The Impact of Rice Lipid on In Vitro Rice Starch Digestibility.

The negative role of lipids in rice starch digestion is well-known; however, the effect of individual native lipids on starch digestibility has not been studied. In this study, native rice lipids, such as triacylglycerols (TAGs), diacylglycerols (DAGs), phosphatidylcholines (PCs) and lysophospholipids (LPLs), were analyzed using liquid chromatography−mass spectrometry (LC-MS) and correlated with in vitro rice starch digestibility. Most of the tested lipids exhibited a negative correlation with the in vitro starch digestibility with the correlations being more pronounced for LPLs. Removal of lipids from rice flour increased the in vitro starch digestibility. Conversely, a lipid extract addition to rice flour reduced the starch digestibility. Addition of 1% pure lysophosphatidylcholine (LPC)16:0, TAG54:6, DAG36:4 or PC36:2 individually to rice flour reduced starch digestibility by different extents in the order of LPC16:0 > TAG54:6 > PC36:2 > DAG36:4. LPC16:0 was the most abundant lipid among all the assessed lipids in the white rice (milled rice), and addition of 1% LPC 16:0 to rice flour reduced glucose release following three hours of in vitro starch digestion by 7.4%. There may be a scope to breed rice with a lipid composition to reach a desired starch digestibility or simply through addition of certain lipids before cooking the rice.

In Vitro Anti-Inflammatory Activity of Essential Oil and ß-Bisabolol Derived from Cotton Gin Trash.

Natural α-bisabolol has been widely used in cosmetics and is sourced mainly from the stems of Candeia trees that have become endangered due to over exploitation. The in vitro anti-inflammatory activity of cotton gin trash (CGT) essential oil and the major terpenoid (ß-bisabolol) purified from the oil were investigated against lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages as well as the 3t3 and HS27 fibroblast cell lines. Nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and interleukin 8 (IL-8) were measured using Greiss reagent, enzyme-linked immunosorbent assay (ELISA), and cytokine bead array (CBA)-flow cytometry. Non-toxic concentrations of CGT oil and ß-bisabolol (1.6-50.0 µg/mL) significantly inhibited the production of the inflammatory mediators in a dose-dependent manner. Maximal inhibition by ß-bisabolol was 55.5% for NO, 62.3% for PGE2, and 45.3% for TNF-α production in RAW cells. ß-Bisabolol induced a level of inhibition similar to an equal concentration of α-bisabolol (50.0 µg/mL), a known anti-inflammatory agent. These results suggest ß-bisabolol exerts similar in vitro effects to known topical anti-inflammatory agents and could therefore be exploited for cosmetic and therapeutic uses. This is the first study to report the in vitro anti-inflammatory activity of ß-bisabolol in CGT essential oil.

Chemical volatiles present in cotton gin trash: A by-product of cotton processing.

Cotton gin trash (CGT), a waste product of cotton gins, make up about 10% of each bale of cotton bolls ginned. The current study investigates high value volatile compounds in CGT to add value to this by-product. The volatile compounds in CGT and different parts of the cotton plant were extracted using various methods, identified by gas chromatography-mass spectrometry (GC-MS) or nuclear magnetic resonance (NMR) spectroscopy, and then quantified by gas chromatography-flame ionisation detection (GC-FID) against available standards. Terpenoids including monoterpenoids and sesquiterpenoids were found to be the most abundant, making up 64.66% (area under peak) of total volatiles extracted by hydro-distillation. The major extractable terpenoids in CGT were α-pinene (13.69-23.05 µg/g), ß-caryophyllene (3.99-74.32 µg/g), α-humulene (2.00-25.71 µg/g), caryophyllene oxide (41.50-102.08 µg/g) and ß-bisabolol (40.05-137.32 µg/g). Recoveries varied between different extraction methods. The terpenoids were found to be more abundant in the calyx (659.12 µg/g) and leaves (627.72 µg/g) than in stalks (112.97 µg/g) and stems (24.24 µg/g) of the cotton plant, indicating the possible biological origin of CGT volatiles. This study is the first to identify and quantify the different terpenoids present in CGT and significantly, ß-bisabolol, an abundant compound (sesquiterpene alcohol) which may have valuable biological prospects. These findings therefore contribute to identifying alternative management strategies and uses of CGT.

Bromoperoxidase Producing Bacillus spp. Isolated from the Hypobranchial Glands of a Muricid Mollusc Are Capable of Tyrian Purple Precursor Biogenesis.

The secondary metabolite Tyrian purple, also known as shellfish purple and royal purple, is a dye with historical importance for humans. The biosynthetic origin of Tyrian purple in Muricidae molluscs is not currently known. A possible role for symbiotic bacteria in the production of tyrindoxyl sulphate, the precursor to Tyrian purple stored in the Australian species, Dicathais orbita, has been proposed. This study aimed to culture bacterial symbionts from the purple producing hypobranchial gland, and screen the isolates for bromoperoxidase genes using molecular methods. The ability of bromoperoxidase positive isolates to produce the brominated indole precursor to Tyrian purple was then established by extraction of the culture, and analysis by liquid chromatography-mass spectrometry (LC-MS). In total, 32 bacterial isolates were cultured from D. orbita hypobranchial glands, using marine agar, marine agar with hypobranchial gland aqueous extracts, blood agar, thiosulphate citrate bile salts sucrose agar, and cetrimide agar at pH 7.2. These included 26 Vibrio spp., two Bacillus spp., one Phaeobacter sp., one Shewanella sp., one Halobacillus sp. and one Pseudoalteromonas sp. The two Bacillus species were the only isolates found to have coding sequences for bromoperoxidase enzymes. LC-MS analysis of the supernatant and cell pellets from the bromoperoxidase producing Bacillus spp. cultured in tryptone broth, supplemented with KBr, confirmed their ability to produce the brominated precursor to Tyrian purple, tyrindoxyl sulphate. This study supports a potential role for symbiotic Bacillus spp. in the biosynthesis of Tyrian purple.

Re-sequencing Resources to Improve Starch and Grain Quality in Rice.

Next-generation sequencing can identify differences in the rice genome that explain the genetic basis of grain quality variation. Differences in rice grain quality are mainly associated with differences in the major component of the grain, starch. Association of rice quality variation with rice genome variation can be conducted at the gene or whole-genome level. Re-sequencing of specific genes or whole genomes can be used depending on the extent to which candidate genes for the traits of interest are known. Amplicon sequencing of genes involved in starch metabolism can help in targeted discovery of the molecular genetic basis of differences in starch related quality attributes. Whole-genome re-sequencing can complement this, when the genetic basis of the trait is expected to be outside the coding region of starch metabolism genes. These approaches have been used successfully to understand the rice genome at specific loci and over the whole genome.

Remobilisation of phosphorus fractions in rice flag leaves during grain filling: Implications for photosynthesis and grain yields.

Phosphorus (P) is translocated from vegetative tissues to developing seeds during senescence in annual crop plants, but the impact of this P mobilisation on photosynthesis and plant performance is poorly understood. This study investigated rice (Oryza sativa L.) flag leaf photosynthesis and P remobilisation in a hydroponic study where P was either supplied until maturity or withdrawn permanently from the nutrient solution at anthesis, 8 days after anthesis (DAA) or 16 DAA. Prior to anthesis, plants received either the minimum level of P in nutrient solution required to achieve maximum grain yield ('adequate P treatment'), or received luxury levels of P in the nutrient solution ('luxury P treatment'). Flag leaf photosynthesis was impaired at 16 DAA when P was withdrawn at anthesis or 8 DAA under adequate P supply but only when P was withdrawn at anthesis under luxury P supply. Ultimately, reduced photosynthesis did not translate into grain yield reductions. There was some evidence plants remobilised less essential P pools (e.g. Pi) or replaceable P pools (e.g. phospholipid-P) prior to remobilisation of P in pools critical to leaf function such as nucleic acid-P and cytosolic Pi. Competition for P between vegetative tissues and developing grains can impair photosynthesis when P supply is withdrawn during early grain filling. A reduction in the P sink strength of grains by genetic manipulation may enable leaves to sustain high rates of photosynthesis until the later stages of grain filling.

Analysis of Lysophospholipid Content in Low Phytate Rice Mutants.

As a fundamental component of nucleic acids, phospholipids, and adenosine triphosphate, phosphorus (P) is critical to all life forms, however, the molecular mechanism of P translocation and distribution in rice grains are still not understood. Here, with the use of five different low phytic acid (lpa) rice mutants, the redistribution in the main P-containing compounds in rice grain, phytic acid (PA), lysophospholipid (LPL), and inorganic P (Pi), was investigated. The lpa mutants showed a significant decrease in PA and phytate-phosphorus (PA-P) concentration with a concomitant increase in Pi concentration. Moreover, defects in the OsST and OsMIK genes result in a great reduction of specific LPL components and LPL-phosphorus (LPL-P) contents in rice grain. In contrast, defective OsMRP5 and Os2-PGK genes led to a significant increase in individual LPL components. The effect of the Os2-PGK gene on the LPL accumulation was validated using breeding lines derived from a cross between KBNT-lpa (Os2-PGK mutation) and Jiahe218. This study demonstrates that these rice lpa mutants lead to the redistribution of Pi in endosperm and modify LPL biosynthesis. Increase LPLs in the endosperm in the lpa mutants may have practical applications in rice breeding to produce "healthier" rice.

Biological Importance of Cotton By-Products Relative to Chemical Constituents of the Cotton Plant.

Although cultivated for over 7000 years, mainly for production of cotton fibre, the cotton plant has not been fully explored for potential uses of its other parts. Despite cotton containing many important chemical compounds, limited understanding of its phytochemical composition still exists. In order to add value to waste products of the cotton industry, such as cotton gin trash, this review focuses on phytochemicals associated with different parts of cotton plants and their biological activities. Three major classes of compounds and some primary metabolites have been previously identified in the plant. Among these compounds, most terpenoids and their derivatives (51), fatty acids (four), and phenolics (six), were found in the leaves, bolls, stalks, and stems. Biological activities, such as anti-microbial and anti-inflammatory activities, are associated with some of these phytochemicals. For example, ß-bisabolol, a sesquiterpenoid enriched in the flowers of cotton plants, may have anti-inflammatory product application. Considering the abundance of biologically active compounds in the cotton plant, there is scope to develop a novel process within the current cotton fibre production system to separate these valuable phytochemicals, developing them into potentially high-value products. This scenario may present the cotton processing industry with an innovative pathway towards a waste-to-profit solution.

Phosphorus remobilization from rice flag leaves during grain filling: an RNA-seq study.

The physiology and molecular regulation of phosphorus (P) remobilization from vegetative tissues to grains during grain filling is poorly understood, despite the pivotal role it plays in the global P cycle. To test the hypothesis that a subset of genes involved in the P starvation response are involved in remobilization of P from flag leaves to developing grains, we conducted an RNA-seq analysis of rice flag leaves during the preremobilization phase (6 DAA) and when the leaves were acting as a P source (15 DAA). Several genes that respond to phosphate starvation, including three purple acid phosphatases (OsPAP3, OsPAP9b and OsPAP10a), were significantly up-regulated at 15 DAA, consistent with a role in remobilization of P from flag leaves during grain filling. A number of genes that have not been implicated in the phosphate starvation response, OsPAP26, SPX-MFS1 (a putative P transporter) and SPX-MFS2, also showed expression profiles consistent with involvement in P remobilization from senescing flag leaves. Metabolic pathway analysis using the KEGG system suggested plastid membrane lipid synthesis is a critical process during the P remobilization phase. In particular, the up-regulation of OsPLDz2 and OsSQD2 at 15 DAA suggested phospholipids were being degraded and replaced by other lipids to enable continued cellular function while liberating P for export to developing grains. Three genes associated with RNA degradation that have not previously been implicated in the P starvation response also showed expression profiles consistent with a role in P mobilization from senescing flag leaves.

Transcriptome of the Australian Mollusc Dicathais orbita Provides Insights into the Biosynthesis of Indoles and Choline Esters.

Dicathais orbita is a mollusc of the Muricidae family and is well known for the production of the expensive dye Tyrian purple and its brominated precursors that have anticancer properties, in addition to choline esters with muscle-relaxing properties. However, the biosynthetic pathways that produce these secondary metabolites in D. orbita are not known. Illumina HiSeq 2000 transcriptome sequencing of hypobranchial glands, prostate glands, albumen glands, capsule glands, and mantle and foot tissues of D. orbita generated over 201 million high quality reads that were de novo assembled into 219,437 contigs. Annotation with reference to the Nr, Swiss-Prot and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases identified candidate-coding regions in 76,152 of these contigs, with transcripts for many enzymes in various metabolic pathways associated with secondary metabolite biosynthesis represented. This study revealed that D. orbita expresses a number of genes associated with indole, sulfur and histidine metabolism pathways that are relevant to Tyrian purple precursor biosynthesis, and many of which were not found in the fully annotated genomes of three other molluscs in the KEGG database. However, there were no matches to known bromoperoxidase enzymes within the D. orbita transcripts. These transcriptome data provide a significant molecular resource for gastropod research in general and Tyrian purple producing Muricidae in particular.

Characterization of Bacterial Communities Associated with the Tyrian Purple Producing Gland in a Marine Gastropod.

Dicathais orbita is a marine mollusc recognised for the production of anticancer compounds that are precursors to Tyrian purple. This study aimed to assess the diversity and identity of bacteria associated with the Tyrian purple producing hypobranchial gland, in comparison with foot tissue, using a high-throughput sequencing approach. Taxonomic and phylogenetic analysis of variable region V1-V3 of 16S rRNA bacterial gene amplicons in QIIME and MEGAN were carried out. This analysis revealed a highly diverse bacterial assemblage associated with the hypobranchial gland and foot tissues of D. orbita. The dominant bacterial phylum in the 16S rRNA bacterial profiling data set was Proteobacteria followed by Bacteroidetes, Tenericutes and Spirochaetes. In comparison to the foot, the hypobranchial gland had significantly lower bacterial diversity and a different community composition, based on taxonomic assignment at the genus level. A higher abundance of indole producing Vibrio spp. and the presence of bacteria with brominating capabilities in the hypobranchial gland suggest bacteria have a potential role in biosynthesis of Tyrian purple in D. orbita.

The contribution of lysophospholipids to pasting and thermal properties of nonwaxy rice starch.

It is known that lysophospholipids (LPLs) may affect rice starch pasting and thermal properties possibly through the formation of an amylose-lipid complex. However, whether these effects of rice LPLs are independent of amylose are still not understood. Here, the diversity of rice flour pasting and thermal properties and their relationship with individual LPL components in native rice endosperm were studied. Several significant correlations between LPLs and pasting properties, such as cool paste viscosity (CPV), breakdown (BD) and consistency (CS) were clearly evident. Thermal properties generally had no relationship with LPLs except for gelatinization enthalpy. Using partial correlation analysis we found that, irrespective of apparent amylose content, CPV and individual LPLs were positively correlated, while BD, CS and other individual LPLs were negatively correlated. This study suggests naturally occurring individual LPLs can contribute to rice flour pasting and thermal properties, either independently or in combination with amylose.

Genotypic variation in lysophospholipids of milled rice.

Phospholipids (PLs) play a prominent role in both grain cellular structure and nutritional function of cereal crops. Their lyso forms (lysophospholipids, LPLs) often combine with cereal starch to form an amylose-lipid complex (ALC), which may influence starch properties. In this study, 20 rice accessions were grown over two seasons at the same location to explore diversity in LPLs of milled rice. Levels of specific LPLs differed significantly among rice genotypes, demonstrating there is a wide diversity in LPLs in rice grain. The main LPL components were lysophosphatidylcholine (LPC) 16:0 (ranging from 3009.7 to 4697.8 µg/g), LPC18:2 (836.6-2182.3 µg/g), lysophosphatidylethanolamine (LPE) 16:0 (625.7-1139.8 µg/g), and LPE18:2 (170.6-481.6 µg/g). Total LPC, total LPE, and total LPL ranged from 4727.1 to 7685.2 µg/g, from 882.8 to 1809.5 µg/g, and from 5609.8 to 9401.1 µg/g, respectively. Although significant (P < 0.001) environment and genotype × environment (G × E) interactions were detected by analysis of variance (ANOVA), these effects accounted for only 0.7-38.9 and 1.8-6.6% of the total variance, respectively. Correlation analysis between LPL components provided insight into the possible LPL biosynthesis pathway in plants. Hierarchical cluster analysis suggested that the 20 rice accessions could be classified into three groups, whereas principal component analysis also identified three groups, with the first two components explaining 57.7 and 16.2% of the total variance. Further genetic studies are needed to identify genes or quantitative trait loci (QTLs) underlying the genetic control of LPLs in rice grain.

Australian wild rice reveals pre-domestication origin of polymorphism deserts in rice genome.

BACKGROUND: Rice is a major source of human food with a predominantly Asian production base. Domestication involved selection of traits that are desirable for agriculture and to human consumers. Wild relatives of crop plants are a source of useful variation which is of immense value for crop improvement. Australian wild rices have been isolated from the impacts of domestication in Asia and represents a source of novel diversity for global rice improvement. Oryza rufipogon is a perennial wild progenitor of cultivated rice. Oryza meridionalis is a related annual species in Australia. RESULTS: We have examined the sequence of the genomes of AA genome wild rices from Australia that are close relatives of cultivated rice through whole genome re-sequencing. Assembly of the resequencing data to the O. sativa ssp. japonica cv. Nipponbare shows that Australian wild rices possess 2.5 times more single nucleotide polymorphisms than in the Asian wild rice and cultivated O. sativa ssp. indica. Analysis of the genome of domesticated rice reveals regions of low diversity that show very little variation (polymorphism deserts). Both the perennial and annual wild rice from Australia show a high degree of conservation of sequence with that found in cultivated rice in the same 4.58 Mbp region on chromosome 5, which suggests that some of the 'polymorphism deserts' in this and other parts of the rice genome may have originated prior to domestication due to natural selection. CONCLUSIONS: Analysis of genes in the 'polymorphism deserts' indicates that this selection may have been due to biotic or abiotic stress in the environment of early rice relatives. Despite having closely related sequences in these genome regions, the Australian wild populations represent an invaluable source of diversity supporting rice food security.

Determination of starch lysophospholipids in rice using liquid chromatography-mass spectrometry (LC-MS).

Acquiring a complete understanding of rice starch lysophospholipids (LPLs), their biosynthetic pathways and genetic diversity, and the influence of genotype by environment interactions has been hampered by the lack of efficient high-throughput extraction and analysis methods. It was hypothesized a single-step aqueous n-propanol extraction combined with liquid chromatography-mass spectrometry (LC-MS) could be employed to analyze starch LPLs in white rice. The investigation found different grinding methods showed little effect on the final LPL detected, and a simple single-step extraction with 75% n-propanol (8 mL/0.15 g) heated at 100 °C for 2 h was as effective as an onerous multistep extraction method. A LC-MS method was optimized to simultaneously quantify 10 major LPLs in rice starch within 15 min. This method enables total and individual starch LPL analysis of a large number of rice samples at little cost. This approach could be applied to starch LPLs in other cereals.

The polymerase chain reaction (PCR): general methods.

The polymerase chain reaction (PCR) converts very low quantities of DNA into very high quantities and is the foundation of many specialized techniques of molecular biology. PCR utilizes components of the cellular machinery of mitotic cell division in vitro which respond predictably to user inputs. This chapter introduces the principles of PCR and discusses practical considerations from target sequence definition through to optimization and application.

Genome walking.

Genome walking is a method for determining the DNA sequence of unknown genomic regions flanking a region of known DNA sequence. The Genome walking has the potential to capture 6-7 kb of sequence in a single round. Ideal for identifying gene promoter regions where only the coding region. Genome walking also has significant utility for capturing homologous genes in new species when there are areas in the target gene with strong sequence conservation to the characterized species. The increasing use of next-generation sequencing technologies will see the principles of genome walking adapted to in silico methods. However, for smaller projects, PCR-based genome walking will remain an efficient method of characterizing unknown flanking sequence.

A method for discovery of genome-wide SNP between any two genotypes from whole-genome re-sequencing data.

Advances in sequencing technologies have aided the discovery of millions of genome-wide DNA polymorphisms such as single-nucleotide polymorphisms (SNPs) and insertion-deletions (InDels) which are an invaluable resource for marker-assisted breeding. Presently available bioinformatics tools assist the discovery of polymorphisms between target genotypes and the reference genome for a range of species. The discovery of polymorphisms between two genotypes within a breeding program is complicated by several factors such as bias in the number of reads from each genotype and residual heterozygosity within each genotype. In this chapter, we describe a novel approach where polymorphisms between a pair of genotypes are discovered from whole-genome re-sequencing data.

Phospholipids in rice: significance in grain quality and health benefits: a review.

Phospholipids (PLs) are a major class of lipid in rice grain. Although PLs are only a minor nutrient compared to starch and protein, they may have both nutritional and functional significance. We have systemically reviewed the literature on the class, distribution and variation of PLs in rice, their relation to rice end-use quality and human health, as well as available methods for analytical profiling. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and their lyso forms are the major PLs in rice. The deterioration of PC in rice bran during storage was considered as a trigger for the degradation of rice lipids with associated rancid flavour in paddy and brown rice. The lyso forms in rice endosperm represent the major starch lipid, and may form inclusion complexes with amylose, affecting the physicochemical properties and digestibility of starch, and hence its cooking and eating quality. Dietary PLs have a positive impact on several human diseases and reduce the side-effects of some drugs. As rice has long been consumed as a staple food in many Asian countries, rice PLs may have significant health benefits for those populations. Rice PLs may be influenced both by genetic (G) and environmental (E) factors, and resolving G×E interactions may allow future exploitation of PL composition and content, thus boosting rice eating quality and health benefits for consumers. We have identified and summarised the different methods used for rice PL analysis, and discussed the consequences of variation in reported PL values due to inconsistencies between methods. This review enhances the understanding of the nature and importance of PLs in rice and outlines potential approaches for manipulating PLs to improve the quality of rice grain and other cereals.

Application of large-scale sequencing to marker discovery in plants.

Advances in DNA sequencing provide tools for efficient large-scale discovery of markers for use in plants. Discovery options include large-scale amplicon sequencing, transcriptome sequencing, gene-enriched genome sequencing and whole genome sequencing. Examples of each of these approaches and their potential to generate molecular markers for specific applications have been described. Sequencing the whole genome of parents identifies all the polymorphisms available for analysis in their progeny. Sequencing PCR amplicons of sets of candidate genes from DNA bulks can be used to define the available variation in these genes that might be exploited in a population or germplasm collection. Sequencing of the transcriptomes of genotypes varying for the trait of interest may identify genes with patterns of expression that could explain the phenotypic variation. Sequencing genomic DNA enriched for genes by hybridization with probes for all or some of the known genes simplifies sequencing and analysis of differences in gene sequences between large numbers of genotypes and genes especially when working with complex genomes. Examples of application of the above-mentioned techniques have been described.