Effect of Different Extraction Methods and Geographical Origins on the Total Phenolic Yield, Composition, and Antimicrobial Activity of Sugarcane Bagasse Extracts.

Several parameters, including particle size, solvent, temperature, and extraction method, affect phenolic compounds' extraction yield from a plant matrix. Considering the wide availability of sugarcane bagasse (SCB), this study analyzed the effect of different extraction methods and geographical origins on the yield, quality, and antimicrobial activity of phenolic compounds from SCB extracts. Samples from three geographical locations (Veracruz, Mexico; Santa Rosa, Texas, USA; and St. Mary, Louisiana, USA) were analyzed. Extraction was performed using an orbital shaker or ultrasonic bath at various times at a fixed temperature of 50°C, with 90% ethanol or methanol. The highest yield (5.91 mg GAE) was obtained using an orbital shaker for 24 h with 90% methanol as the solvent. HPLC-MS identified desferrioxamine b, baicalein, madecassic acid, and podototarin at different concentrations in all three SCB samples. The antimicrobial activity of these compounds was tested against Escherichia coli K12, Bacillus cereus, Enterobacter aerogenes, Streptococcus aureus, and Enterobacter cloacae. The antimicrobial activity was also tested against modifications of the Saccharomyces cerevisiae: the MutL Homolog 1 (MLH1), Slow Growth Suppressor (SGS1), O-6-MethylGuanine-DNA methyltransferase (MGT1), and RADiation sensitive (RAD14), carrying mutations related to different cancer types. In addition, the results were compared with the effect of ampicillin and kanamycin. The SCB extracts showed up to 90% growth inhibition against B. cereus at 200-800 µg/mL and 50% growth inhibition against S. aureus at 800 µg/mL. The inhibitory effect against modified yeast SGS1, RAD14, and MLH1 was 50-80% at 800 µg/mL. The percentage of inhibition and the phenolic compound contents differed depending on the origin of the SCB sample. These findings are promising for using this industrial byproduct to obtain compounds for nutraceutical, food additive, or medical uses.

Effect of glandless cottonseed meal protein and maltodextrin as microencapsulating agents on spray-drying of sugarcane bagasse phenolic compounds.

This study examines the effect of different concentrations of glandless cottonseed meal protein (GCSMP) and maltodextrin (MD) as microencapsulating agents on the shelf life stability of phenolic compounds extracted from sugarcane bagasse (SCB). Sugarcane bagasse showed high antioxidant activity, which remained stable after 30 days of storage at 4°C. The best microencapsulation process was obtained with an MD and GCSMP ratio of 63.6% and 36.4%, respectively. The encapsulating agents' ratio affected the encapsulation efficiency (EE) (p < 0.05), while the spray-drying temperature did not show an effect on the EE of the SCB phenolic compounds (p > 0.05). The antioxidant activity of the microencapsulated phenolic compounds was affected by the MD/GCSMP ratio (p < 0.05). The combination of MD and GCSM showed a higher EE than MD (p < 0.05), while the EE was lower when the ratio of encapsulating agents consisted of either MD or GCSMP alone (p < 0.05). The total phenolic content (TPC) in the microcapsules was not affected by the GCSMP ratio (p < 0.05). The TPC of microencapsulated phenolic compounds was stable up to 100°C for 14 days. GCSMP containing microcapsules showed a corrugated surface compared to a more homogenized surface of MD. The resulting corrugated structure explains the higher EE showed by the GCSMP. PRACTICAL APPLICATION: The use of sugarcane bagasse has been shown to add value to waste from agricultural and industrial sources. Glandless cottonseed meal protein is an excellent protective agent of antioxidants and can be extracted from agricultural waste. The encapsulated antioxidants can be used for the development of healthy, functional foods.

Antimicrobial, Shelf-Life Stability, and Effect of Maltodextrin and Gum Arabic on the Encapsulation Efficiency of Sugarcane Bagasse Bioactive Compounds.

This study shows the effects of maltodextrins and gum arabic as microencapsulation agents on the stability of sugarcane bagasse extracts and the potential use of the extracts as antimicrobial agents. The bioactive compounds in sugarcane bagasse (SCB) were extracted using 90% methanol and an orbital shaker at a fixed temperature of 50 °C, thereby obtaining a yield of the total phenolic content of 5.91 mg GAE/g. The bioactive compounds identified in the by-product were flavonoids, alkaloids, and lignan (-) Podophyllotoxin. The total phenolic content (TPC), antioxidant activity, and shelf-life stability of fresh and microencapsulated TPC were analyzed. This experiment's optimal microencapsulation can be obtained with a ratio of 0.6% maltodextrin (MD)/9.423% gum arabic (GA). Sugarcane bagasse showed high antioxidant activities, which remained stable after 30 days of storage and antimicrobial properties against E. coli, B. cereus, S. aureus, and the modified yeast SGS1. The TPC of the microencapsulated SCB extracts was not affected (p > 0.05) by time or storage temperature due to the combination of MD and GA as encapsulating agents. The antioxidant and antimicrobial capacities of sugarcane bagasse extracts showed their potential use as a source of bioactive compounds for further use as a food additive or nutraceutical. The results are a first step in encapsulating phenolic compounds from SCB as a promising source of antioxidant agents and ultimately a novel resource for functional foods.

Accumulation of bioactive metabolites in cultivated medical Cannabis.

There has been an increased use of medical Cannabis in the United States of America as more states legalize its use. Complete chemical analyses of this material can vary considerably between producers and is often not fully provided to consumers. As phytochemists in a state with legal medical Cannabis we sought to characterize the accumulation of phytochemicals in material grown by licensed commercial producers. We report the development of a simple extraction and analysis method, amenable to use by commercial laboratories for the detection and quantification of both cannabinoids and terpenoids. Through analysis of developing flowers on plants, we can identify sources of variability of floral metabolites due to flower maturity and position on the plant. The terpenoid composition varied by accession and was used to cluster cannabis strains into specific types. Inclusion of terpenoids with cannabinoids in the analysis of medical cannabis should be encouraged, as both of these classes of compounds could play a role in the beneficial medical effects of different cannabis strains.

Correlations of carotenoid content and transcript abundances for fibrillin and carotenogenic enzymes in Capsicum annum fruit pericarp.

The fruits of Capsicum spp. are especially rich sites for carotenoid synthesis and accumulation, with cultivar-specific carotenoid accumulation profiles. Differences in chromoplast structure as well as carotenoid biosynthesis are correlated with distinct carotenoid accumulations and fruit color. In the present study, the inheritance of chromoplast shape, carotenoid accumulation profiles, and transcript levels of four genes were measured. Comparisons of these traits were conducted using fruit from contrasting variants, Costeño Amarillo versus Costeño Red, and from F1 hybrids; crosses between parental lines with novel versions of these traits. Intermediate chromoplast shapes were observed in the F1, but no association between specific carotenoid accumulation and chromoplast shape was detected. Increased total carotenoid content was associated with increased ß-carotene and violaxanthin content. Transcript levels for phytoene synthase (Psy) and ß-carotene hydroxylase (CrtZ-2) were positively correlated with increased levels of specific carotenoids. No correlation was detected between transcript levels of capsanthin/capsorubin synthase (Ccs) and carotenoid composition or chromoplast shape. Transcript levels of fibrillin, were differentially correlated with specific carotenoids, negatively correlated with accumulation of capsanthin, and positively correlated with violaxanthin. The regulation of carotenoid accumulation in chromoplasts in Capsicum fruit continues to be a complex process with multiple steps for control.

Transcriptome analysis of cytoplasmic male sterility and restoration in CMS-D8 cotton.

KEY MESSAGE: A global view of differential expression of genes in CMS-D8 of cotton was presented in this study which will facilitate the understanding of cytoplasmic male sterility in cotton. Cytoplasmic male sterility (CMS) is a maternally inherited trait in higher plants which is incapable of producing functional pollen. However, the male fertility can be restored by one or more nuclear-encoded restorer genes. A genome-wide transcriptome analysis of CMS and restoration in cotton is currently lacking. In this study, Affymetrix GeneChips© Cotton Genome Array containing 24,132 transcripts was used to compare differentially expressed (DE) genes of flower buds at the meiosis stage between CMS and its restorer cotton plants conditioned by the D8 cytoplasm. A total of 458 (1.9 %) of DE genes including 127 up-regulated and 331 down-regulated ones were identified in the CMS-D8 line. Quantitative RT-PCR was used to validate 10 DE genes selected from seven functional categories. The most frequent DE gene group was found to encode putative proteins involved in cell wall expansion, such as pectinesterase, pectate lyase, pectin methylesterase, glyoxal oxidase, polygalacturonase, indole-3-acetic acid-amino synthetase, and xyloglucan endo-transglycosylase. Genes in cytoskeleton category including actin, which plays a key role in cell wall expansion, cell elongation and cell division, were also highly differentially expressed between the fertile and CMS plants. This work represents the first study in utilizing microarray to identify CMS-related genes by comparing overall DE genes between fertile and CMS plants in cotton. The results provide evidence that many CMS-associated genes are mainly involved in cell wall expansion. Further analysis will be required to elucidate the molecular mechanisms of male sterility which will facilitate the development of new hybrid cultivars in cotton.

Carotenoid accumulation in orange-pigmented Capsicum annuum fruit, regulated at multiple levels.

The pericarp of Capsicum fruit is a rich dietary source of carotenoids. Accumulation of these compounds may be controlled, in part, by gene transcription of biosynthetic enzymes. The carotenoid composition in a number of orange-coloured C. annuum cultivars was determined using HPLC and compared with transcript abundances for four carotenogenic enzymes, Psy, LcyB, CrtZ-2, and Ccs determined by qRT-PCR. There were unique carotenoid profiles as well as distinct patterns of transcription of carotenogenic enzymes within the seven orange-coloured cultivars. In one cultivar, 'Fogo', carrying the mutant ccs-3 allele, transcripts were detected for this gene, but no CCS protein accumulated. The premature stop termination in ccs-3 prevented expression of the biosynthetic activity to synthesize the capsanthin and capsorubin forms of carotenoids. In two other orange-coloured cultivars, 'Orange Grande' and 'Oriole', both with wild-type versions of all four carotenogenic enzymes, no transcripts for Ccs were detected and no red pigments accumulated. Finally, in a third case, the orange-coloured cultivar, Canary, transcripts for all four of the wild-type carotenogenic enzymes were readily detected yet no CCS protein appeared to accumulate and no red carotenoids were synthesized. In the past, mutations in Psy and Ccs have been identified as the loci controlling colour in the fruit. Now there is evidence that a non-structural gene may control colour development in Capsicum.

Identification of differentially expressed genes associated with semigamy in Pima cotton (Gossypium barbadense L.) through comparative microarray analysis.

BACKGROUND: Semigamy in cotton is a type of facultative apomixis controlled by an incompletely dominant autosomal gene (Se). During semigamy, the sperm and egg cells undergo cellular fusion, but the sperm and egg nucleus fail to fuse in the embryo sac, giving rise to diploid, haploid, or chimeric embryos composed of sectors of paternal and maternal origin. In this study we sought to identify differentially expressed genes related to the semigamy genotype by implementing a comparative microarray analysis of anthers and ovules between a non-semigametic Pima S-1 cotton and its doubled haploid natural isogenic mutant semigametic 57-4. Selected differentially expressed genes identified by the microarray results were then confirmed using quantitative reverse transcription PCR (qRT-PCR). RESULTS: The comparative analysis between isogenic 57-4 and Pima S-1 identified 284 genes in anthers and 1,864 genes in ovules as being differentially expressed in the semigametic genotype 57-4. Based on gene functions, 127 differentially expressed genes were common to both semigametic anthers and ovules, with 115 being consistently differentially expressed in both tissues. Nine of those genes were selected for qRT-PCR analysis, seven of which were confirmed. Furthermore, several well characterized metabolic pathways including glycolysis/gluconeogenesis, carbon fixation in photosynthetic organisms, sesquiterpenoid biosynthesis, and the biosynthesis of and response to plant hormones were shown to be affected by differentially expressed genes in the semigametic tissues. CONCLUSION: As the first report using microarray analysis, several important metabolic pathways affected by differentially expressed genes in the semigametic cotton genotype have been identified and described in detail. While these genes are unlikely to be the semigamy gene itself, the effects associated with expression changes in those genes do mimic phenotypic traits observed in semigametic plants. A more in-depth analysis of semigamy is necessary to understand its expression and regulation at the genetic and molecular level.

Identification of salt responsive genes using comparative microarray analysis in Upland cotton (Gossypium hirsutum L.).

Salinity negatively impacts plant growth and productivity, and little is known about salt responsive genes in cotton. In this study, an intra-specific backcross population of cotton (Gossypium hirsutum L.) was treated with 200 mM NaCl after which differentially expressed genes were identified by comparison between salt tolerant and susceptible segregant bulks using comparative microarray analysis. Microarray analysis identified 720 salt-responsive genes, of which 695 were down-regulated and only 25 were up-regulated in the salt tolerant bulk. Gene ontology of annotated genes revealed that at least some of the identified salt responsive transcripts belong to pathways known to be associated with salt stress including osmolyte and lipid metabolism, cell wall structure, and membrane synthesis. About 48% of all salt-responsive genes were functionally unknown. Quantitative RT-PCR was used to validate 17 selected salt responsive genes. This work represents the first study in employing microarray to investigate the possible mechanisms of the salt response in cotton. Further analysis of salt-responsive genes associated with salt tolerance in cotton will assist in laying a foundation for molecular manipulation in development of new cultivars with improved salt tolerance.

Comparative expression of miRNA genes and miRNA-based AFLP marker analysis in cultivated tetraploid cottons.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that down-regulate gene expression in a sequence specific manner to control plant growth and development. The identification and characterization of miRNAs are critical steps in finding their target genes and elucidating their functions. The objective of the present study was to assess the genetic variation of miRNA genes through expression comparisons and miRNA-based AFLP marker analysis. Seven miRNAs were first selected for RT-PCR and four for quantitative RT-PCR analysis that showed considerably high or differential expression levels in early stages of boll development. Except for miR160a, differential gene expression of miR171, 390a, and 396a was detected in early developing bolls at one or more timepoints between two cultivated cotton cultivars, Pima Phy 76 (Gossypium barbadense) and Acala 1517-99 (Gossypium hirsutum). Our further work demonstrated that genetic diversity of miRNA genes can be assessed by miRNA-AFLP analysis using primers designed from 22 conserved miRNA genes in combination with AFLP primers. Homologous miRNA genes can be also identified and isolated for sequencing and confirmation using this homology-based genotyping approach. This strategy offers an alternative to isolating a full length of miRNA genes and their up-stream and down-stream sequences. The significance of the expression and sequence differences of miRNAs between cotton species or genotypes needs further studies.

A root-specific bZIP transcription factor is responsive to water deficit stress in tepary bean (Phaseolus acutifolius) and common bean (P. vulgaris).

Root cDNA libraries were differentially screened to isolate water deficit-responsive transcripts in the relatively drought-resistant plant tepary bean (Phaseolus acutifolius). A novel root-specific, water deficit-responsive transcript was identified and predicted to encode a bZIP transcription factor. The orthologous form of this gene was isolated from the drought-sensitive P. vulgaris and the patterns of expression of these genes compared. These genes have predicted amino acid sequences in the bZIP domain that are 64% similar to a soybean bZIP protein. There were three amino acid differences between the P. acutifolius bZIP and the P. vulgaris gene product. Both species transcribed this gene in a root-specific and water deficit-responsive manner. The cell-specific pattern of expression for the gene was determined using in situ hybridization and immunolocalization. Two tissues in the root accumulated the protein: epidermis and phloem. The nuclear localization of this protein was determined by electron microscopy. The bZIP protein accumulated in the nuclei of both the epidermal cell and the vascular cell in response to water deficit stress in both species in a similar manner.