Unraveling transcriptomics of sorghum grain carotenoids: a step forward for biofortification.

BACKGROUND: Sorghum (Sorghum bicolor [L.] Moench) is a promising target for pro-vitamin A biofortification as it is a global staple crop, particularly in regions where vitamin A deficiency is prevalent. As with most cereal grains, carotenoid concentrations are low in sorghum, and breeding could be a feasible strategy to increase pro-vitamin A carotenoids to biologically relevant concentrations. However, there are knowledge gaps in the biosynthesis and regulation of sorghum grain carotenoids, which can limit breeding effectiveness. The aim of this research was to gain an understanding of the transcriptional regulation of a priori candidate genes in carotenoid precursor, biosynthesis, and degradation pathways. RESULTS: We used RNA sequencing of grain to compare the transcriptional profile of four sorghum accessions with contrasting carotenoid profiles through grain development. Most a priori candidate genes involved in the precursor MEP, carotenoid biosynthesis, and carotenoid degradation pathways were found to be differentially expressed between sorghum grain developmental stages. There was also differential expression of some of the a priori candidate genes between high and low carotenoid content groups at each developmental time point. Among these, we propose geranyl geranyl pyrophosphate synthase (GGPPS), phytoene synthase (PSY), and phytoene desaturase (PDS) as promising targets for pro-vitamin A carotenoid biofortification efforts in sorghum grain. CONCLUSIONS: A deeper understanding of the controls underlying biosynthesis and degradation of sorghum grain carotenoids is needed to advance biofortification efforts. This study provides the first insights into the regulation of sorghum grain carotenoid biosynthesis and degradation, suggesting potential gene targets to prioritize for molecular breeding.

Characterization of grain carotenoids in global sorghum germplasm to guide genomics-assisted breeding strategies.

BACKGROUND: Crop biofortification is a successful strategy to ameliorate Vitamin A deficiency. Sorghum is a good candidate for vitamin A biofortification, as it is a staple food in regions with high prevalence of vitamin A deficiency. ß-carotene-the main provitamin A carotenoid-is below the target concentration in sorghum grain, therefore biofortification breeding is required. Previous studies found evidence that sorghum carotenoid variation is oligogenic, suggesting that marker-assisted selection can be an appropriate biofortification method. However, we hypothesize that sorghum carotenoids have both oligogenic and polygenic components of variation. Genomics-assisted breeding could accelerate breeding efforts, but there exists knowledge gaps in the genetics underlying carotenoid variation, as well as appropriate germplasm to serve as donors. RESULTS: In this study, we characterized carotenoids in 446 accessions from the sorghum association panel and carotenoid panel using high-performance liquid chromatography, finding high carotenoid accessions not previously identified. Genome-wide association studies conducted with 345 accessions, confirmed that zeaxanthin epoxidase is a major gene underlying variation for not only zeaxanthin, but also lutein and ß-carotene. High carotenoid lines were found to have limited genetic diversity, and originated predominantly from only one country. Potential novel genetic diversity for carotenoid content was identified through genomic predictions in 2,495 accessions of unexplored germplasm. Oligogenic variation of carotenoids was confirmed, as well as evidence for polygenic variation, suggesting both marker-assisted selection and genomic selection can facilitate breeding efforts. CONCLUSIONS: Sorghum vitamin A biofortification could be beneficial for millions of people who rely on it as a dietary staple. Carotenoid content in sorghum is low, but high heritability suggests that increasing concentrations through breeding is possible. Low genetic diversity among high carotenoid lines might be the main limitation for breeding efforts, therefore further germplasm characterization is needed to assess the feasibility of biofortification breeding. Based on germplasm here evaluated, most countries' germplasm lacks high carotenoid alleles, thus pre-breeding will be needed. A SNP marker within the zeaxanthin epoxidase gene was identified as a good candidate for use in marker-assisted selection. Due to the oligogenic and polygenic variation of sorghum grain carotenoids, both marker-assisted selection and genomic selection can be employed to accelerate breeding efforts.

Antimicrobial Activity of Sorghum Phenolic Extract on Bovine Foodborne and Mastitis-Causing Pathogens.

Antimicrobial resistance in bacterial pathogens associated with bovine mastitis and human foodborne illnesses from contaminated food and water have an impact on animal and human health. Phenolic compounds have antimicrobial properties and some specialty sorghum grains are high in phenolic compounds, and the grain extract may have the potential as a natural antimicrobial alternative. The study's objective was to determine antimicrobial effects of sorghum phenolic extract on bacterial pathogens that cause bovine mastitis and human foodborne illnesses. Bacterial pathogens tested included Escherichia coli, Salmonella Typhimurium, Campylobacter jejuni, Campylobacter coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Klebsiella oxytoca, Staphylococcus aureus, and Enterococcus faecalis. Antibacterial activities of sorghum phenolic extracts were determined by agar-well diffusion assay. Sorghum phenolic extract was added to the wells in concentrations of 0, 100, 200, 500, 1000, or 4000 µg/mL. The control wells did not receive phenolic extract. Plates were incubated for 18-24 h, and the diameter of each zone of inhibition was measured. The results indicated that sorghum phenolic extract had inhibitory effects on Staphylococcus aureus, Enterococcus faecalis, Campylobacter jejuni, and Campylobacter coli.

Advancing provitamin A biofortification in sorghum: Genome-wide association studies of grain carotenoids in global germplasm.

Vitamin A deficiency is one of the most prevalent nutritional deficiencies worldwide. Sorghum [Sorghum bicolor L. (Moench)] is a major cereal crop consumed by millions of people in regions with high vitamin A deficiency. We quantified carotenoid concentrations in a diverse sorghum panel using high-performance liquid chromatography and conducted a genome-wide association study (GWAS) of grain carotenoids to identify genes underlying carotenoid variation. There was moderate variation for ß-carotene (00.8 µg g-1 ), lutein (0.3-9.4 µg g-1 ), and zeaxanthin (0.2-9.1 µg g-1 ), but ß-cryptoxanthin and α-carotene were nearly undetectable. Genotype had the largest effect size, at 81% for zeaxanthin, 62% for ß-carotene, and 53% for lutein. Using multiple models, GWAS identified several significant associations between carotenoids and single nucleotide polymorphisms (SNPs), some of which colocalized with known carotenoid genes that have not been previously implicated in carotenoid variation. Several of the candidate genes identified have also been identified in maize (Zea mays L.) and Arabidopsis (Arabidopsis thaliana) carotenoid GWAS studies. Notably, an SNP inside the putative ortholog of maize zeaxanthin epoxidase (ZEP) had the most significant association with zeaxanthin and with the ratio between lutein and zeaxanthin, suggesting that ZEP is a major gene controlling sorghum carotenoid variation. Overall findings suggest there is oligogenic inheritance for sorghum carotenoids and suitable variation for marker-assisted selection. The high carotenoid germplasm and significant associations identified in this study can be used in biofortification efforts to improve the nutritional quality of sorghum.

Genetic architecture of kernel composition in global sorghum germplasm.

BACKGROUND: Sorghum [Sorghum bicolor (L.) Moench] is an important cereal crop for dryland areas in the United States and for small-holder farmers in Africa. Natural variation of sorghum grain composition (protein, fat, and starch) between accessions can be used for crop improvement, but the genetic controls are still unresolved. The goals of this study were to quantify natural variation of sorghum grain composition and to identify single-nucleotide polymorphisms (SNPs) associated with variation in grain composition concentrations. RESULTS: In this study, we quantified protein, fat, and starch in a global sorghum diversity panel using near-infrared spectroscopy (NIRS). Protein content ranged from 8.1 to 18.8%, fat content ranged from 1.0 to 4.3%, and starch content ranged from 61.7 to 71.1%. Durra and bicolor-durra sorghum from Ethiopia and India had the highest protein and fat and the lowest starch content, while kafir sorghum from USA, India, and South Africa had the lowest protein and the highest starch content. Genome-wide association studies (GWAS) identified quantitative trait loci (QTL) for sorghum protein, fat, and starch. Previously published RNAseq data was used to identify candidate genes within a GWAS QTL region. A putative alpha-amylase 3 gene, which has previously been shown to be associated with grain composition traits, was identified as a strong candidate for protein and fat variation. CONCLUSIONS: We identified promising sources of genetic material for manipulation of grain composition traits, and several loci and candidate genes that may control sorghum grain composition. This survey of grain composition in sorghum germplasm and identification of protein, fat, and starch QTL contributes to our understanding of the genetic basis of natural variation in sorghum grain nutritional traits.

Persistent organic pollutants and biomarkers of diabetes risk in a cohort of Great Lakes sport caught fish consumers.

BACKGROUND: Exposure to persistent organic pollutants (POPs) is associated with increased diabetes risk, although the mechanism of action is not well delineated. METHODS: We investigated established diabetes biomarkers that could implicate potential mechanistic pathways, including C-reactive protein (CRP), a marker of systemic inflammation; gamma glutamyl transferase (GGT), a liver enzyme associated with oxidative stress; and adiponectin, an adipokine modulating glucose regulation and fatty acid oxidation. These biomarkers as well as hemoglobin A1c (HA1c), and POPs [polychlorinated biphenyls (PCBs), p,p-dichlorodiphenyldichloroethylene (DDE) and polybrominated diphenyl ethers (PBDEs)] were measured in a cohort of Great Lakes sport caught fish (GLSCF) consumers. We examined associations of POPs and fish consumption with HA1c and incident diabetes, and evaluated mediation and moderation by the diabetes biomarkers. RESULTS: Odds of incident diabetes were elevated with exposure to DDE and PCBs. DDE and PCB 118 were positively, and fish meals were inversely, associated with HA1c. CRP was inversely associated with saltwater and total fish meals, particularly in persons with higher adiposity, but did not mediate the associations of fish meals with HA1c. There were few associations of POPs with adiponectin, CRP and GGT, with the exception of positive associations of PCB 118 with GGT, PBDEs with GGT in older persons, and PBDEs with adiponectin. Adiponectin, CRP and GGT did not mediate associations of DDE and PCBs with HA1c or incident diabetes. However, the association of DDE with HA1c was stronger in persons with higher CRP, GGT and BMI, and lower adiponectin, while the association of PCB 118 with HA1c was stronger in persons with higher GGT. CONCLUSIONS: These findings suggest that adiponectin, CRP and GGT did not mediate effects of POPs on diabetes or HA1c. However, POPs may have stronger effects on blood glucose in persons at higher risk for diabetes.

Genome-wide association study of grain polyphenol concentrations in global sorghum [Sorghum bicolor (L.) Moench] germplasm.

Identifying natural variation of health-promoting compounds in staple crops and characterizing its genetic basis can help improve human nutrition through crop biofortification. Some varieties of sorghum, a staple cereal crop grown worldwide, have high concentrations of proanthocyanidins and 3-deoxyanthocyanidins, polyphenols with antioxidant and anti-inflammatory properties. We quantified total phenols, proanthocyanidins, and 3-deoxyanthocyanidins in a global sorghum diversity panel (n = 381) using near-infrared spectroscopy (NIRS), and characterized the patterns of variation with respect to geographic origin and botanical race. A genome-wide association study (GWAS) with 404,628 SNP markers identified novel quantitative trait loci for sorghum polyphenols, some of which colocalized with homologues of flavonoid pathway genes from other plants, including an orthologue of maize (Zea mays) Pr1 and a homologue of Arabidopsis (Arabidopsis thaliana) TT16. This survey of grain polyphenol variation in sorghum germplasm and catalog of flavonoid pathway loci may be useful to guide future enhancement of cereal polyphenols.

Dissecting genome-wide association signals for loss-of-function phenotypes in sorghum flavonoid pigmentation traits.

Genome-wide association studies are a powerful method to dissect the genetic basis of traits, although in practice the effects of complex genetic architecture and population structure remain poorly understood. To compare mapping strategies we dissected the genetic control of flavonoid pigmentation traits in the cereal grass sorghum by using high-resolution genotyping-by-sequencing single-nucleotide polymorphism markers. Studying the grain tannin trait, we find that general linear models (GLMs) are not able to precisely map tan1-a, a known loss-of-function allele of the Tannin1 gene, with either a small panel (n = 142) or large association panel (n = 336), and that indirect associations limit the mapping of the Tannin1 locus to Mb-resolution. A GLM that accounts for population structure (Q) or standard mixed linear model that accounts for kinship (K) can identify tan1-a, whereas a compressed mixed linear model performs worse than the naive GLM. Interestingly, a simple loss-of-function genome scan, for genotype-phenotype covariation only in the putative loss-of-function allele, is able to precisely identify the Tannin1 gene without considering relatedness. We also find that the tan1-a allele can be mapped with gene resolution in a biparental recombinant inbred line family (n = 263) using genotyping-by-sequencing markers but lower precision in the mapping of vegetative pigmentation traits suggest that consistent gene-level resolution will likely require larger families or multiple recombinant inbred lines. These findings highlight that complex association signals can emerge from even the simplest traits given epistasis and structured alleles, but that gene-resolution mapping of these traits is possible with high marker density and appropriate models.

Increased adiposity, dysregulated glucose metabolism and systemic inflammation in Galectin-3 KO mice.

Obesity and type 2 diabetes are associated with increased production of Galectin-3 (Gal-3), a protein that modulates inflammation and clearance of glucose adducts. We used Lean and Diet-induced Obese (DIO) WT and Gal-3 KO mice to investigate the role of Gal-3 in modulation of adiposity, glucose metabolism and inflammation. Deficiency of Gal-3 lead to age-dependent development of excess adiposity and systemic inflammation, as indicated by elevated production of acute-phase proteins, number of circulating pro-inflammatory Ly6C(high) monocytes and development of neutrophilia, microcytic anemia and thrombocytosis in 20-week-old Lean and DIO male Gal-3 KO mice. This was associated with impaired fasting glucose, heightened response to a glucose tolerance test and reduced adipose tissue expression of adiponectin, Gal-12, ATGL and PPARγ, in the presence of maintained insulin sensitivity and hepatic expression of gluconeogenic enzymes in 20-week-old Gal-3 KO mice compared to their diet-matched WT controls. Expression of PGC-1α and FGF-21 in the liver of Lean Gal-3 KO mice was comparable to that observed in DIO animals. Impaired fasting glucose and altered responsiveness to a glucose load preceded development of excess adiposity and systemic inflammation, as demonstrated in 12-week-old Gal-3 KO mice. Finally, a role for the microflora in mediating the fasting hyperglycemia, but not the excessive response to a glucose load, of 12-week-old Gal-3 KO mice was demonstrated by administration of antibiotics. In conclusion, Gal-3 is an important modulator of glucose metabolism, adiposity and inflammation.

Adipose tissue-specific modulation of galectin expression in lean and obese mice: evidence for regulatory function.

OBJECTIVE: Galectins (Gal) exert many activities, including regulation of inflammation and adipogenesis. We evaluated modulation of Gal-1, -3, -9 and -12 in visceral (VAT) and subcutaneous (SAT) adipose tissue in mice. DESIGN AND METHODS: We used two mouse models of obesity, high-fat diet induced obesity (DIO) and ob/ob mice. We also evaluated the response of Gal-1 KO mice to DIO. RESULTS: Both age and diet modulated expression of galectins, with DIO mice having higher serum Gal-1 and Gal-3 versus lean mice after 13-17 weeks of high-fat diet. In DIO mice there was a progressive increase in expression of Gal-1 and Gal-9 in SAT, whereas Gal-3 increased in both VAT and SAT. Expression of Gal-12 declined over time in VAT of DIO mice, similar to adiponectin. Obesity lead to increased production of Gal-1 in adipocytes, whereas the increased Gal-3 and Gal-9 of obesity mostly derived from the stromovascular fraction. Expression of Gal-12 was restricted to adipocytes. There was increased production of Gal-3 and Gal-9, but not Gal-1, in CD11c(-) and CD11c(+) macrophages from VAT of DIO versus lean mice. Expression of Gal-1, -3 and -12 in VAT and SAT of ob/ob mice followed a trend comparable to DIO mice. Rosiglitazone reduced serum Gal-1, but not Gal-3 and modulated expression of Gal-3 in VAT and Gal-9 and Gal-12 in SAT of DIO mice. High-fat feeding lead to increased adiposity in Gal-1 KO versus WT mice, with loss of correlation between leptin and adiposity and no alterations in glucose and insulin levels. CONCLUSIONS: Obesity leads to differential modulation of Gal-1, 3, 9 and 12 in VAT and SAT, with Gal-1 acting as a modulator of adiposity.

Obesity and IL-6 interact in modulating the response to endotoxemia in mice.

Obesity is associated with elevated levels of IL-6. High IL-6 is prognostic of mortality in sepsis, while controversial data link obesity to sepsis outcome. We used Lean and diet-induced obese (DIO) WT and IL-6 KO mice to investigate the interaction between obesity and IL-6 in endotoxemia. Circulating levels of IL-6 were significantly higher in WT DIO versus WT Lean mice receiving LPS (2.5 µg/mouse, ip). Obesity lead to greater weight loss in response to LPS, with IL-6 deficiency being partially protective. Plasma TNFα, IFNγ, Galectin-3 and leptin were significantly elevated in response to LPS and were each differentially affected by obesity and/or IL-6 deficiency. Plasma Galectin-1 and adiponectin were significantly suppressed by LPS, with obesity and IL-6 deficiency modulating the response. However, LPS comparably increased IL-10 levels in each group. Leukopenia with relative neutrophilia and thrombocytopenia developed in each group after injection of LPS, with obesity and genotype affecting the kinetics, but not the magnitude, of the response. Hepatic induction of the acute-phase protein SAA by LPS was not affected by obesity or IL-6 deficiency, although baseline levels were highest in WT DIO mice. Injection of LPS significantly increased hepatic mRNA expression of PAI-1 in Lean WT and Lean KO mice, while it suppressed the high baseline levels observed in the liver of DIO WT and DIO KO mice. Thus, both IL-6 and obesity modulate the response to endotoxemia, suggesting a complex interaction that needs to be considered when evaluating the effect of obesity on the outcome of septic patients.

Rosiglitazone improves survival and hastens recovery from pancreatic inflammation in obese mice.

Obesity increases severity of acute pancreatitis (AP) by unclear mechanisms. We investigated the effect of the PPAR-gamma agonist rosiglitazone (RGZ, 0.01% in the diet) on severity of AP induced by administration of IL-12+ IL-18 in male C57BL6 mice fed a low fat (LFD) or high fat diet (HFD), under the hypothesis that RGZ would reduce disease severity in HFD-fed obese animals. In both LFD and HFD mice without AP, RGZ significantly increased body weight and % fat mass, with significant upregulation of adiponectin and suppression of erythropoiesis. In HFD mice with AP, RGZ significantly increased survival and hastened recovery from pancreatic inflammation, as evaluated by significantly improved pancreatic histology, reduced saponification of visceral adipose tissue and less severe suppression of erythropoiesis at Day 7 post-AP. This was associated with significantly lower circulating and pancreas-associated levels of IL-6, Galectin-3, osteopontin and TIMP-1 in HFD + RGZ mice, particularly at Day 7 post-AP. In LFD mice with AP, RGZ significantly worsened the degree of intrapancreatic acinar and fat necrosis as well as visceral fat saponification, without affecting other parameters of disease severity or inflammation. Induction of AP lead to major suppression of adiponectin levels at Day 7 in both HFD and HFD + RGZ mice. In conclusion, RGZ prevents development of severe AP in obese mice even though it significantly increases adiposity, indicating that obesity can be dissociated from AP severity by improving the metabolic and inflammatory milieu. However, RGZ worsens selective parameters of AP severity in LFD mice.

Role of IL-6 in the resolution of pancreatitis in obese mice.

Obesity increases severity of acute pancreatitis and risk of pancreatic cancer. Pancreatitis and obesity are associated with elevated IL-6, a cytokine involved in inflammation and tumorigenesis. We studied the role of IL-6 in the response of lean and obese mice to pancreatitis induced by IL-12 + IL-18. Lean and diet-induced obese (DIO) WT and IL-6 KO mice and ob/ob mice pretreated with anti-IL-6 antibodies were evaluated at Days 1, 7, and 15 after induction of pancreatitis. Prolonged elevation of IL-6 in serum and visceral adipose tissue was observed in DIO versus lean WT mice, whereas circulating sIL-6R declined in DIO but not lean mice with pancreatitis. The severe inflammation and lethality of DIO mice were also observed in IL-6 KO mice. However, the delayed resolution of neutrophil infiltration; sustained production of CXCL1, CXCL2, and CCL2; prolonged activation of STAT-3; and induction of MMP-7 in the pancreas, as well as heightened induction of serum amylase A of DIO mice, were blunted significantly in DIO IL-6 KO mice. In DIO mice, production of OPN and TIMP-1 was increased for a prolonged period, and this was mediated by IL-6 in the liver but not the pancreas. Results obtained in IL-6 KO mice were confirmed in ob/ob mice pretreated with anti-IL-6 antibodies. In conclusion, IL-6 does not contribute to the increased severity of pancreatitis of obese mice but participates in delayed recovery from acute inflammation and may favor development of a protumorigenic environment through prolonged activation of STAT-3, induction of MMP-7, and sustained production of chemokines.

Hematological and acute-phase responses to diet-induced obesity in IL-6 KO mice.

Obesity is associated with chronic inflammation and elevated levels of IL-6. The role of IL-6 in induction of acute-phase proteins and modulation of hematological responses has been demonstrated in models of inflammation and aging, but not in obesity. We hypothesized that IL-6 is necessary to regulate the acute-phase response and hematological changes associated with diet-induced obesity (DIO) in mice. Feeding a 60%kcal/fat diet for 13 weeks to C57BL6 WT male mice induced a significant increase in IL-6 expression in visceral adipose tissue (VAT), but not liver, compared to mice fed chow diet. Significantly elevated IL-6 levels were present in the peritoneal lavage fluid, but not plasma, of DIO compared to lean mice. A comparable degree of obesity, hepatomegaly, hyperleptinemia, VAT inflammation and insulin resistance was observed in DIO WT and IL-6 KO mice compared to WT and KO mice fed chow diet. Significant leukocytosis was observed in DIO WT but not DIO KO mice compared to lean groups. A significant reduction in platelet counts, without alterations in platelet size, percentage of circulating reticulated platelets and number of bone marrow megakaryocytes, was present in DIO KO mice compared to each other group. Hepatic expression of thrombopoietin was comparable in each group, with DIO WT and KO mice having reduced VAT expression compared to lean mice. Lean KO mice had significantly elevated plasma levels of thrombopoietin compared to each other group, whereas liver-associated thrombopoietin levels were comparable in each group. Deficiency of IL-6 resulted in blunted hepatic induction of the acute-phase protein serum amyloid A-1, whereas expression of hepcidin-1 and -2, LPS-binding protein, ceruloplasmin, plasminogen activator inhibitor-1 and thrombospondin-1 was IL-6-independent. In conclusion, in the absence of overt metabolic alterations, IL-6 modulates leukocytosis, thrombopoiesis and induction of SAA-1, but not other acute-phase proteins in obese mice.

Role of leptin receptor-induced STAT3 signaling in modulation of intestinal and hepatic inflammation in mice.

Leptin-deficient ob/ob mice are resistant to dextran sulfate sodium (DSS)-induced colitis and Concanavalin A (Con A)-induced hepatitis. However, the signal transduction pathways involved have not been identified. The present study investigated the effect of leptin-induced STAT3 signaling in the DSS and Con A models. Mice carrying a leptin receptor (LEPR) gene mutant for Y1138 (s/s mice), with abrogated leptin-induced STAT3 signaling, were compared with wild-type (WT) and LEPR-deficient db/db mice. Administration of DSS to s/s mice resulted in a clinical score and colon shortening of intermediate severity compared with disease induced in WT and db/db mice-the latter group having the lowest disease severity. A comparable degree of inflammatory infiltrate and epithelial damage was observed in the colon of WT and s/s mice, and these parameters were reduced in db/db mice. Levels of IFN-gamma, IL-6, IL-10, and TNF-alpha were comparable in the colon of s/s and db/db mice, and a similar trend was observed for CXCL2. s/s and WT mice developed severe liver disease in response to Con A, whereas db/db mice were protected. However, Con A-induced serum IL-6 and TNF-alpha levels in s/s mice mimicked levels observed in db/db rather than WT mice. In conclusion, lack of leptin-induced STAT3 signaling is associated with reduced cytokine production following DSS and Con A administration, but it appears to sensitize mice to the effects of proinflammatory mediators.