Manipulating assimilate availability provides insight into the genes controlling grain size in sorghum.

Variation in grain size, a major determinant of grain yield and quality in cereal crops, is determined by both the plant's genetic potential and the available assimilate to fill the grain in the absence of stress. This study investigated grain size variation in response to variation in assimilate supply in sorghum using a diversity panel (n = 837) and a backcross-nested association mapping population (n = 1421) across four experiments. To explore the effects of genetic potential and assimilate availability on grain size, the top half of selected panicles was removed at anthesis. Results showed substantial variation in five grain size parameters with high heritability. Artificial reduction in grain number resulted in a general increase in grain weight, with the extent of the increase varying across genotypes. Genome-wide association studies identified 44 grain size quantitative trait locus (QTL) that were likely to act on assimilate availability and 50 QTL that were likely to act on genetic potential. This finding was further supported by functional enrichment analysis and co-location analysis with known grain number QTL and candidate genes. RNA interference and overexpression experiments were conducted to validate the function of one of the identified gene, SbDEP1, showing that SbDEP1 positively regulates grain number and negatively regulates grain size by controlling primary branching in sorghum. Haplotype analysis of SbDEP1 suggested a possible role in racial differentiation. The enhanced understanding of grain size variation in relation to assimilate availability presented in this study will benefit sorghum improvement and have implications for other cereal crops.

CRISPR-knockout of ß-kafirin in sorghum does not recapitulate the grain quality of natural mutants.

MAIN CONCLUSION: When gene editing was applied to knockout beta-kafirin, there was a compensatory increase of gamma-kafirin which does not occur in domesticated null varieties, so enhanced grain quality was not achieved. Sorghum bicolor is an important animal feedstock cereal crop throughout Australia and the southern United States, where its use as a food product is limited by issues with low calorific and nutritive value. Qualities such as reduced digestibility and low essential amino acid content are directly attributed to the kafirin grain storage proteins, the major components of protein bodies within the endosperm. Specifically, the ß- and γ-kafirins have few protease cleavage sites and high levels of cysteine residues which lead to a highly cross-linked shell of intra- and inter-molecular disulphide linkages that encapsulate the more digestible α- and δ-kafirins in the core of the protein bodies. Naturally occurring ß-kafirin mutants exist and are known to have improved grain quality, with enhanced protein contents and digestibility, traits which are often attributed to the lack of this cysteine-rich kafirin in the mature grain. However, when CRISPR/Cas9 editing was used to create ß-kafirin knockout lines, there was no improvement to grain quality in the Tx430 background, although they did have unique protein composition and changes to protein body morphology in the vitreous endosperm. One explanation of the divergence in quality traits found the lines lacking ß-kafirin are due to a drastic increase of γ-kafirin which was only found in the gene edited lines. This study highlights that in some germplasm, there is a level of redundancy between the peripheral kafirins, and that improvement of grain protein digestibility cannot be achieved by simply removing the ß-kafirin protein in all genetic backgrounds.

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.

Endogenous U6 promoters improve CRISPR/Cas9 editing efficiencies in Sorghum bicolor and show potential for applications in other cereals.

KEY MESSAGE: Endogenous U6 promoters increase CRISPR/Cas9 editing efficiency in sorghum and may be useful for gene editing applications in other cereals.

Hotter, drier, CRISPR: the latest edit on climate change.

KEY MESSAGE: Integrating CRISPR/Cas9 genome editing into modern breeding programs for crop improvement in cereals. Global climate trends in many agricultural regions have been rapidly changing over the past decades, and major advances in global food systems are required to ensure food security in the face of these emerging challenges. With increasing climate instability due to warmer temperatures and rising CO2 levels, the productivity of global agriculture will continue to be negatively impacted. To combat these growing concerns, creative approaches will be required, utilising all the tools available to produce more robust and tolerant crops with increased quality and yields under more extreme conditions. The integration of genome editing and transgenics into current breeding strategies is one promising solution to accelerate genetic gains through targeted genetic modifications, producing crops that can overcome the shifting climate realities. This review focuses on how revolutionary genome editing tools can be directly implemented into breeding programs for cereal crop improvement to rapidly counteract many of the issues affecting agriculture production in the years to come.

Genomic interventions for sustainable agriculture.

Agricultural production faces a Herculean challenge to feed the increasing global population. Food production systems need to deliver more with finite land and water resources while exerting the least negative influence on the ecosystem. The unpredictability of climate change and consequent changes in pests/pathogens dynamics aggravate the enormity of the challenge. Crop improvement has made significant contributions towards food security, and breeding climate-smart cultivars are considered the most sustainable way to accelerate food production. However, a fundamental change is needed in the conventional breeding framework in order to respond adequately to the growing food demands. Progress in genomics has provided new concepts and tools that hold promise to make plant breeding procedures more precise and efficient. For instance, reference genome assemblies in combination with germplasm sequencing delineate breeding targets that could contribute to securing future food supply. In this review, we highlight key breakthroughs in plant genome sequencing and explain how the presence of these genome resources in combination with gene editing techniques has revolutionized the procedures of trait discovery and manipulation. Adoption of new approaches such as speed breeding, genomic selection and haplotype-based breeding could overcome several limitations of conventional breeding. We advocate that strengthening varietal release and seed distribution systems will play a more determining role in delivering genetic gains at farmer's field. A holistic approach outlined here would be crucial to deliver steady stream of climate-smart crop cultivars for sustainable agriculture.

Large-scale GWAS in sorghum reveals common genetic control of grain size among cereals.

Grain size is a key yield component of cereal crops and a major quality attribute. It is determined by a genotype's genetic potential and its capacity to fill the grains. This study aims to dissect the genetic architecture of grain size in sorghum. An integrated genome-wide association study (GWAS) was conducted using a diversity panel (n = 837) and a BC-NAM population (n = 1421). To isolate genetic effects associated with genetic potential of grain size, rather than the genotype's capacity to fill the grains, a treatment of removing half of the panicle was imposed during flowering. Extensive and highly heritable variation in grain size was observed in both populations in 5 field trials, and 81 grain size QTL were identified in subsequent GWAS. These QTL were enriched for orthologues of known grain size genes in rice and maize, and had significant overlap with SNPs associated with grain size in rice and maize, supporting common genetic control of this trait among cereals. Grain size genes with opposite effect on grain number were less likely to overlap with the grain size QTL from this study, indicating the treatment facilitated identification of genetic regions related to the genetic potential of grain size. These results enhance understanding of the genetic architecture of grain size in cereal, and pave the way for exploration of underlying molecular mechanisms and manipulation of this trait in breeding practices.

Up-regulation of lipid biosynthesis increases the oil content in leaves of Sorghum bicolor.

Synthesis and accumulation of the storage lipid triacylglycerol in vegetative plant tissues has emerged as a promising strategy to meet the world's future need for vegetable oil. Sorghum (Sorghum bicolor) is a particularly attractive target crop given its high biomass, drought resistance and C4 photosynthesis. While oilseed-like triacylglycerol levels have been engineered in the C3 model plant tobacco, progress in C4 monocot crops has been lagging behind. In this study, we report the accumulation of triacylglycerol in sorghum leaf tissues to levels between 3 and 8.4% on a dry weight basis depending on leaf and plant developmental stage. This was achieved by the combined overexpression of genes encoding the Zea mays WRI1 transcription factor, Umbelopsis ramanniana UrDGAT2a acyltransferase and Sesamum indicum Oleosin-L oil body protein. Increased oil content was visible as lipid droplets, primarily in the leaf mesophyll cells. A comparison between a constitutive and mesophyll-specific promoter driving WRI1 expression revealed distinct changes in the overall leaf lipidome as well as transitory starch and soluble sugar levels. Metabolome profiling uncovered changes in the abundance of various amino acids and dicarboxylic acids. The results presented here are a first step forward towards the development of sorghum as a dedicated biomass oil crop and provide a basis for further combinatorial metabolic engineering.

Discovering new alleles for yellow spot resistance in the Vavilov wheat collection.

KEY MESSAGE: GWAS detected 11 yellow spot resistance QTL in the Vavilov wheat collection. Promising adult-plant resistance loci could provide a sustainable genetic solution to yellow spot in modern wheat varieties. Yellow spot, caused by the fungal pathogen Pyrenophora tritici-repentis (Ptr), is the most economically damaging foliar disease of wheat in Australia. Genetic resistance is considered to be the most sustainable means for disease management, yet the genomic regions underpinning resistance to Ptr, particularly adult-plant resistance (APR), remain vastly unknown. In this study, we report results of a genome-wide association study using 295 accessions from the Vavilov wheat collection which were extensively tested for response to Ptr infections in glasshouse and field trials at both seedling an adult growth stages. Combining phenotypic datasets from multiple experiments in Australia and Russia with 25,286 genome-wide, high-quality DArTseq markers, we detected a total of 11 QTL, of which 5 were associated with seedling resistance, 3 with all-stage resistance, and 3 with APR. Interestingly, the novel APR QTL were effective even in the presence of host sensitivity gene Tsn1. These genomic regions could offer broad-spectrum yellow spot protection, not just to ToxA but also other pathogenicity or virulence factors. Vavilov wheat accessions carrying APR QTL combinations displayed enhanced levels of resistance highlighting the potential for QTL stacking through breeding. We propose that the APR genetic factors discovered in our study could be used to improve resistance levels in modern wheat varieties and contribute to the sustainable control of yellow spot.

Expression patterns of the native Shrunken-2 promoter in Sorghum bicolor visualised through use of the GFP reporter gene.

KEY MESSAGE: The AGPase large subunit (shrunken-2) promoter was demonstrated to be active in the placentochalaza and endosperm of developing grain as well as the root tips in transgenic sorghum. The temporal and spatial expression patterns of the Sorghum bicolor Shrunken-2 (Sh2) promoter were evaluated using the green fluorescence protein reporter gene (gfp) in transgenic sorghum, within the context of upregulating starch biosynthesis in the developing grain. GFP fluorescence was analysed throughout development in various tissue types using confocal laser scanning microscopy techniques. Sh2 promoter activity was first detected in the placentochalaza region of the developing caryopsis and apoplasm adjacent to the nucellar epidermis at 7 days post anthesis (dpa) where fluorescence remained relatively constant until 17 dpa. Fluorescence in this region weakened by 20 dpa and disappeared by 25 dpa. Expression was also detected in the developing endosperm, but not until 12 dpa, continuing until 25 dpa. Whilst the endosperm expression was expected, the fluorescence detected in the placentochalaza was completely unexpected. Although transcript presence does not mean the resulting biochemistry is also present, these preliminary findings may suggest alternate spatial activity of ADP-glucose pyrophosphorylase prior to uptake by the developing grain. Sh2 promoter activity was also unexpectedly detected in the root tips at all developmental time points. Sh2 promoter activity was not detected in any reproductive floral tissue (both pre and post anthesis) or in pollen. Similarly, no expression was detected in leaf tissue at any stage.

Domestication and the storage starch biosynthesis pathway: signatures of selection from a whole sorghum genome sequencing strategy.

Next-generation sequencing of complete genomes has given researchers unprecedented levels of information to study the multifaceted evolutionary changes that have shaped elite plant germplasm. In conjunction with population genetic analytical techniques and detailed online databases, we can more accurately capture the effects of domestication on entire biological pathways of agronomic importance. In this study, we explore the genetic diversity and signatures of selection in all predicted gene models of the storage starch synthesis pathway of Sorghum bicolor, utilizing a diversity panel containing lines categorized as either 'Landraces' or 'Wild and Weedy' genotypes. Amongst a total of 114 genes involved in starch synthesis, 71 had at least a single signal of purifying selection and 62 a signal of balancing selection and others a mix of both. This included key genes such as STARCH PHOSPHORYLASE 2 (SbPHO2, under balancing selection), PULLULANASE (SbPUL, under balancing selection) and ADP-glucose pyrophosphorylases (SHRUNKEN2, SbSH2 under purifying selection). Effectively, many genes within the primary starch synthesis pathway had a clear reduction in nucleotide diversity between the Landraces and wild and weedy lines indicating that the ancestral effects of domestication are still clearly identifiable. There was evidence of the positional rate variation within the well-characterized primary starch synthesis pathway of sorghum, particularly in the Landraces, whereby low evolutionary rates upstream and high rates downstream in the metabolic pathway were expected. This observation did not extend to the wild and weedy lines or the minor starch synthesis pathways.

Total transcriptome, proteome, and allergome of Johnson grass pollen, which is important for allergic rhinitis in subtropical regions.

BACKGROUND: Genomic data are lacking for many allergen sources. To circumvent this limitation, we implemented a strategy to reveal the repertoire of pollen allergens of a grass with clinical importance in subtropical regions, where an increasing proportion of the world's population resides. OBJECTIVE: We sought to identify and immunologically characterize the allergenic components of the Panicoideae Johnson grass pollen (JGP; Sorghum halepense). METHODS: The total pollen transcriptome, proteome, and allergome of JGP were documented. Serum IgE reactivities with pollen and purified allergens were assessed in 64 patients with grass pollen allergy from a subtropical region. RESULTS: Purified Sor h 1 and Sor h 13 were identified as clinically important allergen components of JGP with serum IgE reactivity in 49 (76%) and 28 (43.8%), respectively, of patients with grass pollen allergy. Within whole JGP, multiple cDNA transcripts and peptide spectra belonging to grass pollen allergen families 1, 2, 4, 7, 11, 12, 13, and 25 were identified. Pollen allergens restricted to subtropical grasses (groups 22-24) were also present within the JGP transcriptome and proteome. Mass spectrometry confirmed the IgE-reactive components of JGP included isoforms of Sor h 1, Sor h 2, Sor h 13, and Sor h 23. CONCLUSION: Our integrated molecular approach revealed qualitative differences between the allergenic components of JGP and temperate grass pollens. Knowledge of these newly identified allergens has the potential to improve specific diagnosis and allergen immunotherapy treatment for patients with grass pollen allergy in subtropical regions and reduce the burden of allergic respiratory disease globally.

Genetic differentiation analysis for the identification of complementary parental pools for sorghum hybrid breeding in Ethiopia.

KEY MESSAGE: The potential for exploiting heterosis for sorghum hybrid production in Ethiopia with improved local adaptation and farmers preferences has been investigated and populations suitable for initial hybrid development have been identified. Hybrids in sorghum have demonstrated increased productivity and stability of performance in the developed world. In Ethiopia, the uptake of hybrid sorghum has been limited to date, primarily due to poor adaptation and absence of farmer's preferred traits in existing hybrids. This study aimed to identify complementary parental pools to develop locally adapted hybrids, through an analysis of whole genome variability of 184 locally adapted genotypes and introduced hybrid parents (R and B). Genetic variability was assessed using genetic distance, model-based STRUCTURE analysis and pair-wise comparison of groups. We observed a high degree of genetic similarity between the Ethiopian improved inbred genotypes and a subset of landraces adapted to lowland agro-ecology with the introduced R lines. This coupled with the genetic differentiation from existing B lines, indicated that these locally adapted genotype groups are expected to have similar patterns of heterotic expression as observed between introduced R and B line pools. Additionally, the hybrids derived from these locally adapted genotypes will have the benefit of containing farmers preferred traits. The groups most divergent from introduced B lines were the Ethiopian landraces adapted to highland and intermediate agro-ecologies and a subset of lowland-adapted genotypes, indicating the potential for increased heterotic response of their hybrids. However, these groups were also differentiated from the R lines, and hence are different from the existing complementary heterotic pools. This suggests that although these groups could provide highly divergent parental pools, further research is required to investigate the extent of heterosis and their hybrid performance.

Transgenic, high-protein sorghums display promise in poultry diets in an initial comparison.

This study aimed to compare the inclusion of transgenic sorghums against commercially available sorghums on growth performance in broiler chickens. Isonitrogenous and isoenergetic diets were offered to a total 288 male Ross 308 broiler chickens from 14 to 35 d posthatch. Three dietary treatments were diets based on transgenic sorghums with a mean protein content of 154.7 g/kg and 5 treatments were based on commercially available sorghum hybrids with a mean protein content of 90.6 g/kg. Soybean meal inclusions in the commercial sorghum diets averaged 215 g/kg, which was reduced to 171 g/kg in the transgenic sorghum diets because of the higher protein contents. Overall growth performance was highly satisfactory, and commercial sorghums supported 2.55% (2,330 vs. 2,272 g/bird; P = 0.010) more weight gains and 2.74% (2,929 vs. 2,851 g/bird; P = 0.012) higher feed intakes; however, the transgenic sorghums supported a fractionally better FCR (1.255 vs 1.257; P = 0.826). There were no statistical differences in apparent jejunal and ileal starch and protein (N) digestibility coefficients between treatments. The transgenic sorghum diets generated slightly, but significantly, higher AME:GE ratios and AMEn, but the commercial sorghum diets generated 6.33% (235 vs. 221 g/kg; P < 0.001) greater breast meat yields. Apparent ileal digestibility coefficients of 16 amino acids averaged 0.839 and 0.832 for transgenic and commercial sorghum-based diets, respectively, without any significant differences in individual amino acids. This outcome suggests amino acid digestibilities of the transgenic sorghums may be inherently higher than commercial hybrid sorghums as the 25.7% higher average soybean meal inclusions would have advantaged amino acid digestibilities in commercial sorghum diets. The possibility that the digestibilities of amino acids in the kafirin component of transgenic sorghums was enhanced by modifications to the structure of kafirin protein bodies is discussed. In conclusion, transgenic sorghums with higher protein concentrations led to 20.5% reduction of soybean meal inclusions in broiler diets, and this change did not compromise feed conversion efficiency compared to standard commercial hybrid sorghums.

Integrating stay-green and PIN-FORMED genes: PIN-FORMED genes as potential targets for designing climate-resilient cereal ideotypes.

Plant architecture modification (e.g. short-stature crops) is one of the key outcomes of modern crop breeding for high-yielding crop varieties. In cereals, delayed senescence, or stay-green, is an important trait that enables post-anthesis drought stress adaptation. Stay-green crops can prolong photosynthetic capacity during grain-filling period under post-anthesis drought stress, which is essential to ensure grain yield is not impacted under drought stress conditions. Although various stay-green quantitative trait loci have been identified in cereals, the underlying molecular mechanisms regulating stay-green remain elusive. Recent advances in various gene-editing technologies have provided avenues to fast-track crop improvement, such as the breeding of climate-resilient crops in the face of climate change. We present in this viewpoint the focus on using sorghum as the model cereal crop, to study PIN-FORMED (PIN) auxin efflux carriers as means to modulate plant architecture, and the potential to employ it as an adaptive strategy to address the environmental challenges posed by climate uncertainties.

Regioselective stilbene O-methylations in Saccharinae grasses.

O-Methylated stilbenes are prominent nutraceuticals but rarely produced by crops. Here, the inherent ability of two Saccharinae grasses to produce regioselectively O-methylated stilbenes is reported. A stilbene O-methyltransferase, SbSOMT, is first shown to be indispensable for pathogen-inducible pterostilbene (3,5-bis-O-methylated) biosynthesis in sorghum (Sorghum bicolor). Phylogenetic analysis indicates the recruitment of genus-specific SOMTs from canonical caffeic acid O-methyltransferases (COMTs) after the divergence of Sorghum spp. from Saccharum spp. In recombinant enzyme assays, SbSOMT and COMTs regioselectively catalyze O-methylation of stilbene A-ring and B-ring respectively. Subsequently, SOMT-stilbene crystal structures are presented. Whilst SbSOMT shows global structural resemblance to SbCOMT, molecular characterizations illustrate two hydrophobic residues (Ile144/Phe337) crucial for substrate binding orientation leading to 3,5-bis-O-methylations in the A-ring. In contrast, the equivalent residues (Asn128/Asn323) in SbCOMT facilitate an opposite orientation that favors 3'-O-methylation in the B-ring. Consistently, a highly-conserved COMT is likely involved in isorhapontigenin (3'-O-methylated) formation in wounded wild sugarcane (Saccharum spontaneum). Altogether, our work reveals the potential of Saccharinae grasses as a source of O-methylated stilbenes, and rationalize the regioselectivity of SOMT activities for bioengineering of O-methylated stilbenes.

Expression pattern of the alpha-kafirin promoter coupled with a signal peptide from Sorghum bicolor L. Moench.

Regulatory sequences with endosperm specificity are essential for foreign gene expression in the desired tissue for both grain quality improvement and molecular pharming. In this study, promoters of seed storage α-kafirin genes coupled with signal sequence (ss) were isolated from Sorghum bicolor L. Moench genomic DNA by PCR. The α-kafirin promoter (α-kaf) contains endosperm specificity-determining motifs, prolamin-box, the O2-box 1, CATC, and TATA boxes required for α-kafirin gene expression in sorghum seeds. The constructs pMB-Ubi-gfp and pMB-kaf-gfp were microprojectile bombarded into various sorghum and sweet corn explants. GFP expression was detected on all explants using the Ubi promoter but only in seeds for the α-kaf promoter. This shows that the α-kaf promoter isolated was functional and demonstrated seed-specific GFP expression. The constructs pMB-Ubi-ss-gfp and pMB-kaf-ss-gfp were also bombarded into the same explants. Detection of GFP expression showed that the signal peptide (SP)::GFP fusion can assemble and fold properly, preserving the fluorescent properties of GFP.

Highly efficient sorghum transformation.

A highly efficient microprojectile transformation system for sorghum (Sorghum bicolor L.) has been developed by using immature embryos (IEs) of inbred line Tx430. Co-bombardment was performed with the neomycin phosphotransferase II (nptII) gene and the green fluorescent protein (gfp) gene, both under the control of the maize ubiquitin1 (ubi1) promoter. After optimization of both tissue culture media and parameters of microprojectile transformation, 25 independent transgenic events were obtained from 121 bombarded IEs. The average transformation frequency (the total number of independent transgenic events divided by the total number of bombarded IEs) was 20.7% in three independent experiments. Transgenic events were confirmed by both PCR screening and Southern hybridization of genomic DNA from primary transgenics (T0). More than 90% of transformants were fertile and displayed normal morphology in a containment glasshouse. Co-transformation rate of the nptII and gfp genes was 72% in these experiments. The segregation of nptII and gfp in T1 progenies was observed utilizing fluorescence microscopy and geneticin selection of seedlings indicating both were inherited in the T1 generation. The transformation procedure, from initiating IEs to planting putative transgenic plantlets in the glasshouse, was completed within 11-16 weeks, and was approximately threefold more efficient than the previously reported best sorghum transformation system.