SWATH-MS-Based Proteomics Reveals the Regulatory Metabolism of Amaryllidaceae Alkaloids in Three Lycoris Species.

Alkaloids are a class of nitrogen-containing alkaline organic compounds found in nature, with significant biological activity, and are also important active ingredients in Chinese herbal medicine. Amaryllidaceae plants are rich in alkaloids, among which galanthamine, lycorine, and lycoramine are representative. Since the difficulty and high cost of synthesizing alkaloids have been the major obstacles in industrial production, particularly the molecular mechanism underlying alkaloid biosynthesis is largely unknown. Here, we determined the alkaloid content in Lycoris longituba, Lycoris incarnata, and Lycoris sprengeri, and performed a SWATH-MS (sequential window acquisition of all theoretical mass spectra)-based quantitative approach to detect proteome changes in the three Lycoris. A total of 2193 proteins were quantified, of which 720 proteins showed a difference in abundance between Ll and Ls, and 463 proteins showed a difference in abundance between Li and Ls. KEGG enrichment analysis revealed that differentially expressed proteins are distributed in specific biological processes including amino acid metabolism, starch, and sucrose metabolism, implicating a supportive role for Amaryllidaceae alkaloids metabolism in Lycoris. Furthermore, several key genes collectively known as OMT and NMT were identified, which are probably responsible for galanthamine biosynthesis. Interestingly, RNA processing-related proteins were also abundantly detected in alkaloid-rich Ll, suggesting that posttranscriptional regulation such as alternative splicing may contribute to the biosynthesis of Amaryllidaceae alkaloids. Taken together, our SWATH-MS-based proteomic investigation may reveal the differences in alkaloid contents at the protein levels, providing a comprehensive proteome reference for the regulatory metabolism of Amaryllidaceae alkaloids.

Study on the homology of the genomes of tetraploid Asiatic lilies (Lilium) using FISH.

Asiatic lily cultivars, bred by hybridization and (or) chromosome doubling of species of section Sinomartagon of Lilium, are diploid, triploid, or tetraploid, but the homology of the genomes among species of section Sinomartagon and Asiatic lilies remains unclear. In the present research, two tetraploid Asiatic cultivars were analyzed, using 45S rDNA as probe, for their FISH karyotypes and their chromosomal association, anaphase I, telophase II, and pollen viability were surveyed to assess the multivalent segregation. Chromosomal assortment of six progenies of the two tetraploid cultivars were also investigated. The results showed that the tetraploid cultivars had similar FISH karyotypes, they predominantly formed multivalents, and these were equally separated because their anaphase I, telophase II, and pollen viability were similar to those of diploid species. Apart from minor variations, FISH karyotypes of progenies were similar to each other and to their parents. Based on these results and considering the high crossability among species of section Sinomartagon and (or) Asiatic lilies, we concluded that species of section Sinomartagon and their resulting cultivars share a common genome; thus, polyploidy Asiatic lilies are autopolyploid.

Development of Cymbidium ensifolium genic-SSR markers and their utility in genetic diversity and population structure analysis in cymbidiums.

BACKGROUND: Cymbidium is a genus of 68 species in the orchid family, with extremely high ornamental value. Marker-assisted selection has proven to be an effective strategy in accelerating plant breeding for many plant species. Analysis of cymbidiums genetic background by molecular markers can be of great value in assisting parental selection and breeding strategy design, however, in plants such as cymbidiums limited genomic resources exist. In order to obtain efficient markers, we deep sequenced the C. ensifolium transcriptome to identify simple sequence repeats derived from gene regions (genic-SSR). RESULT: The 7,936 genic-SSR markers were identified. A total of 80 genic-SSRs were selected, and primers were designed according to their flanking sequences. Of the 80 genic-SSR primer sets, 62 were amplified in C. ensifolium successfully, and 55 showed polymorphism when cross-tested among 9 Cymbidium species comprising 59 accessions. Unigenes containing the 62 genic-SSRs were searched against Non-redundant (Nr), Gene Ontology database (GO), eukaryotic orthologous groups (KOGs) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The search resulted in 53 matching Nr sequences, of which 39 had GO terms, 18 were assigned to KOGs, and 15 were annotated with KEGG. Genetic diversity and population structure were analyzed based on 55 polymorphic genic-SSR data among 59 accessions. The genetic distance averaged 0.3911, ranging from 0.016 to 0.618. The polymorphic index content (PIC) of 55 polymorphic markers averaged 0.407, ranging from 0.033 to 0.863. A model-based clustering analysis revealed that five genetic groups existed in the collection. Accessions from the same species were typically grouped together; however, C. goeringii accessions did not always form a separate cluster, suggesting that C. goeringii accessions were polyphyletic. CONCLUSION: The genic-SSR identified in this study constitute a set of markers that can be applied across multiple Cymbidium species and used for the evaluation of genetic relationships as well as qualitative and quantitative trait mapping studies. Genic-SSR's coupled with the functional annotations provided by the unigenes will aid in mapping candidate genes of specific function.

"Structure-function" analysis using starches isolated from Lycoris chinensis bulbs of different developmental stages.

In this study, Lycoris chinensis bulbs of four developmental stages were compared for starch characteristics. Based on correlation analysis and hierarchical cluster analysis, the relationships among 36 traits were discussed. Compared to commonly consumed starches, L. chinensis starch had higher amylose content (33.4-43.2 %) and weight-average molar mass (36410-82,781 kDa), lower gelatinization temperature (61.8-68.1 °C), gel hardness (19.0-39.5 g) and viscosities. Among developmental stages, starches varied significantly in characteristics. As compared to juvenile stage (S1), mature bulbs (S4) had higher amylose content, lower gelatinization temperature, weight-average molar mass and degree of polymorphism. Correlation analysis revealed that the molecular weight-related traits had significantly positive correlations to gelatinization temperature (Tp, p < 0.05), positive but weak correlations to traits of particle size distribution, significantly negative correlations to AAC and many parameters of viscosity properties (p < 0.05). Based on the results of correlation analysis and hierarchical cluster analysis, the 36 traits of starch characteristics were proposed to be divided into three groups: particle size-related traits, molecular weight-related traits and AAC-related traits. The information presented in the current study are useful for future studies on starches of Lycoris and other bulb species, and instructive for future studies in investigating the "Structure-Function" relationship in starch.

Characterization of starch extracted from seeds of Cycas revoluta.

Introduction: Starch is major component in the big seeds of Cycas revoluta, however the characteristics of Cycas revoluta remain unknown. Methods: In this study, the physicochemical and structural properties of two starch samples extracted from Cycad revoluta seeds were systematically investigated, using various techniques. Results: The amylose contents of the two samples were 34.3 % and 35.5%, respectively. The spherical-truncated shaped starch granules possessed A-type crystallinity, and had an average diameter less than 15 µm. Compared to most commonly consumed cereal and potato starch, Cycad revoluta starch showed distinctive characteristics. For physicochemical properties, in the process of gelatinization, the Cycad revoluta starch showed similar viscosity profile to starches of some potato varieties, but Cycad revoluta starch had higher gelatinization temperature. Upon cooling, Cycad revoluta starch formed harder gels than rice starch. For structure, the molecular weight (indexed by Mw, Mn and Rz values), branching degree and the branch chain length distribution were determined. Discussion: The results suggested that Cycad revoluta starch were different in structure from the main-stream starches. Notable differences in some starch traits between the two samples were recorded, which could be attributed to environmental factors. In general, this study provides useful information on the utilization of Cycad revoluta starch in both food and non-food industries.

Structural and physicochemical properties of bracken fern (Pteridium aquilinum) starch.

Introduction: Bracken fern (Pteridium aquilinum) starch is a non-mainstream, litter-researched starch, thus the starch characteristics remain largely unknown. Methods: The structural and physicochemical properties of two bracken starches were systematically investigated, by use of various techniques that routinely applied in starch analysis. Results and Discussion: The starches had amylose contents of 22.6 and 24.7%, respectively. The starch granules possessed C-type polymorph with D (4,3) ranging from 18.6 to 24.5 µm. During gelatinization event, the bracken starches showed lower viscosity than typical for rice starch, and lower gelatinization temperature than typical for cereal starches. After gelatinization event, bracken starches formed much softer and sticky gel than rice and potato starch. The molecular weight and branching degree (indexed by Mw, Mn and Rz values) of bracken starches were much higher than starches of many other sources. The branch chain length distributions showed that the bracken starches were structurally similar to some rice varieties (e. g. BP033, Beihan 1#), as reflected by proportions of A, B1, B2, and B3 chains. Notable differences in some starch traits between the two bracken starches were recorded, e. g. amylose content, gel hardness, gelatinization temperature and traits of structural properties. This study provides useful information on the utilization of bracken starch in both food and non-food industries.

Starch properties of high and low amylose proso millet (Panicum miliaceum L.) genotypes are differentially affected by varying salt and pH.

Variation in salt content and in pH are common in starch-based foods and can affect starch properties and final product texture. Fifteen accessions of proso millet starch with diverse amylose content were selected to investigate single factor and interaction effects of pH and NaCl on thermal, pasting, and textural properties. Pasting properties and gelatinization temperatures were markedly altered by salt addition. Changes in pH only had substantial effects on ΔH, but other properties were generally stable under different pH conditions. From two-way ANOVA, interactive effects of salt and pH were found to affect ΔH. The response of starch of different genotypes in terms of thermal and pasting properties differed under the same pH and salinity conditions. The reason is likely that ions in the starch-water system performed the roles of both reducing water activity and building of hydrogen bonds, which will have opposite effects on starch gelatinization.

Diversity analysis of starch physicochemical properties in 95 proso millet (Panicum miliaceum L.) accessions.

In this study, 95 accessions of proso millet (Panicum miliaceum L.) were characterized for starch physicochemical properties, including apparent amylose content (AAC), gel textural properties, Rapid Visco Analyzer (RVA) pasting viscosity properties, thermal and retrogradation properties. Based on genotypic data, the genetic diversity and inter-relationship of these starch traits were analyzed. Diverse starch quality was found, for example, AAC ranged from 0 to 32.3%, gelatinization temperature (GT) varied from 71.5 to 79.0 ℃, and RVA profile showed distinct patterns among proso millet of different AAC types. Interestingly, high AAC proso millet usually had GT lower than that of low AAC proso millet, which is different from the findings in rice starch. Many starch traits were significantly correlated and most of the 18 tested traits could be classified as either AAC-related traits or GT-related traits. In summary, the information presented here will be useful for further development of proso millet products.

Characterization of morphology and physicochemical properties of native starches isolated from 12 Lycoris species.

In this study, starch was isolated from 13 genotypes of 12 Lycoris species, and the morphology, granule size distribution and physicochemical properties, including apparent amylose content (AAC), Rapid Visco Analyzer (RVA) pasting properties, textural properties, thermal and retrogradation properties were characterized. The majority of starch granules of the 13 Lycoris genotypes were oval in shape, and granule size followed a normal distribution with a mean diameter of 20-30 µm. Contrary to previously published findings, the XRD results revealed that lycoris starches had either C-type or CA-type crystallinity. All lycoris starches showed high AAC varying from 25.6% to 32.7%, and low gelatinization temperature (GT) ranging from 58.8 to 69.7℃. Inter-relationships among 18 starch quality traits were analyzed based on correlation analysis. The present study provides information on lycoris starch characteristics which should serve as a useful guide for later studies on lycoris starch utilization in food and non-food industries.

Genetic variation in starch physicochemical properties of Chinese foxtail millet (Setaria italica Beauv.).

In this study, 34 Chinese foxtail millet germplasm accessions (cultivars and breeding lines) were characterized for starch physicochemical properties including apparent amylose content, and gel textural, RVA pasting, thermal and retrogradation properties. The relationships among 21 starch parameters were discussed based on correlation analysis and hierarchical cluster analysis. Generally, this set of germplasm presented a surprisingly low genetic diversity in starch properties. The apparent amylose content ranged from 18.3% to 25.3% with a CV value of 5.0%, and gelatinization temperature ranged from 72.1 to 73.8 °C with a CV value of 0.5%. AAC was negatively correlated only with ΔHg (p < 0.01) while GT only had a strong relationship with To and Tc of thermal properties (p < 0.001). The 21 measured starch parameters could be separated into three groups based on cluster analysis. This work should provide useful information for breeding improved quality foxtail millet.

Genotypic diversity and environmental stability of starch physicochemical properties in the USDA rice mini-core collection.

The USDA rice mini-core collection consists of 217 accessions representative of a world-wide germplam bank. We investigated its genotypic diversity in starch physicochemical properties and the effects of genotype, environment and G×E interaction in this study. High genotypic diversity was found in all 18 measured starch quality traits in the mini-core rice in two location-years in China. Genotype, environment and G×E effects on these traits were analysed using 115 common accessions successfully produced in both environments. Thermal properties (To, Tp and Tc) were very stable whereas most other traits differed significantly between environments. However, when these accessions were divided into five subgroups based on amylose content, environment was found to have differential effects. G×E interaction also played a significant role in determining the starch traits. These findings will provide guidance for selection from the diverse genotypes in the USDA mini-core collection for cultivation and for developing cultivars with desired cooking and eating quality.

Association Analysis of Markers Derived from Starch Biosynthesis Related Genes with Starch Physicochemical Properties in the USDA Rice Mini-Core Collection.

Rice eating and cooking quality is largely determined by starch physicochemical properties. The diverse accessions in the USDA rice mini-core collection (URMC) facilitate extensive association analysis of starch physicochemical properties with molecular markers specific to starch biosynthesis related genes. To identify significant trait-marker associations that can be utilized in rice breeding programs for improved starch quality, we conducted two association analyses between 26 molecular markers derived from starch biosynthesis related genes and 18 parameters measured of starch physicochemical properties in two sets of the mini-core accessions successfully grown in two environments in China. Many significant trait-marker associations (P < 0.001) were detected in both association analyses. Five markers of Waxy gene, including the (CT)n repeats, the G/T SNP of intron 1, the 23 bp sequence duplication (InDel) of exon 2, the A/C SNP of exon 6, and the C/T SNP of exon 10, were found to be primarily associated with starch traits related to apparent amylose content (AAC), and two markers targeting the 4,329-4,330 bp GC/TT SNPs and 4,198 bp G/A SNP of SSIIa gene were mainly associated with traits related to gelatinization temperature (GT). Two new haplotypes were found in the mini-core collection based on the combinations of the 23 bp InDel and three SNPs (G/T of intron 1, A/C of exon 6, and C/T of exon 10) of Waxy gene. Furthermore, our analyses indicated that the (CT)n polymorphisms of Waxy gene had a non-negligible effect on AAC related traits, as evidenced by significant variation in AAC related traits among rice accessions with the same Waxy SNPs but different (CT) n repeats. As the five Waxy markers and the two SSIIa markers showed consistent major effects on starch quality traits across studies, these markers should have priority for utilization in marker-assisted breeding.