Tuning the mechanical properties of pectin films with polyphenol-rich plant extracts.

The mechanical properties of pectin films enhanced with polyphenol-rich fruit extracts were investigated. The scavenging and reducing activity of plant extracts incorporated into the pectin films were determined using bench assays, and their antioxidant activity was correlated with a high presence of polyphenols, which were predominantly comprised of flavonoids and anthocyanins. The pectin films generated from the extracts exhibited a range of mechanical properties; tensile strength (4.99 MPa - 6.91 MPa), elongation at break (45.8 % - 52.3 %), and stiffness (1835 g mm-1 - 2765 g mm-1). To investigate the underlying relationships between plant extract composition and mechanical properties, Projection to Latent Structures (PLS) models were developed. The PLS models revealed that extracts containing high sugar and polyphenol content increase the tensile strength and moisture content of films. The elongation at break of the films was improved or diminished depending on the profile of sugar, acids, and polyphenols in the fruit extracts. Furthermore, the structures and concentration of anthocyanins and flavonoids were identified to strongly influenced the elongation at break differences. By modifying the concentration of sugars, organic acids, and polyphenols, the mechanical properties of pectin-based films can be tuned for tailored applications as food packaging materials.

Qualitative Analysis of Polyphenols in Glycerol Plant Extracts Using Untargeted Metabolomics.

Glycerol is a reliable solvent for extracting polyphenols from food and waste products. There has been an increase in the application of glycerol over benchmark alcoholic solvents such as ethanol and methanol for natural product generation because of its non-toxic nature and high extraction efficiency. However, plant extracts containing a high glycerol concentration are unsuitable for mass spectrometry-based investigation utilising electrospray ionization, inhibiting the ability to analyse compounds of interest. In this investigation, a solid phase extraction protocol is outlined for removing glycerol from plant extracts containing a high concentration of glycerol and their subsequent analysis of polyphenols using ultra-performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry. Using this method, glycerol-based extracts of Queen Garnet Plum (Prunus salicina) were investigated and compared to ethanolic extracts. Anthocyanins and flavonoids in high abundance were found in both glycerol and ethanol extracts. The polyphenol metabolome of Queen Garnet Plum was 53% polyphenol glycoside derivatives and 47% polyphenols in their aglycone forms. Furthermore, 56% of the flavonoid derivates were found to be flavonoid glycosides, and 44% were flavonoid aglycones. In addition, two flavonoid glycosides not previously found in Queen Garnet Plum were putatively identified: Quercetin-3-O-xyloside and Quercetin-3-O-rhamnoside.

Lipidomics reveals associations between rice quality traits.

INTRODUCTION: Lipids are a diverse group of macromolecules that occur in rice grains and are known to impact rice grain properties. Identifying the relationships between specific lipids and traits of quality is important to improve varietal selection for high quality rice. OBJECTIVES: Using untargeted lipidomics, this study aims to understand the role of lipids on different traits of quality by identifying the genotypic effect of lipids and their impact on traits of cooking and eating quality of a rice mapping population. METHODS: Lipids from milled rice grains of three sets of rice samples were screened by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) in the positive ionisation mode. Lipid features were putatively identified using analytical standards and online databases. Multivariate statistics were carried out to identify the lipid profile of varieties across three experiments. Correlation analysis was carried out between lipid features and 12 quality traits across a rice mapping population that segregates for grain physical and texture-associated traits. RESULTS: Thousands of features in rice grain lipids were detected, and were grouped into six categories-fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids and prenol lipids. A strong genotypic basis for the lipid profile was observed among the four varieties grown under five nitrogen treatments. Clear differentiation in lipid profiles between waxy and non-waxy rice was observed. Strong correlations were observed for putative lipids that form the amylose-lipid complex and with amylose content and viscosity parameters. CONCLUSIONS: This study demonstrates the strength of untargeted lipidomics in putatively determining features that differentiate varieties from each other, and reveals the role of specific lipids on the physical and textural quality of rice.

Quality evaluation, fatty acid analysis and untargeted profiling of volatiles in Cambodian rice.

This study provides the first investigation of the physical traits, pasting properties and volatile compounds of Cambodian rice cultivars, including traditional, improved, and improved traditional varieties, allowing for their differentiation as high and low quality rice. Analysis of the grain quality traits illustrates interesting features of traditional varieties and correlations between traits that assist with understanding texture. Untargeted profiling of volatile compounds shows that high quality fragrant varieties not only contain 2-acetyl-1-pyrroline but also several other compounds, including aldehydes, alcohols and 2-alkylfurans that contribute to overall aroma. Moreover, low odour threshold volatile compounds, which can be derived from the oxidation of unsaturated fatty acids, were more abundant in the fragrant varieties. The percentage area of both oleic and linoleic acid were found to be significantly different among the rice varieties tested. Such findings suggest that unsaturated fatty acids in milled rice contribute to rice fragrance, and thereby to overall quality.

Metabolomics and genomics combine to unravel the pathway for the presence of fragrance in rice.

Since it was first characterised in 1983, 2-acetyl-1-pyrroline (2AP) has been considered to be the most important aroma compound in rice. In this study, we show four other amine heterocycles: 6-methyl, 5-oxo-2,3,4,5-tetrahydropyridine (6M5OTP), 2-acetylpyrrole, pyrrole and 1-pyrroline, that correlate strongly with the production of 2AP, and are present in consistent proportions in a set of elite aromatic rice varieties from South East Asia and Australia as well as in a collection of recombinant inbred lines (RILs) derived from indica Jasmine-type varieties, Australian long grain varieties (temperate japonica) and Basmati-type rice (Grp V). These compounds were detected through untargeted metabolite profiling by two-dimensional gas chromatography-time-of-flight mass spectrometry (GC × GC-TOF-MS), and their identity were confirmed by comparison with authentic standards analysed using gas chromatography mass spectrometry (GC-MS) and High Resolution GC × GC-TOF-MS (GC × GC HRT-4D). Genome-wide association analysis indicates that all compounds co-localised with a single quantitative trait locus (QTL) that harbours the FGR gene responsible for the production of GABA. Together, these data provide new insights into the production of 2AP, and evidence for understanding the pathway leading to the accumulation of aroma in fragrant rice.

The molecular structural features controlling stickiness in cooked rice, a major palatability determinant.

The stickiness of cooked rice is important for eating quality and consumer acceptance. The first molecular understanding of stickiness is obtained from leaching and molecular structural characteristics during cooking. Starch is a highly branched glucose polymer. We find (i) the molecular size of leached amylopectin is 30 times smaller than that of native amylopectin while (ii) that of leached amylose is 5 times smaller than that of native amylose, (iii) the chain-length distribution (CLD: the number of monomer units in a chain on the branched polymer) of leached amylopectin is similar to native amylopectin while (iv) the CLD of leached amylose is much narrower than that of the native amylose, and (v) mainly amylopectin, not amylose, leaches out of the granule and rice kernel during cooking. Stickiness is found to increase with decreasing amylose content in the whole grain, and, in the leachate, with increasing total amount of amylopectin, the proportion of short amylopectin chains, and amylopectin molecular size. Molecular adhesion mechanisms are put forward to explain this result. This molecular structural mechanism provides a new tool for rice breeders to select cultivars with desirable palatability by quantifying the components and molecular structure of leached starch.

Instrumental measurement of cooked rice texture by dynamic rheological testing and its relation to the fine structure of rice starch.

Increasing demands for better instrumental methods to evaluate cooked rice texture is driving innovations in rice texture research. This study characterized cooked rice texture by descriptive sensory analysis and two instrumental methods (texture profile analysis (TPA) and dynamic rheological testing) using a set of 18 varieties of rice with a wide range in amylose content (0-30%). The panellists' results indicated that hardness and stickiness were the two most discriminating attributes among 13 tested textural attributes. The consistency coefficient (K(*)) and loss tangent (tan δ) from a dynamic frequency sweep were used to compare with hardness and stickiness tested by TPA and sensory panellists, showing that using K(*) to express hardness, and tan δ to express stickiness, are both statistically and mechanistically meaningful. The instrumental method is rationalized in terms of starch structural differences between rices: a higher proportion of both amylose and long amylopectin branches with DP 70-100 causes a more elastic and less viscous texture, which is readily understood in terms of polymer dynamics in solution.

Identification of stable QTLs causing chalk in rice grains in nine environments.

KEY MESSAGE: A novel QTL cluster for chalkiness on Chr04 was identified using single environment analysis and joint mapping across 9 environments in Asia and South American. QTL NILs showed that each had a significant effect on chalk. Chalk in rice grains leads to a significant loss in the proportion of marketable grains in a harvested crop, leading to a significant financial loss to rice farmers and traders. To identify the genetic basis of chalkiness, two sets of recombinant inbred lines (RILs) derived from reciprocal crosses between Lemont and Teqing were used to find stable QTLs for chalkiness. The RILs were grown in seven locations in Asia and Latin American and in two controlled environments in phytotrons. A total of 32 (21) and 46 (22) QTLs for DEC and PGWC, most of them explaining more than 10% of phenotypic variation, were detected based on single environment analysis in T/L (L/T) population, respectively. Seven (2) and 7 (3) QTLs for DEC and PGWC were identified in the T/L (L/T) population using joined analysis across all environments, respectively. Six major QTLs clusters were found on five chromosomes: 1, 2, 4, 5 and 11. The biggest cluster at id4007289-RM252 on Chr04 was a novelty, including 16 and 4 QTLs detected by single environment analysis and joint mapping across all environments, respectively. The detected digenic epistatic QTLs explained up to 13% of phenotypic variation, suggesting that epistasis play an important role in the genetic control of chalkiness in rice. QTL NILs showed that each QTL cluster had a significant effect on chalk. These chromosomal regions could be targets for MAS, fine mapping and map-based cloning for low chalkiness breeding.

The importance of amylose and amylopectin fine structure for textural properties of cooked rice grains.

Statistically and causally meaningful relationships are established between starch molecular structure (the molecular distribution of branched starch and the chain length distribution of debranched starch) and texture (hardness and stickiness) of cooked rice grains. The amounts of amylose chains with degree of polymerization (DP) 100-20,000, and of long amylopectin chains, positively correlated with hardness, while amylopectin chains with DP<70 and amylose molecular size both showed negative correlations with hardness (p<0.05). There was also a significant negative correlation between stickiness and the amounts of long amylopectin chains (p<0.01). For rices with similar amylose content, the amount of amylose chains with DP 1000-2000 positively correlated with hardness while size negatively correlated with hardness (p<0.05). This indicates for the first time that, regardless of amylose content, rice varieties with smaller amylose molecular sizes and with higher proportions of long amylose chains have a harder texture after cooking.

Planthopper "adaptation" to resistant rice varieties: changes in amino acid composition over time.

The brown planthopper, Nilaparvata lugens, shows considerable geographic and temporal variability in its response to varieties of cultivated rice. N. lugens has repeatedly "adapted" to resistant rice varieties; however, the physiological changes underlying planthopper adaptation are poorly understood. Endosymbionts within planthoppers, such as yeast-like endosymbionts (YLS) could play a role as they produce essential amino acids for planthoppers. We used a full factorial study to determine how natal rice variety, exposed rice variety, YLS presence, and the number of reared generations affected nymphal development, planthopper total nitrogen content, and planthopper hydrolyzed amino acid profiles. Nymphal development was strongly influenced by a four-way interaction between the exposed rice variety, natal rice variety, number of reared generations, and YLS presence. While symbiosis improved nymphal performance in the 8th generation, it appeared to be a drain on nymphs in the 11th generation, when the aposymbiotic nymphs actually showed higher performance than the symbiotic nymphs. This suggests that the symbiotic relationship may be acting beneficially in one generation while acting as a drain during another generation. Aposymbiotic planthoppers reared for 11 generations had a higher proportional concentration of rare amino acids than those reared for 8 generations, indicating that the planthopper itself appears to improve its ability to acquire rare amino acids. Therefore, the change in amino acid composition of planthoppers suggests an underlying change in protein expression or amino acid metabolism over time.

Impact of down-regulation of starch branching enzyme IIb in rice by artificial microRNA- and hairpin RNA-mediated RNA silencing.

The inactivation of starch branching IIb (SBEIIb) in rice is traditionally associated with elevated apparent amylose content, increased peak gelatinization temperature, and a decreased proportion of short amylopectin branches. To elucidate further the structural and functional role of this enzyme, the phenotypic effects of down-regulating SBEIIb expression in rice endosperm were characterized by artificial microRNA (amiRNA) and hairpin RNA (hp-RNA) gene silencing. The results showed that RNA silencing of SBEIIb expression in rice grains did not affect the expression of other major isoforms of starch branching enzymes or starch synthases. Structural analyses of debranched starch showed that the doubling of apparent amylose content was not due to an increase in the relative proportion of amylose chains but instead was due to significantly elevated levels of long amylopectin and intermediate chains. Rices altered by the amiRNA technique produced a more extreme starch phenotype than those modified using the hp-RNA technique, with a greater increase in the proportion of long amylopectin and intermediate chains. The more pronounced starch structural modifications produced in the amiRNA lines led to more severe alterations in starch granule morphology and crystallinity as well as digestibility of freshly cooked grains. The potential role of attenuating SBEIIb expression in generating starch with elevated levels of resistant starch and lower glycaemic index is discussed.

Maintaining the yield of edible rice in a warming world.

High temperature increases the amount of chalk in rice (Oryza sativa L.) grains, which causes grains to break during polishing, lowering the amount of rice for consumption. Here, we examined the effect of elevated temperature on substrate supply to the panicle, the capacity of the panicle to produce edible grains, and underlying factors affecting yield of edible grain in two varieties. During grain-filling, substrate supply followed a bell shaped curve, and high temperature significantly shortened supply time. The rate of grain-filling did not change and paddy yield fell in both varieties. In high temperature, yield loss in IR8 was due to lighter grains relative to those grown in cool temperature, but in IR60, it was due to the early sacrifice of 30% of the spikelets. The yield of edible rice was zero for IR8 and ~60% for IR60 for the high temperature treatments, and 100% for IR60 and 70% for IR8 in the cool temperature. IR60 differs from IR8 in regulation of substrate supply, architecture of the panicles and the capacity of the panicles to alter sink size in response to the stress and these factors may be responsible for the difference in edible rice in the two varieties.

The origin and evolution of fragrance in rice (Oryza sativa L.).

Fragrance in the grain is one of the most highly valued grain quality traits in rice, yet the origin and evolution of the betaine aldehyde dehydrogenase gene (BADH2) underlying this trait remains unclear. In this study, we identify eight putatively nonfunctional alleles of the BADH2 gene and show that these alleles have distinct geographic and genetic origins. Despite multiple origins of the fragrance trait, a single allele, badh2.1, is the predominant allele in virtually all fragrant rice varieties today, including the widely recognized Basmati and Jasmine types. Haplotype analysis allowed us to establish a single origin of the badh2.1 allele within the Japonica varietal group and demonstrate the introgression of this allele from Japonica to Indica. Basmati-like accessions were nearly identical to the ancestral Japonica haplotype across a 5.3-Mb region flanking BADH2 regardless of their fragrance phenotype, demonstrating a close evolutionary relationship between Basmati varieties and the Japonica gene pool. These results clarify the relationships among fragrant rice varieties and challenge the traditional assumption that the fragrance trait arose in the Indica varietal group.

Not just a grain of rice: the quest for quality.

A better understanding of the factors that contribute to the overall grain quality of rice (Oryza sativa) will lay the foundation for developing new breeding and selection strategies for combining high quality, with high yield. This is necessary to meet the growing global demand for high quality rice while offering producing countries additional opportunities for generating higher export revenues. Several recent developments in genetics, genomics, metabolomics and phenomics are enhancing our understanding of the pathways that determine several quality traits. New research strategies, as well as access to the draft of the rice genome, will not only advance our understanding of the molecular mechanisms that lead to quality rice but will also pave the way for efficient and targeted grain improvement.

Is there a second fragrance gene in rice?

Aromatic rice is highly prized by most rice consumers, and many countries cultivate traditional and improved aromatic varieties. 2-Acetyl-1-pyrroline (2AP) is the major aromatic compound in rice, and is believed to accumulate because of an eight-base-pair (8-bp) deletion in an allele at the fragrance locus. In this study, 2AP was quantified and the presence or absence of the fragrance allele (fgr) was determined in 464 samples of traditional varieties of rice from the T.T. Chang Genetic Resources Centre at the International Rice Research Institute. It was shown that a number of aromatic varieties, primarily from South and South-East Asia, do not carry the 8-bp deletion, but 2AP was identified in both raw and cooked rice of these varieties. We suggest that the 8-bp deletion in fgr is not the only cause of aroma, and at least one other mutation drives the accumulation of 2AP. The amount of 2AP in most uniform fgr genotypes was not significantly different from that in aromatic nfgr genotypes, but several fgr genotypes, primarily from South Asia, reproducibly accumulated exceptionally large amounts of 2AP. We suggest that the mutation leading to 2AP in aromatic nfgr varieties possibly originated several times and, through either domestication or evolution, the fgr gene and other alleles leading to 2AP have combined in South Asia, leading to several highly aromatic traditional varieties. The identification of multiple mutations for 2AP will enable rice breeding programmes to select actively for multiple genetic sources of 2AP, leading to the development of highly aromatic and, consequently, high-quality varieties of rice.

Plant metabolomics and its potential application for human nutrition.

With the growing interest in the use of metabolomic technologies for a wide range of biological targets, food applications related to nutrition and quality are rapidly emerging. Metabolomics offers us the opportunity to gain deeper insights into, and have better control of, the fundamental biochemical basis of the things we eat. So doing will help us to design modified breeding programmes aimed at better quality produce; optimised food processing strategies and ultimately, improved (micro)nutrient bioavailability and bioefficacy. A better understanding of the pathways responsible for the biosynthesis of nutritionally relevant metabolites is key to gaining more effective control of the absence/level of presence of such components in our food. Applications of metabolomic technologies in both applied and fundamental science strategies are therefore growing rapidly in popularity. Currently, the world has two highly contrasting nutrition-related problems--over-consumption and under-nourishment. Dramatic increases in the occurrence of overweight individuals and obesity in developed countries are in staggering contrast to the still-familiar images of extreme malnutrition in many parts of the developing world. Both problems require a modified food supply, achieved through highly contrasting routes. For each, metabolomics has a future role to play and this review shall deal with this key dichotomy and illustrate where metabolomics may have a future part to play. In this short overview, attention is given to how the various technologies have already been exploited in a plant-based food context related to key issues such as biofortification, bioprotectants and the general link between food composition and human health. Research on key crops such as rice and tomato are used as illustration of potentially broader application across crop species. Although the focus is clearly on food supply, some attention is given to the complementary field of research, nutrigenomics, where similar technologies are being applied to understand nutrition better from the human side.

Gelatinization temperature of rice explained by polymorphisms in starch synthase.

The cooking quality of rice is associated with the starch gelatinization temperature (GT). Rice genotypes with low GT have probably been selected for their cooking quality by humans during domestication. We now report polymorphisms in starch synthase IIa (SSIIa) that explain the variation in rice starch GT. Sequence analysis of the eight exons of SSIIa identified significant polymorphism in only exon 8. These single nucleotide polymorphisms (SNPs) were determined in 70 diverse genotypes of rice. Two SNPs could classify all 70 genotypes into either high GT or low GT types which differed in GT by 8 degrees C. 'A' rather than 'G' at base 2412 determined whether a methionine or valine was present at the corresponding amino acid residue in SSIIa, whilst two adjacent SNPs at bases 2543 and 2544 coded for either leucine (GC) or phenylalanine (TT). Rice varieties with high GT starch had a combination of valine and leucine at these residues. In contrast, rice varieties with low GT starch had a combination of either methionine and leucine or valine and phenylalanine at these same residues. At least two distinct polymorphisms have apparently been selected for their desirable cooking qualities in the domestication of rice.

Improved methods for the structural analysis of the amylose-rich fraction from rice flour.

Cooking and sensory properties of rice are largely determined by the amylose content and structure. For relationships between functional and structural properties, a more accurate method to determine the structure of amylose is required. Here we calibrate size exclusion chromatography (SEC) columns, using Mark-Houwink parameters for linear starch and pullulan standards, to obtain the true molecular weight distribution of linear starch. When the molecular weight distribution is reported relative to pullulan, rather than the actual molecular weight which is readily obtained from universal calibration, it is seen that the molecular weights of longer amylose chains are greatly underestimated. We validate the SEC method to enable the measurement of the hydrodynamic volume distribution of the starch by examining reproducibility and recovery. Analysis of the starch in the sample pre- and post-SEC shows that 20% of the carbohydrate is not recovered. Comparison of the weight-average degree of polymerization, X(w), of (undebranched) starch of pre- and post-SEC is made using iodine binding as well as Berry plots of data from multi-angle laser light scattering (MALLS). These both show that current SEC techniques for starch analysis lead to significant loss of high molecular weight material. Indeed, for the systems studied here, the values for X(w) after SEC are about three times lower than those before SEC. Iodine-starch complexes of pre- and post-SEC samples reveals that the SEC techniques give reliable data for the amylose fraction but not for amylopectin. We address reports in the literature suggesting that the conventional isoamylase method for debranching starch would lead to incomplete debranching and thus incorrect molecular weight distributions. However, it is shown using (1)H NMR that isoamylase can completely debranch the amylose (to within the detection limit of 0.5%), and by SEC that successive incubation with isoamylase, alpha-amylase, and beta-amylase can degrade the amylose-rich fraction completely to maltose. We develop a method to obtain a hot water soluble fraction (HWSF), rich in undamaged amylose molecules, directly from rice flour, avoiding the structural degradation of previous techniques. With appropriate sample handling, the formation of associations between starch chains is minimized. With the combination of calibrated and validated SEC methods, and an improved extraction of amylose from rice, the X(w) for both HWSF and debranched HWSF are found to be much larger than has previously been reported.

Fluorescence recovery after photobleaching as a probe of diffusion in starch systems.

The diffusion coefficients of dextran probes of various molecular weights in starch solutions over a wide concentration range were carried out using fluorescent recovery after photobleaching (FRAP), combined with a confocal microscope and tracer probe diffusion. The technique is simple to implement and can be carried out using increasingly common microscopy apparatus, giving access to a wide variety of new structural and kinetic information. The data can be rationalized in terms of the effects of probe molecular weight and on matrix starch concentration and structure. This provides a new tool to investigate the behavior of systems where starch is an ingredient that contributes to the processing and textural properties of food.

Mechanistic information from analysis of molecular weight distributions of starch.

A methodology is developed for interpreting the molecular weight distributions of debranched amylopectin, based on techniques developed for quantitatively and qualitatively finding mechanistic information from the molecular weight distributions of synthetic polymers. If the only events occurring are random chain growth and stoppage (i.e., the rates are independent of degree of polymerization over the range in question), then the number of chains of degree of polymerization N, P(N), is linear in ln P(N) with a negative slope, where the slope gives the ratio of the stoppage and growth rates. This starting point suggests that mechanistic inferences can be made from a plot of lnP against N. Application to capillary electrophoresis data for the P(N) of debranched starch from across the major taxa, from bacteria (Escherichia coli), green algae (Chlamydomonas reinhardtii), mammals (Bos), and flowering plants (Oryza sativa, rice; Zea mays, maize; Triticum aestivum, wheat; Hordeum vulgare, barley; and Solanum tuberosum, potato), gives insights into the biosynthetic pathways, showing the differences and similarities of the alpha-1,4-glucans produced by the various species. Four characteristic regions for storage starch from the higher plants are revealed: (1) an initial increasing region corresponding to the formation of new branches, (2) a linear ln P region with negative slope, indicating random growth and stoppage, (3) a region corresponding to the formation of the crystalline lamellae and subsequent elongation of chains, and (4) a second linear ln P with negative slope region. Each region can be assigned to specific enzymatic processes in starch synthesis, including determining the ranges of degrees of polymerization which are subject to random and nonrandom processes.