Tong, C 2016, 'Lysophospholipids in rice (Oryza sativa L.): identification, genetic diversity and their relations to grain qualities', PhD thesis, Southern Cross University, Lismore, NSW.
Copyright C Tong 2016
At present, functional lipids in rice have been paid increasing attention as their importance in rice quality and health benefit being clearly recognized. However, phospholipids (PLs) in rice grain are still a class of underutilised lipid as technological restriction which shows important functions and good nutrition. The lyso-forms of PLs (lysophospholipids, LPLs) in rice endosperm often combine with starch to form an amylose-lipid complex, significantly affecting starch qualities. Data of rice endosperm LPLs generated previously describe the range of variation for LPLs composition, but lack a comprehensive composition profile and poor link to genotype × environment and phosphorus metabolism. Improvement of grain quality and promotion of rice consumption could be greatly enhanced provided that the underlying control and regulatory mechanism of rice LPLs are elucidated. Therefore, it is necessary to establish a reliable and quick method to extract and analyse different LPLs components, which may facilitate dissecting the genetic variation and its underlying genetic controls. Detailed research on rice LPLs may also contribute to developing new cultivars with added values suited to consumer demands and nutritional food security. In the present study, a rapid and effective approach was developed to extract and quantify rice endosperm LPLs. The genetic diversity of rice individual LPLs, contributions of native LPLs to rice eating quality and phosphorus metabolism have been clarified. The main results are summarized as follows:
1. A single-step aqueous n-propanol extraction combined with liquid chromatography mass spectrometry (HPLC-MS) was developed and employed to analyse starch LPLs in white rice. It has been showed that different grinding methods have little effect on the final LPL detection, and the simple single-step extraction with 75% n-propanol (8 mL/0.15 g) heated at 100oC for two hours was as effective as an onerous multi-step extraction method. Ten major LPLs in rice starch were simultaneously quantified within 15 minutes by an optimized HPLC-MS method. This method enables total and individual starch LPLs analysis of a large number of rice samples at little cost and could be applied to starch LPLs in other cereals.
2. Levels of specific LPLs in 20 accessions differed significantly among rice genotypes, demonstrating there is a wide diversity in starch LPLs in rice grain. The main LPL components were components were Lysophosphatidylcholine (LPC) 16:0 (ranging from 3009.7-4697.8 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 only accounted for 0.7-38.9% and 1.8-6.6% of the total variance, respectively. Positive relations was revealed by correlation analysis between LPC14:0 and LPE14:0, LPC16:0 and LPE16:0, LPC18:1 and LPE18:1, LPC18:2 and LPE18:2, LPC18:3 and LPE18:3, possibly indicating the biosynthesis of fatty acids was prior to the combination of a certain proportion of aminoalcohols in the PL biosynthesis. It provided an insight into the possible LPL biosynthesis pathway in plants. Hierarchical cluster analysis suggested the 20 rice accessions could be classified into three groups while principal component analysis also identified three groups with the first two components explaining 57.7 and 16.2% of the total variance. 22 quantitative trait loci (QTLs) (P<0.001) distributed on chromosomes except Chr.3 and Chr.7 via association test were identified, providing an abundant and necessary information for next starch LPLs metabolic and genetic architecture analysis in rice.
3. Rice flour pasting and thermal properties are crucial assessment indices for rice eating and cooking quality and industry applications. The diversity of rice flour pasting and thermal properties and their relationship with individual LPL components native in rice endosperm were demonstrated. 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, it was been found that, irrespective of apparent amylose content, CPV and individual LPLs were positively correlated, while BD, CS and other individual LPLs were negatively correlated. These results suggested natural individual LPLs could contribute to rice flour pasting and thermal properties, either independently or in combination with amylose.
4. It is known that improvement of phosphorus (P) acquisition and utilization efficiency is contributed to rice nutrients and qualities. However, the molecular mechanism of P transportation and distribution in rice seeds are still not understood. Here, with the use of 5 different low phytic acids (lpa) rice mutants, the changes in the main P-contained compounds, i.e. phytic acid (PA), LPL, and inorganic P (Pi) in rice seed were studied. The lpa mutants showed a significant decrease in PA and phytate-phosphorus (PA-P) concentration while increase in Pi concentration. Moreover, defects in the OsST and OsMIK genes resulted in a great reduction of specific LPLs components and LPL-phosphorus (LPL-P) contents in rice grain. Contrarily, defective OsMRP5 and Os2-PGK genes led to a significant increase of individual LPLs components except LPC14:0. To validate the effect of Os2-PGK gene on the LPLs accumulation, nine inbreeding lines derived from the cross KBNT-lpa and Jiahe 218 were used to investigate the LPLs contents. It was confirmed that the breeding lines with lpa mutation still had higher total LPL concentration than the normal lines. This study suggests a relationship between PA and PL biosynthesis in rice grain possibly derived from the Pi allocation and re-distribution in rice seeds.