoring and smart farming. The system achieves video rate frame transfer and exposure times down to a few hundred microseconds in typical daylight conditions with ∼63,000 spatial points and 32 spectral channels across the 470nm to 700nm wavelength range. We designed portable, ruggedized opto-mechanics to allow for imaging from an airborne platform. To ensure successful data collection prior to flight, imaging speed and signal-to-noise ratio was characterized for imaging a variety of land covers from the air. The system was validated by performing a series of observations including: Liriope Muscari plants under a range of water-stress conditions in a controlled laboratory experiment and field observations of sorghum plants in a variety of soil conditions. Finally, we collected data from a series of engineering flights and present reassembled images and spectral sampling of rural and urban landscapes.

Genetic modification of PIN genes induces causal mechanisms of stay-green drought adaptation phenotype.

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.

Regulation of dhurrin pathway gene expression during Sorghum bicolor development.

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. 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.

Low-field magnetic resonance imaging of roots in intact clayey and silty soils.

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.

The Sorghum bicolor reference genome: improved assembly, gene annotations, a transcriptome atlas, and signatures of genome organization.

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.

Transcriptome Profiling of Tiller Buds Provides New Insights into PhyB Regulation of Tillering and Indeterminate Growth in Sorghum.

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.

3D Sorghum Reconstructions from Depth Images Identify QTL Regulating Shoot Architecture.

Dissecting the genetic basis of complex traits is aided by frequent and nondestructive 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 (Sorghum bicolor), an important grain, forage, and bioenergy crop, at multiple developmental time points from a greenhouse-grown recombinant inbred line population. A semiautomated 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 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 quantitative trait loci 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.

Photosynthetic leaf area modulates tiller bud outgrowth in sorghum.

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.

CONSTANS is a photoperiod regulated activator of flowering in sorghum.

BACKGROUND: Sorghum genotypes used for grain production in temperate regions are photoperiod insensitive and flower early avoiding adverse environments during the reproductive phase. In contrast, energy sorghum hybrids are highly photoperiod sensitive with extended vegetative phases in long days, resulting in enhanced biomass accumulation. SbPRR37 and SbGHD7 contribute to photoperiod sensitivity in sorghum by repressing expression of SbEHD1 and FT-like genes, thereby delaying flowering in long days with minimal influence in short days (PNAS_108:16469-16474, 2011; Plant Genome_in press, 2014). The GIGANTEA (GI)-CONSTANS (CO)-FLOWERING LOCUS T (FT) pathway regulates flowering time in Arabidopsis and the grasses (J Exp Bot_62:2453-2463, 2011). In long day flowering plants, such as Arabidopsis and barley, CONSTANS activates FT expression and flowering in long days. In rice, a short day flowering plant, Hd1, the ortholog of CONSTANS, activates flowering in short days and represses flowering in long days. RESULTS: Quantitative trait loci (QTL) that modify flowering time in sorghum were identified by screening Recombinant Inbred Lines (RILs) derived from BTx642 and Tx7000 in long days, short days, and under field conditions. Analysis of the flowering time QTL on SBI-10 revealed that BTx642 encodes a recessive CONSTANS allele containing a His106Tyr substitution in B-box 2 known to inactivate CONSTANS in Arabidopsis thaliana. Genetic analysis characterized sorghum CONSTANS as a floral activator that promotes flowering by inducing the expression of EARLY HEADING DATE 1 (SbEHD1) and sorghum orthologs of the maize FT genes ZCN8 (SbCN8) and ZCN12 (SbCN12). The floral repressor PSEUDORESPONSE REGULATOR PROTEIN 37 (PRR37) inhibits sorghum CONSTANS activity and flowering in long days. CONCLUSION: Sorghum CONSTANS is an activator of flowering that is repressed post-transcriptionally in long days by the floral inhibitor PRR37, contributing to photoperiod sensitive flowering in Sorghum bicolor, a short day plant.

Stay-green alleles individually enhance grain yield in sorghum under drought by modifying canopy development and water uptake patterns.

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.

Drought adaptation of stay-green sorghum is associated with canopy development, leaf anatomy, root growth, and water uptake.

Stay-green sorghum plants exhibit greener leaves and stems during the grain-filling period under water-limited conditions compared with their senescent counterparts, resulting in increased grain yield, grain mass, and lodging resistance. Stay-green has been mapped to a number of key chromosomal regions, including Stg1, Stg2, Stg3, and Stg4, but the functions of these individual quantitative trait loci (QTLs) remain unclear. The objective of this study was to show how positive effects of Stg QTLs on grain yield under drought can be explained as emergent consequences of their effects on temporal and spatial water-use patterns that result from changes in leaf-area dynamics. A set of four Stg near-isogenic lines (NILs) and their recurrent parent were grown in a range of field and semicontrolled experiments in southeast Queensland, Australia. These studies showed that the four Stg QTLs regulate canopy size by: (1) reducing tillering via increased size of lower leaves, (2) constraining the size of the upper leaves; and (3) in some cases, decreasing the number of leaves per culm. In addition, they variously affect leaf anatomy and root growth. The multiple pathways by which Stg QTLs modulate canopy development can result in considerable developmental plasticity. The reduction in canopy size associated with Stg QTLs reduced pre-flowering water demand, thereby increasing water availability during grain filling and, ultimately, grain yield. The generic physiological mechanisms underlying the stay-green trait suggest that similar Stg QTLs could enhance post-anthesis drought adaptation in other major cereals such as maize, wheat, and rice.

Coincident light and clock regulation of pseudoresponse regulator protein 37 (PRR37) controls photoperiodic flowering in sorghum.

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.

Digital genotyping of sorghum - a diverse plant species with a large repeat-rich genome.

BACKGROUND: Rapid acquisition of accurate genotyping information is essential for all genetic marker-based studies. For species with relatively small genomes, complete genome resequencing is a feasible approach for genotyping; however, for species with large and highly repetitive genomes, the acquisition of whole genome sequences for the purpose of genotyping is still relatively inefficient and too expensive to be carried out on a high-throughput basis. Sorghum bicolor is a C4 grass with a sequenced genome size of ~730 Mb, of which ~80% is highly repetitive. We have developed a restriction enzyme targeted genome resequencing method for genetic analysis, termed Digital Genotyping (DG), to be applied to sorghum and other grass species with large repeat-rich genomes. RESULTS: DG templates are generated using one of three methylation sensitive restriction enzymes that recognize a nested set of 4, 6 or 8 bp GC-rich sequences, enabling varying depth of analysis and integration of results among assays. Variation in sequencing efficiency among DG markers was correlated with template GC-content and length. The expected DG allele sequence was obtained 97.3% of the time with a ratio of expected to alternative allele sequence acquisition of >20:1. A genetic map aligned to the sorghum genome sequence with an average resolution of 1.47 cM was constructed using 1,772 DG markers from 137 recombinant inbred lines. The DG map enhanced the detection of QTL for variation in plant height and precisely aligned QTL such as Dw3 to underlying genes/alleles. Higher-resolution NgoMIV-based DG haplotypes were used to trace the origin of DNA on SBI-06, spanning Ma1 and Dw2 from progenitors to BTx623 and IS3620C. DG marker analysis identified the correct location of two miss-assembled regions and located seven super contigs in the sorghum reference genome sequence. CONCLUSION: DG technology provides a cost-effective approach to rapidly generate accurate genotyping data in sorghum. Currently, data derived from DG are used for many marker-based analyses, including marker-assisted breeding, pedigree and QTL analysis, genetic map construction, map-based gene cloning and association studies. DG in combination with whole genome resequencing is dramatically accelerating all aspects of genetic analysis of sorghum, an important genetic reference for C4 grass species.

Epicuticular wax accumulation and regulation of wax pathway gene expression during bioenergy Sorghum stem development.

Bioenergy sorghum is a drought-tolerant high-biomass C4 grass targeted for production on annual cropland marginal for food crops due primarily to abiotic constraints. To better understand the overall contribution of stem wax to bioenergy sorghum's resilience, the current study characterized sorghum stem cuticular wax loads, composition, morphometrics, wax pathway gene expression and regulation using vegetative phase Wray, R07020, and TX08001 genotypes. Wax loads on sorghum stems (~103-215 µg/cm2) were much higher than Arabidopsis stem and leaf wax loads. Wax on developing sorghum stem internodes was enriched in C28/30 primary alcohols (~65%) while stem wax on fully developed stems was enriched in C28/30 aldehydes (~80%). Scanning Electron Microscopy showed minimal wax on internodes prior to the onset of elongation and that wax tubules first appear associated with cork-silica cell complexes when internode cell elongation is complete. Sorghum homologs of genes involved in wax biosynthesis/transport were differentially expressed in the stem epidermis. Expression of many wax pathway genes (i.e., SbKCS6, SbCER3-1, SbWSD1, SbABCG12, SbABCG11) is low in immature apical internodes then increases at the onset of stem wax accumulation. SbCER4 is expressed relatively early in stem development consistent with accumulation of C28/30 primary alcohols on developing apical internodes. High expression of two SbCER3 homologs in fully elongated internodes is consistent with a role in production of C28/30 aldehydes. Gene regulatory network analysis aided the identification of sorghum homologs of transcription factors that regulate wax biosynthesis (i.e., SbSHN1, SbWRI1/3, SbMYB94/96/30/60, MYS1) and other transcription factors that could regulate and specify expression of the wax pathway in epidermal cells during cuticle development.

Transcriptional regulation of the raffinose family oligosaccharides pathway in Sorghum bicolor reveals potential roles in leaf sucrose transport and stem sucrose accumulation.

Bioenergy sorghum hybrids are being developed with enhanced drought tolerance and high levels of stem sugars. Raffinose family oligosaccharides (RFOs) contribute to plant environmental stress tolerance, sugar storage, transport, and signaling. To better understand the role of RFOs in sorghum, genes involved in myo-inositol and RFO metabolism were identified and relative transcript abundance analyzed during development. Genes involved in RFO biosynthesis (SbMIPS1, SbInsPase, SbGolS1, SbRS) were more highly expressed in leaves compared to stems and roots, with peak expression early in the morning in leaves. SbGolS, SbRS, SbAGA1 and SbAGA2 were also expressed at high levels in the leaf collar and leaf sheath. In leaf blades, genes involved in myo-inositol biosynthesis (SbMIPS1, SbInsPase) were expressed in bundle sheath cells, whereas genes involved in galactinol and raffinose synthesis (SbGolS1, SbRS) were expressed in mesophyll cells. Furthermore, SbAGA1 and SbAGA2, genes that encode neutral-alkaline alpha-galactosidases that hydrolyze raffinose, were differentially expressed in minor vein bundle sheath cells and major vein and mid-rib vascular and xylem parenchyma. This suggests that raffinose synthesized from sucrose and galactinol in mesophyll cells diffuses into vascular bundles where hydrolysis releases sucrose for long distance phloem transport. Increased expression (>20-fold) of SbAGA1 and SbAGA2 in stem storage pith parenchyma of sweet sorghum between floral initiation and grain maturity, and higher expression in sweet sorghum compared to grain sorghum, indicates these genes may play a key role in non-structural carbohydrate accumulation in stems.

High planting density induces the expression of GA3-oxidase in leaves and GA mediated stem elongation in bioenergy sorghum.

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.

Shade signals alter the expression of circadian clock genes in newly-formed bioenergy sorghum internodes.

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.

Maturity2, a novel regulator of flowering time in Sorghum bicolor, increases expression of SbPRR37 and SbCO in long days delaying flowering.

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. Eight sorghum genotypes previously identified as recessive for Ma2 contained the mutated version of Sobic.002G302700 present in 80M (ma2) and one additional putative recessive ma2 allele was identified in diverse sorghum accessions.

Developmental dynamics of stem starch accumulation in Sorghum bicolor.

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, ß-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.