R3-MYB repressor Mybr97 is a candidate gene associated with the Anthocyanin3 locus and enhanced anthocyanin accumulation in maize.

KEY MESSAGE: Anthocyanin3 inhibits the anthocyanin and monolignol pathways in maize. Transposon-tagging, RNA-sequencing, and GST-pulldown assays determine Anthocyanin3 may be R3-MYB repressor gene Mybr97. Anthocyanins are colorful molecules receiving recent attention due to their numerous health benefits and applications as natural colorants and nutraceuticals. Purple corn is being investigated as a more economical source of anthocyanins. Anthocyanin3 (A3) is a known recessive intensifier of anthocyanin pigmentation in maize. In this study, anthocyanin content was elevated 100-fold in recessive a3 plants. Two approaches were used to discover candidates involved with the a3 intense purple plant phenotype. First, a large-scale transposon-tagging population was created with a Dissociation (Ds) insertion in the nearby Anthocyanin1 gene. A de novo a3-m1::Ds mutant was generated, and the transposon insertion was found to be located in the promoter of Mybr97, which has homology to R3-MYB repressor CAPRICE in Arabidopsis. Second, a bulked segregant RNA-sequencing population found expression differences between pools of green A3 plants and purple a3 plants. All characterized anthocyanin biosynthetic genes were upregulated in a3 plants along with several genes of the monolignol pathway. Mybr97 was highly downregulated in a3 plants, suggesting its role as a negative regulator of the anthocyanin pathway. Photosynthesis-related gene expression was reduced in a3 plants through an unknown mechanism. Numerous transcription factors and biosynthetic genes were also upregulated and need further investigation. Mybr97 may inhibit anthocyanin synthesis by associating with basic helix-loop helix transcription factors like Booster1. Overall, Mybr97 is the most likely candidate gene for the A3 locus. A3 has a profound effect on the maize plant and has many favorable implications for crop protection, human health, and natural colorant production.

Roles of Vegetable Surface Properties and Sanitizer Type on Annual Disease Burden of Rotavirus Illness by Consumption of Rotavirus-Contaminated Fresh Vegetables: A Quantitative Microbial Risk Assessment.

Enteric viruses are often detected in water used for crop irrigation. One concern is foodborne viral disease via the consumption of fresh produce irrigated with virus-contaminated water. Although the food industry routinely uses chemical sanitizers to disinfect post-harvest fresh produce, it remains unknown how sanitizer and fresh produce properties affect the risk of viral illness through fresh produce consumption. A quantitative microbial risk assessment model was conducted to estimate (i) the health risks associated with consumption of rotavirus (RV)-contaminated fresh produce with different surface properties (endive and kale) and (ii) how risks changed when using peracetic acid (PAA) or a surfactant-based sanitizer. The modeling results showed that the annual disease burden depended on the combination of sanitizer and vegetable type when vegetables were irrigated with RV-contaminated water. Global sensitivity analyses revealed that the most influential factors in the disease burden were RV concentration in irrigation water and postharvest disinfection efficacy. A postharvest disinfection efficacy of higher than 99% (2-log10 ) was needed to decrease the disease burden below the World Health Organization (WHO) threshold, even in scenarios with low RV concentrations in irrigation water (i.e., river water). All scenarios tested here with at least 99.9% (3-log10 ) disinfection efficacy had a disease burden lower than the WHO threshold, except for the endive treated with PAA. The disinfection efficacy for the endive treated with PAA was only about 80%, leading to a disease burden 100 times higher than the WHO threshold. These findings should be considered and incorporated into future models for estimating foodborne viral illness risks.

Prospects for economical natural colorants: insights from maize.

KEY MESSAGE: Anthocyanin pigments from maize offer a natural yet economical alternative to artificial dyes. Breeding for optimal colorant production requires understanding and integrating all facets of anthocyanin chemistry and genetics research. Replacing artificial dyes with natural colorants is becoming increasingly popular in foods and beverages. However, natural colorants are often expensive, have lower stability, and reduced variability in hue. Purple corn is rich in anthocyanins and offers a scalable and affordable alternative to synthetic dyes ranging in color from orange to reddish-purple. This diversity is attributable to differences in anthocyanin composition and concentration. Here we review the chemistry, biosynthesis, and genetics of purple corn and outline key factors associated with the feasibility of producing an economical source of natural colorants. Anthocyanin compositional modifications including acylation, methylation, and polymerization with flavan-3-ols can influence color stability and hue, yet there is more to learn regarding the genetic factors responsible for these modifications. Activators and repressors of anthocyanin biosynthesis structural genes as well as factors controlling trafficking and storage largely control anthocyanin yield. Further knowledge of these mechanisms will allow breeders to apply molecular strategies that accelerate the production of purple corn hybrids to meet growing demands for natural colorants.

Individual and interactive effects of herbivory on plant fitness: endopolyploidy as a driver of genetic variation in tolerance and resistance.

Previous studies have shown a causal link between mammalian herbivory, tolerance, and chemical defense in Arabidopsis thaliana, driven by the process of endoreduplication (replication of the genome without mitosis). Removal of the apical meristem by mammalian herbivores lowers auxin, which triggers entry into the endocycle. Increasing chromosome number through endoreduplication, and therefore gene copy number, provides a means of increasing gene expression promoting rapid regrowth rates, higher defensive chemistry and enhanced fitness. Here, we assess whether insect leaf-feeding elicits the same compensatory response as the removal of apical dominance. Insect feeding has been shown to downregulate auxin production, which should trigger endoreduplication. Results here support this contention; insect leaf-feeding by Trichoplusia ni elicited a compensatory response similar to that of mammalian herbivores-an ecotype-specific response consistent with the level of endoreduplication. The interactive effects of mammalian and insect herbivory were also assessed to determine whether interactions were additive (pairwise) or non-additive (diffuse) on tolerance (fitness). Specifically, results indicate that herbivory is either diffuse (a significant clipping × T. ni interaction) or pairwise (no significant interaction between clipping and T. ni herbivory), dependent upon plant genotype and compensatory ability. In general, herbivore-induced changes in plant quality appear to be responsible for the observed differences in herbivory and fitness compensation. We discuss the importance of evaluating endoreduplication among plants within a population to avoid masking the association between tolerance and resistance and the fitness consequences of multi-herbivore interactions.

Targeted Metabolomic and Transcriptomic Analyses of "Red Russian" Kale (Brassicae napus var. pabularia) Following Methyl Jasmonate Treatment and Larval Infestation by the Cabbage Looper (Trichoplusia ni Hübner).

Methyl jasmonate (MeJA), synthesized in the jasmonic acid (JA) pathway, has been found to upregulate glucosinolate (GS) biosynthesis in plant species of the Brassicaceae family. Exogenous application of MeJA has shown to increase tissue GS concentrations and the formation of myrosinase-mediated GS hydrolysis products (GSHPs). In vitro and in vivo assays have demonstrated the potential health-promoting effects of certain GSHPs. MeJA is also known to elicit and induce genes associated with defense mechanisms to insect herbivory in Brassica species. To investigate the relationship between MeJA-induced GS biosynthesis and insect defense, three treatments were applied to "Red Russian" kale (Brassicae napus var. pabularia) seedlings: (1) a 250 µM MeJA leaf spray treatment; (2) leaf infestation with larvae of the cabbage looper (Trichoplusia ni (Hübner)); (3) control treatment (neither larval infestation nor MeJA application). Samples of leaf tissue from the three treatments were then assayed for changes in GS and GSHP concentrations, GS gene biosynthesis expression, and myrosinase activity. Major differences were observed between the three treatments in the levels of GS accumulation and GS gene expression. The insect-damaged samples showed significantly lower aliphatic GS accumulation, while both MeJA and T. ni infestation treatments induced greater accumulation of indolyl GS. The gene expression levels of CYP81F4, MYB34, and MYB122 were significantly upregulated in samples treated with MeJA and insects compared to the control group, which explained the increased indolyl GS concentration. The results suggest that the metabolic changes promoted by MeJA application and the insect herbivory response share common mechanisms of induction. This work provides potentially useful information for kale pest control and nutritional quality.

Molecular constraints on resistance-tolerance trade-offs.

Plants have numerous mechanisms to cope with the negative effects of herbivory, including plant resistance, structural and chemical traits that reduce damage, and plant tolerance, the ability to compensate for tissues lost. It has been argued that resistance and tolerance represent alternate strategies and thus there should be a trade-off between resistance and tolerance. However, resistance and tolerance are controlled via the same molecular pathway, the oxidative pentose phosphate pathway and the process of endoreduplication. Endoreduplication is the replication of the genome without mitosis, which leads to an increase in cellular chromosome number. Increasing chromosome number and therefore gene copy number provides a means of increasing gene expression that has been shown to enhance compensation following herbivory. By measuring glucosinolate levels and seed production following the removal of apical dominance in genotypes of Arabidopsis thaliana we show that there is a positive association between tolerance and induced chemical defense. Similarly, the direct association between tolerance and resistance is demonstrated by genetically manipulating the endoreduplication pathway. By overexpressing ILP1, a positive regulator of endoreduplication, and thus compensation, we experimentally increased glucosinolate production and tolerance in the Col-0 genotype. We suggest that many herbaceous plants that endoreduplicate (~90%) would show a positive relationship between compensation and chemical defense, given that the molecular pathways are shared in common. We discuss these findings in light of contrasting results on measures of tolerance and resistance, given that the true relationship can be masked by ignoring genetic variation in endoreduplication and the timing of chemical measurement.

Cultivar-Specific Changes in Primary and Secondary Metabolites in Pak Choi (Brassica Rapa, Chinensis Group) by Methyl Jasmonate.

Glucosinolates, their hydrolysis products and primary metabolites were analyzed in five pak choi cultivars to determine the effect of methyl jasmonate (MeJA) on metabolite flux from primary metabolites to glucosinolates and their hydrolysis products. Among detected glucosinolates (total 14 glucosinolates; 9 aliphatic, 4 indole and 1 aromatic glucosinolates), indole glucosinolate concentrations (153-229%) and their hydrolysis products increased with MeJA treatment. Changes in the total isothiocyanates by MeJA were associated with epithiospecifier protein activity estimated as nitrile formation. Goitrin, a goitrogenic compound, significantly decreased by MeJA treatment in all cultivars. Changes in glucosinolates, especially aliphatic, significantly differed among cultivars. Primary metabolites including amino acids, organic acids and sugars also changed with MeJA treatment in a cultivar-specific manner. A decreased sugar level suggests that they might be a carbon source for secondary metabolite biosynthesis in MeJA-treated pak choi. The result of the present study suggests that MeJA can be an effective agent to elevate indole glucosinolates and their hydrolysis products and to reduce a goitrogenic compound in pak choi. The total glucosinolate concentration was the highest in "Chinese cabbage" in the control group (32.5 µmol/g DW), but indole glucosinolates increased the greatest in "Asian" when treated with MeJA.

Effect of Leaf Surface Chemical Properties on Efficacy of Sanitizer for Rotavirus Inactivation.

The use of sanitizers is essential for produce safety. However, little is known about how sanitizer efficacy varies with respect to the chemical surface properties of produce. To answer this question, the disinfection efficacies of an oxidant-based sanitizer and a new surfactant-based sanitizer for porcine rotavirus (PRV) strain OSU were examined. PRV was attached to the leaf surfaces of two kale cultivars with high epicuticular wax contents and one cultivar of endive with a low epicuticular wax content and then treated with each sanitizer. The efficacy of the oxidant-based sanitizer correlated with leaf wax content as evidenced by the 1-log10 PRV disinfection on endive surfaces (low wax content) and 3-log10 disinfection of the cultivars with higher wax contents. In contrast, the surfactant-based sanitizer showed similar PRV disinfection efficacies (up to 3 log10) that were independent of leaf wax content. A statistical difference was observed with the disinfection efficacies of the oxidant-based sanitizer for suspended and attached PRV, while the surfactant-based sanitizer showed similar PRV disinfection efficacies. Significant reductions in the entry and replication of PRV were observed after treatment with either disinfectant. Moreover, the oxidant-based-sanitizer-treated PRV showed sialic acid-specific binding to the host cells, whereas the surfactant-based sanitizer increased the nonspecific binding of PRV to the host cells. These findings suggest that the surface properties of fresh produce may affect the efficacy of virus disinfection, implying that food sanitizers should be carefully selected for the different surface characteristics of fresh produce. IMPORTANCE: Food sanitizer efficacies are affected by the surface properties of vegetables. This study evaluated the disinfection efficacies of two food sanitizers, an oxidant-based sanitizer and a surfactant-based sanitizer, on porcine rotavirus strain OSU adhering to the leaf epicuticular surfaces of high- and low-wax-content cultivars. The disinfection efficacy of the oxidant-based sanitizer was affected by the surface properties of the vegetables, while the surfactant-based sanitizer was effective for both high- and low-wax leafy vegetable cultivars. This study suggests that the surface properties of vegetables may be an important factor that interacts with disinfection with food sanitizers of rotaviruses adhering to fresh produce.

Transcriptome and Metabolome Analyses of Glucosinolates in Two Broccoli Cultivars Following Jasmonate Treatment for the Induction of Glucosinolate Defense to Trichoplusia ni (Hübner).

Lepidopteran larvae growth is influenced by host plant glucosinolate (GS) concentrations, which are, in turn, influenced by the phytohormone jasmonate (JA). In order to elucidate insect resistance biomarkers to lepidopteran pests, transcriptome and metabolome analyses following JA treatments were conducted with two broccoli cultivars, Green Magic and VI-158, which have differentially induced indole GSs, neoglucobrassicin and glucobrassicin, respectively. To test these two inducible GSs on growth of cabbage looper (Trichoplusia ni), eight neonate cabbage looper larvae were placed onto each of three plants per JA treatments (0, 100, 200, 400 µM) three days after treatment. After five days of feeding, weight of larvae and their survival rate was found to decrease with increasing JA concentrations in both broccoli cultivars. JA-inducible GSs were measured by high performance liquid chromatography. Neoglucobrassicin in Green Magic and glucobrassicin in VI-158 leaves were increased in a dose-dependent manner. One or both of these glucosinolates and/or their hydrolysis products showed significant inverse correlations with larval weight and survival (five days after treatment) while being positively correlated with the number of days to pupation. This implies that these two JA-inducible glucosinolates can influence the growth and survival of cabbage looper larvae. Transcriptome profiling supported the observed changes in glucosinolate and their hydrolysis product concentrations following JA treatments. Several genes related to GS metabolism differentiate the two broccoli cultivars in their pattern of transcriptional response to JA treatments. Indicative of the corresponding change in indole GS concentrations, transcripts of the transcription factor MYB122, core structure biosynthesis genes (CYP79B2, UGT74B1, SUR1, SOT16, SOT17, and SOT18), an indole glucosinolate side chain modification gene (IGMT1), and several glucosinolate hydrolysis genes (TGG1, TGG2, and ESM1) were significantly increased in Green Magic (statistically significant in most cases at 400 µM) while UGT74B1 and MYB122 were significantly increased in VI-158. Therefore, these metabolite and transcript biomarker results indicate that transcriptome profiling can identify genes associated with the formation of two different indole GS and their hydrolysis products. Therefore, these metabolite and transcript biomarkers could be useful in an effective marker-assisted breeding strategy for resistance to generalist lepidopteran pests in broccoli and potentially other Brassica vegetables.

Genetic analysis of glucosinolate variability in broccoli florets using genome-anchored single nucleotide polymorphisms.

KEY MESSAGE: The identification of genetic factors influencing the accumulation of individual glucosinolates in broccoli florets provides novel insight into the regulation of glucosinolate levels in Brassica vegetables and will accelerate the development of vegetables with glucosinolate profiles tailored to promote human health. Quantitative trait loci analysis of glucosinolate (GSL) variability was conducted with a B. oleracea (broccoli) mapping population, saturated with single nucleotide polymorphism markers from a high-density array designed for rapeseed (Brassica napus). In 4 years of analysis, 14 QTLs were associated with the accumulation of aliphatic, indolic, or aromatic GSLs in floret tissue. The accumulation of 3-carbon aliphatic GSLs (2-propenyl and 3-methylsulfinylpropyl) was primarily associated with a single QTL on C05, but common regulation of 4-carbon aliphatic GSLs was not observed. A single locus on C09, associated with up to 40 % of the phenotypic variability of 2-hydroxy-3-butenyl GSL over multiple years, was not associated with the variability of precursor compounds. Similarly, QTLs on C02, C04, and C09 were associated with 4-methylsulfinylbutyl GSL concentration over multiple years but were not significantly associated with downstream compounds. Genome-specific SNP markers were used to identify candidate genes that co-localized to marker intervals and previously sequenced Brassica oleracea BAC clones containing known GSL genes (GSL-ALK, GSL-PRO, and GSL-ELONG) were aligned to the genomic sequence, providing support that at least three of our 14 QTLs likely correspond to previously identified GSL loci. The results demonstrate that previously identified loci do not fully explain GSL variation in broccoli. The identification of additional genetic factors influencing the accumulation of GSL in broccoli florets provides novel insight into the regulation of GSL levels in Brassicaceae and will accelerate development of vegetables with modified or enhanced GSL profiles.

Variation in chilling tolerance for photosynthesis and leaf extension growth among genotypes related to the C4 grass Miscanthus ×giganteus.

The goal of this study was to identify cold-tolerant genotypes within two species of Miscanthus related to the exceptionally chilling-tolerant C4 biomass crop accession: M. ×giganteus 'Illinois' (Mxg) as well as in other Mxg genotypes. The ratio of leaf elongation at 10 °C/5 °C to that at 25 °C/25 °C was used to identify initially the 13 most promising Miscanthus genotypes out of 51 studied. Net leaf CO2 uptake (A sat) and the maximum operating efficiency of photosystem II (ФPSII) were measured in warm conditions (25 °C/20 °C), and then during and following a chilling treatment of 10 °C/5 °C for 11 d. Accessions of M. sacchariflorus (Msa) showed the smallest decline in leaf elongation on transfer to chilling conditions and did not differ significantly from Mxg, indicating greater chilling tolerance than diploid M. sinensis (Msi). Msa also showed the smallest reductions in A sat and ФPSII, and greater chilling-tolerant photosynthesis than Msi, and three other forms of Mxg, including new triploid accessions and a hexaploid Mxg 'Illinois'. Tetraploid Msa 'PF30153' collected in Gifu Prefecture in Honshu, Japan did not differ significantly from Mxg 'Illinois' in leaf elongation and photosynthesis at low temperature, but was significantly superior to all other forms of Mxg tested. The results suggested that the exceptional chilling tolerance of Mxg 'Illinois' cannot be explained simply by the hybrid vigour of this intraspecific allotriploid. Selection of chilling-tolerant accessions from both of Mxg's parental species, Msi and Msa, would be advisable for breeding new highly chilling-tolerant Mxg genotypes.

Optimization of methyl jasmonate application to broccoli florets to enhance health-promoting phytochemical content.

BACKGROUND: Spray treatment of methyl jasmonate (MeJA) has been shown to increase glucosinolate (GS) concentrations and health-promoting activity in Brassica vegetables. Since there is no reported standardized protocol, several MeJA treatment studies have been conducted to maximize human health bioactivity using the F1 broccoli cultivar 'Green Magic'. RESULTS: Foliar MeJA application 4 days prior to harvest of broccoli at commercial maturity resulted in enhanced total GS concentrations. Although a single application of 250 µmol L(-1) MeJA maximized GS concentrations in broccoli florets, two days of consecutive treatments (4 and 3 days prior to harvest) of 250 µmol L(-1) MeJA further enhanced neoglucobrassicin concentrations and floret extract quinone reductase (QR)-inducing activity. With increasing concentrations of MeJA in spray applications to broccoli florets, concentrations of the glucosinolates glucoraphanin, gluconasturtiin and neoglucobrassicin and the isothiocyanate sulforaphane as well as anticancer and anti-inflammatory bioactivities as measured by QR induction and inhibition of nitric oxide (NO) production respectively were significantly increased. Concentrations of these phytochemicals showed strong positive correlations with QR-inducing and NO-inhibitory activities. CONCLUSION: These application protocols were found to maximize GS and GS hydrolysis product concentrations and putatively enhance the health-promoting properties of broccoli heads for consumers.

Pre-harvest methyl jasmonate treatment enhances cauliflower chemoprotective attributes without a loss in postharvest quality.

Methyl jasmonate (MeJA) treatment can significantly increase glucosinolate (GS) concentrations in Brassica vegetables and potentially enhance anticancer bioactivity. Although MeJA treatment may promote ethylene biosynthesis, which can be detrimental to postharvest quality, there are no previous reports of its effect on cauliflower postharvest quality. To address this, cauliflower curds in field plots were sprayed with either 0.1 % Triton X-100 (control) or 500 µM MeJA solutions four days prior to harvest, then stored at 4 °C. Tissue subsamples were collected after 0, 10, 20, and 30 days of postharvest storage and assayed for visual color change, ethylene production, GS concentrations, and extract quinone reductase inductive activity. MeJA treatment increased curd GS concentrations of glucoraphanin, glucobrassicin, and neoglucobrassicin by 1.5, 2.4, and 4.6-fold over controls, respectively. MeJA treated cauliflower showed significantly higher quinone reductase activity, a biomarker for anticancer bioactivity, without reducing visual color and postharvest quality for 10 days at 4 °C storage.

Increasing aleurone layer number and pericarp yield for elevated nutrient content in maize.

The bran is a nutritive fraction of the maize (Zea mays L.) kernel containing micronutrients, quality protein, and antioxidants beneficial for human health. Bran consists of two major components: aleurone and pericarp. Increasing this nutritive fraction would therefore have implications on biofortification of maize. Since quantification of these two layers is difficult, the goals of this study were to develop efficient techniques for analyzing these layers and to develop molecular markers for pericarp and aleurone yield. Two populations with various characteristics were genotyped using genotyping-by-sequencing. The first was a yellow corn population with contrasting pericarp thicknesses. The second was a blue corn population segregating for Intensifier1 alleles. Both populations segregated for the multiple aleurone layer (MAL) trait that is known to increase aleurone yield. In this study, it was found that MALs are mostly determined by a locus on chromosome 8, but several minor loci are also involved. The inheritance of MALs was complex and seemingly more additive than dominant. In the blue corn population, anthocyanin content increased 20 to 30% with the addition of MALs demonstrating its effectiveness at increasing aleurone yield. Elemental analysis was performed on MAL lines and indicated a role of MALs in increasing iron content in the grain. Iron content was increased 17.5% in the MAL lines over the single aleurone layer lines and 35.5% over the recurrent parent, Mo17. Zinc content was increased 15.5% in the MAL lines compared to the recurrent parent. QTL analyses are presented in this study on many pericarp, aleurone, and grain quality traits. Molecular markers were also tested for the MAL locus on chromosome 8, and candidate genes are discussed. Results of this study may assist plant breeders enhancing anthocyanin content and other beneficial phytonutrients in maize.

A framework genetic map for Miscanthus sinensis from RNAseq-based markers shows recent tetraploidy.

BACKGROUND: Miscanthus (subtribe Saccharinae, tribe Andropogoneae, family Poaceae) is a genus of temperate perennial C4 grasses whose high biomass production makes it, along with its close relatives sugarcane and sorghum, attractive as a biofuel feedstock. The base chromosome number of Miscanthus (x = 19) is different from that of other Saccharinae and approximately twice that of the related Sorghum bicolor (x = 10), suggesting large-scale duplications may have occurred in recent ancestors of Miscanthus. Owing to the complexity of the Miscanthus genome and the complications of self-incompatibility, a complete genetic map with a high density of markers has not yet been developed. RESULTS: We used deep transcriptome sequencing (RNAseq) from two M. sinensis accessions to define 1536 single nucleotide variants (SNVs) for a GoldenGate™ genotyping array, and found that simple sequence repeat (SSR) markers defined in sugarcane are often informative in M. sinensis. A total of 658 SNP and 210 SSR markers were validated via segregation in a full sibling F1 mapping population. Using 221 progeny from this mapping population, we constructed a genetic map for M. sinensis that resolves into 19 linkage groups, the haploid chromosome number expected from cytological evidence. Comparative genomic analysis documents a genome-wide duplication in Miscanthus relative to Sorghum bicolor, with subsequent insertional fusion of a pair of chromosomes. The utility of the map is confirmed by the identification of two paralogous C4-pyruvate, phosphate dikinase (C4-PPDK) loci in Miscanthus, at positions syntenic to the single orthologous gene in Sorghum. CONCLUSIONS: The genus Miscanthus experienced an ancestral tetraploidy and chromosome fusion prior to its diversification, but after its divergence from the closely related sugarcane clade. The recent timing of this tetraploidy complicates discovery and mapping of genetic markers for Miscanthus species, since alleles and fixed differences between paralogs are comparable. These difficulties can be overcome by careful analysis of segregation patterns in a mapping population and genotyping of doubled haploids. The genetic map for Miscanthus will be useful in biological discovery and breeding efforts to improve this emerging biofuel crop, and also provide a valuable resource for understanding genomic responses to tetraploidy and chromosome fusion.

Assessing the diversity of anthocyanin composition in various tissues of purple corn (Zea mays L.).

Anthocyanins are natural pigments used in various foods, beverages, textiles, and nutraceuticals. Anthocyanins in the grain of purple corn (Zea mays L., Poaceae) have been a focus of many studies, but not much is known about anthocyanins in other maize tissues. In this study, purple corn variety Apache Red Cob was crossed to genetic stock 320 N, which is recessive for anthocyanin 3. The result was intense anthocyanin production in portions of the plant not normally pigmented. Anthocyanin extracts from anthers, cob glumes, husks, kernels, leaf sheaths, seedlings, silks, and tassels were assessed using UHPLC. A previously undescribed pigment produced in anthers was determined by NMR to be anthocyanidin 3-6″-phenylacetylglucoside. Multivariate analysis classified maize anthocyanins into 8 major compositional profiles. Results of this study show that maize produces anthocyanins abundantly in non-grain portions of the plant and that maize anthocyanin extracts have numerous applications due to the diversity in pigment profiles and hues.

Linking anthocyanin diversity, hue, and genetics in purple corn.

While maize with anthocyanin-rich pericarp (purple corn) is rising in popularity as a source of natural colorant for foods and beverages, information on color range and stability-factors associated with anthocyanin decorations and compositional profiles-is currently limited. Furthermore, to maximize the scalability and meet growing demands, both anthocyanin concentrations and agronomic performance must improve in purple corn varieties. Using the natural anthocyanin diversity present in a purple corn landrace, Apache Red, we generated a population with variable flavonoid profiles-flavanol-anthocyanin condensed forms (0-83%), acylated anthocyanins (2-72%), pelargonidin-derived anthocyanins (5-99%), C-glycosyl flavone co-pigments up to 1904 µg/g, and with anthocyanin content up to 1598 µg/g. Each aspect of the flavonoid profiles was found to play a role in either the resulting extract hue or intensity. With genotyping-by-sequencing of this population, we mapped aspects of the flavonoid profile. Major quantitative trait loci (QTLs) for anthocyanin type were found near loci previously identified only in aleurone-pigmented maize varieties [Purple aleurone1 (Pr1) and Anthocyanin acyltransferase1 (Aat1)]. A QTL near P1 (Pericarp color1) was found for both flavone content and flavanol-anthocyanin condensed forms. A significant QTL associated with peonidin-derived anthocyanins near a candidate S-adenosylmethionine-dependent methyltransferase was also identified, warranting further investigation. Mapping total anthocyanin content produced signals near Aat1, the aleurone-associated bHLH R1 (Colored1), the plant color-associated MYB, Pl1 (Purple plant1), the aleurone-associated recessive intensifier, In1 (Intensifier1), and several previously unidentified candidates. This population represents one of the most anthocyanin diverse pericarp-pigmented maize varieties characterized to date. Moreover, the candidates identified here will serve as branching points for future research studying the genetic and molecular processes determining anthocyanin profile in pericarp.

A natural colorant system from corn: Flavone-anthocyanin copigmentation for altered hues and improved shelf life.

Anthocyanins are a major source of natural red colorants but currently face difficulties matching the hue range, stability, and affordability of synthetic options. Purple corn offers an FDA and EFSA-approved economical source of anthocyanin-based colorants. A C-glycosyl flavone and anthocyanin copigmentation system consisting of a flavone-rich anthocyanin-poor line and two anthocyanin-rich flavone-poor lines containing either pelargonidin or cyanidin-derived anthocyanins is described. This system offers a broad hue range and can improve stability. Cyanidin-rich model beverages had better stability than pelargonidin-rich beverages over time, but the addition of flavone-rich extract to both resulted in significantly longer half-lives (up to 50% longer). Flavone copigments produced hyperchromic and bathochromic shifts in both. A protective effect from flavone copigmentation was observed for glycosides. In contrast acylated forms displayed significantly shorter half-lives. Results suggest that corn C-glycosyl flavone-rich extracts could serve as a color enhancing and stabilizing agent for anthocyanin colorants.

Adhesion and removal of E. coli K12 as affected by leafy green produce epicuticular wax composition, surface roughness, produce and bacterial surface hydrophobicity, and sanitizers.

Contaminated leafy vegetables have been associated with high-profile outbreaks causing severe illnesses. A good understanding of the interactions between human pathogen and produce is important for developing improved food safety control strategies. Currently, the role played by produce surface physiochemical characteristics in such interactions is not well-understood. This work was performed to examine the effects of produce physiochemical characteristics, including surface roughness, epicuticular wax composition, and produce and bacteria surface hydrophobicity on attachment and removal of vegetative bacteria. Escherichia coli K12 was used as a model microorganism to evaluate attachment to and removal from five leafy green vegetables after washing with selected sanitizers. A detailed epicuticular wax component analysis was conducted and the changes of wax composition after sanitation were also evaluated. The results showed that E. coli K12 removal is positively correlated with alkanes, ketones, and total wax content on leaf surfaces. Vegetables with high surface wax content had less rough leaf surfaces and more bacterial removal than the low wax produce. Produce surface roughness positively correlated to E. coli K12 adhesion and negatively correlated to removal. The cells preferentially attached to cut vegetable surfaces, with up to 1.49 times more attachment than on leaf adaxial surfaces.

An AMA1/MSP119 Adjuvanted Malaria Transplastomic Plant-Based Vaccine Induces Immune Responses in Test Animals.

Malaria is a tropical human disease, caused by protozoan parasites, wherein a significant number of the world's population is at risk. Annually, more than 219 million new cases are reported. Although there are prevention treatments, there are no highly and widely effective licensed anti-malarial vaccines available for use. Opportunities for utilization of plant-based vaccines as novel platforms for developing safe, reliable, and affordable treatments offer promise for developing such a vaccine against malaria. In this study, a Malchloroplast candidate vaccine was designed, composed of segments of AMA1 and MSP1 proteins, two epitopes of Plasmodium falciparum, along with a GK1 peptide from Taenia solium as adjuvant, and this was expressed in tobacco chloroplasts. Transplastomic tobacco lines were generated using biolistic transformation, and these were confirmed to carry the synthetic gene construct. Expression of the synthetic GK1 peptide was confirmed using RT-PCR and Western blots. Furthermore, the GK1 peptide was detected by HPLC at levels of up to 6 µg g-1 dry weight of tobacco leaf tissue. The plant-derived Malchloroplast candidate vaccine was subsequently tested in BALB/c female mice following subcutaneous administration, and was found to elicit specific humoral responses. Furthermore, components of this candidate vaccine were recognized by antibodies in Plasmodium falciparum malaria patients and were immunogenic in test mice. Thus, this study provided a 'proof of concept' for a promising plant-based candidate subunit vaccine against malaria.