Detection and subcellular localization of dehydrin-like proteins in quinoa (Chenopodium quinoa Willd.) embryos

The aim of this study was to characterize the dehydrin content in mature embryos of two quinoa cultivars, Sajama and Baer La Unión. Cultivar Sajama grows at 3600-4000 m altitude and is adapted to the very arid conditions characteristic of the salty soils of the Bolivian Altiplano, with less than 250 mm of annual rain and a minimum temperature of -1 degrees C. Cultivar Baer La Unión grows at sea-level regions of central Chile and is adapted to more humid conditions (800 to 1500 mm of annual rain), fertile soils, and temperatures above 5 degrees C. Western blot analysis of embryo tissues from plants growing under controlled greenhouse conditions clearly revealed the presence of several dehydrin bands (at molecular masses of approximately 30, 32, 50, and 55 kDa), which were common to both cultivars, although the amount of the 30 and 32 kDa bands differed. Nevertheless, when grains originated from their respective natural environments, three extra bands (at molecular masses of approximately 34, 38, and 40 kDa), which were hardly visible in Sajama, and another weak band (at a molecular mass of approximately 28 kDa) were evident in Baer La Unión. In situ immunolocalization microscopy detected dehydrin-like proteins in all axis and cotyledon tissues. At the subcellular level, dehydrins were detected in the plasma membrane, cytoplasm and nucleus. In the cytoplasm, dehydrins were found associated with mitochondria, rough endoplasmic reticulum cisternae, and proplastid membranes. The presence of dehydrins was also recognized in the matrix of protein bodies. In the nucleus, dehydrins were associated with the euchromatin. Upon examining dehydrin composition and subcellular localization in two quinoa cultivars belonging to highly contrasting environments, we conclude that most dehydrins detected here were constitutive components of the quinoa seed developmental program, but some of them (specially the 34, 38, and 40 kDa bands) may reflect quantitative molecular differences associated with the adaptation of both cultivars to contrasting environmental conditions.     

藜麦及其资源开发利用

藜麦Chenopodium quinoa Willd.英文名:quinoa,原产于南美洲安第斯山区,是印加土著居民的主要传统食物,至今已有5 000~7 000多年的利用和种植历史。古代印加人将它称之为"粮食之母"。藜麦在20世纪80年代,被美国宇航局用于宇航员的太空食品。联合国粮农组织认为藜麦是唯一的单一植物即可满足人体基本营养需求的食物,正式推荐藜麦为最适宜人类的完美的全营养食品。本文对藜麦的植物形态、生态特性、营养价值以及在我国种植展望作了综合报道。     

Triterpene saponins from Chenopodium quinoa Willd

Twenty triterpene saponins (1-20) have been isolated from different parts of Chenopodium quinoa (flowers, fruits, seed coats, and seeds) and their structures have been elucidated by analysis of chemical and spectroscopic data including 1D- and 2D-NMR. Four compounds (1-4) were identified: 3beta-[(O-beta-d-glucopyranosyl-(1-->3)-alpha-l-arabinopyranosyl)oxy]-23-oxo-olean-12-en-28-oic acid beta-d-glucopyranoside (1), 3beta-[(O-beta-d-glucopyranosyl-(1-->3)-alpha-l-arabinopyranosyl)oxy]-27-oxo-olean-12-en-28-oic acid beta-d-glucopyranoside (2), 3-O-alpha-l-arabinopyranosyl serjanic acid 28-O-beta-d-glucopyranosyl ester (3), and 3-O-beta-d-glucuronopyranosyl serjanic acid 28-O-beta-d-glucopyranosyl ester (4). The following known compounds have not previously been reported as saponin constituents from the flowers and the fruits of this plant: two bidesmosides of serjanic acid (5,6), four bidesmosides of oleanolic acid (7-10), five bidesmosides of phytolaccagenic acid (11-15), four bidesmosides of hederagenin (16-19), and one bidesmoside of 3beta,23,30-trihydroxy olean-12-en-28-oic acid (20). The cytotoxicity of these saponins and their aglycones was tested in HeLa cells. Induction of apoptosis in Caco-2 cells by bidesmosidic saponins 1-4 and their aglycones I-III was determined by flow cytometric DNA analysis. The saponins with an aldehyde group were most active. The relationships between structure and cytotoxic activity of saponins and their aglycones are discussed.     

Salt tolerance mechanisms in quinoa (Chenopodium quinoa Willd.)

In the face of diminishing fresh water resources and increasing soil salinisation it is relevant to evaluate the potential of halophytic plant species to be cultivated in arid and semi-arid regions, where the productivity of most crop plants is markedly affected. Quinoa is a facultative halophytic plant species with the most tolerant varieties being able to cope with salinity levels as high as those present in sea water. This characteristic has aroused the interest in the species, and a number of studies have been performed with the aim of elucidating the mechanisms used by quinoa in order to cope with high salt levels in the soil at various stages of plant development. In quinoa key traits seem to be an efficient control of Na⁺ sequestration in leaf vacuoles, xylem Na⁺ loading, higher ROS tolerance, better K⁺ retention, and an efficient control over stomatal development and aperture. The purpose of this review is to give an overview on the existing knowledge of the salt tolerance of quinoa, to discuss the potential of quinoa for cultivation in salt-affected regions and as a basis for further research in the field of plant salt tolerance.     

Distribution of minerals in quinoa (Chenopodium quinoa Willd.) seeds

The distribution of minerals in quinoa (Chenopodium quinoa Willd.) seed was examined using energy dispersive X-ray microanalysis (EDX) in combination with scanning electron microscopy (SEM). Phosphorus, K, and Mg coincided in localization in embryonic tissue. Since phytin globoids have been known to localize in protein bodies in embryonic cells of quinoa seed, it is thought that P is attributed to phytic acid and that K and Mg form to phytate. Calcium and K were present in the pericarp, where the cell wall is thickly developed, suggesting that these minerals are associated with pectin. Sulfur occurred in embryonic tissues, which would be derived from sulfur amino acid residues of storage proteins concentrated in the tissues. Abrasion of quinoa seeds resulted particularly in decrease in Ca content.     

Effect of salinity on composition, viability and germination of seeds of Chenopodium quinoa Willd

Salinity influences plant growth, seed yield and seed quality even of halophytic crops such as Chenopodium quinoa. Plant growth, total seed yield, number of seeds, fresh weight and dry weight of seeds, were all significantly reduced in the presence of salinity. Only at high salinity did the content of proteins (as well as total N) increase significantly in the seeds whereas the content of total carbohydrates (as well as total C) decrease. Aside from that the capacity for germination was diminished by a reduced seed size and a disproportionate reduction of the volume of the perisperm. However, the reduced capacity seemed to be compensated by an accelerated germination owing to high Na and Cl concentrations leading to a low water potential in the walls of the plant ovary. At high salinity the passage of NaCl to the seed interior was hindered by the seed cover. There was an obvious gradient between potentially toxic (Na and Cl) and essentially needed elements (K, Mg, Ca, P and S) across the seed coat of salt treated plants and also a significant change of the distribution of elements in the embryo. The results indicate a highly protected seed interior leading to a high salinity resistance of quinoa seeds.     

Breeding quinoa (Chenopodium quinoa Willd.): potential and perspectives

Quinoa (Chenopodium quinoa Willd.) originated in the Andean region of South America; this species is associated with exceptional grain nutritional quality and is highly valued for its ability to tolerate abiotic stresses. However, its introduction outside the Andes has yet to take off on a large scale. In the Andes, quinoa has until recently been marginally grown by small-scale Andean farmers, leading to minor interest in the crop from urban consumers and the industry. Quinoa breeding programs were not initiated until the 1960s in the Andes, and elsewhere from the 1970s onwards. New molecular tools available for the existing quinoa breeding programs, which are critically examined in this review, will enable us to tackle the limitations of allotetraploidy and genetic specificities. The recent progress, together with the declaration of “The International Year of the Quinoa” by the Food and Agriculture Organization of the United Nations, anticipates a bright future for this ancient species.     

Biological activities and chemistry of saponins from Chenopodium quinoa Willd.

Chenopodium quinoa Willd. is a valuable food source which has gained importance in many countries of the world. The plant contains various bitter-tasting saponins which present an important antinutritional factor. Various triterpene saponins have been reported in C. quinoa including both monodesmosidic and bidesmosidic triterpene saponins of oleanolic acid, hederagenin, phytolaccagenic acid, and serjanic acid as the major aglycones and other aglycones as 3β-hydroxy-23-oxo-olean-12-en-28-oic acid, 3β-hydroxy-27-oxo-olean-12-en-28-oic acid, and 3β, 23α, 30β-trihydroxy-olean-12-en-28-oic acid. A tridesmosidic saponin of hederagenin has also been reported. Here we review the occurrence, analysis, chemical structures, and biological activity of triterpene saponins of C. quinoa. In particular, the mode of action of the mono- and bidesmosidic triterpene saponins and aglycones are discussed.     

Simple sequence repeat marker development and genetic mapping in quinoa (Chenopodium quinoa Willd.)

Quinoa is a regionally important grain crop in the Andean region of South America. Recently quinoa has gained international attention for its high nutritional value and tolerances of extreme abiotic stresses. DNA markers and linkage maps are important tools for germplasm conservation and crop improvement programmes. Here we report the development of 216 new polymorphic SSR (simple sequence repeats) markers from libraries enriched for GA, CAA and AAT repeats, as well as 6 SSR markers developed from bacterial artificial chromosome-end sequences (BES-SSRs). Heterozygosity (H) values of the SSR markers ranges from 0.12 to 0.90, with an average value of 0.57. A linkage map was constructed for a newly developed recombinant inbred lines (RIL) population using these SSR markers. Additional markers, including amplified fragment length polymorphisms (AFLPs), two 11S seed storage protein loci, and the nucleolar organizing region (NOR), were also placed on the linkage map. The linkage map presented here is the first SSR-based map in quinoa and contains 275 markers, including 200 SSR. The map consists of 38 linkage groups (LGs) covering 913 cM. Segregation distortion was observed in the mapping population for several marker loci, indicating possible chromosomal regions associated with selection or gametophytic lethality. As this map is based primarily on simple and easily-transferable SSR markers, it will be particularly valuable for research in laboratories in Andean regions of South America.     

金藜麦耐盐性分析及营养评价

对我国沿海地区新收集种质资源金藜麦(Chenopodium quinoa Willd.)进行了耐盐性及营养品质评价。结果表明:金藜麦在对盐胁迫相对敏感的芽期和苗期表现出相对较高的耐盐性;子粒蛋白质含量为14.2%,蛋白营养价值优于牛奶以及小麦、水稻、玉米、大豆等作物;子粒中富含维生素B、E等以及钙、锰、铁、铜、锌等矿质元素,特别是钙含量高达190.16 mg/100g,是小米钙含量的35倍;且金藜麦子粒含有丰富的必需脂肪酸,如亚油酸(3.58 g/100g)和亚麻酸(0.44 g/100g),天然抗氧化剂维生素E含量为7.66 mg/100g。这些研究结果表明,新收集的金藜麦种质资源具有较高的营养价值和耐盐性,将为我国藜麦研究和种植提供重要的种质资源。     

Crop/weed gene flow:Chenopodium quinoa Willd. andC. berlandieri Moq.

Introduction of the Andean grain chenopod (Chenopodium quinoa) into North America placed this crop within the distributional range of a related wild species,C. berlandieri. This wild species, native to the North American flora, is cross-compatible withC. Quinoa. Isozyme analysis of progeny fromC. berlandieri plants growing within and at the periphery of theC. Quinoa fields, combined with fertility assessment and phenetic comparison among putative hybrids and parental types, indicates that over 30% of progeny from wild plants growing as weeds withC. quinoa in 1987 were crop/weed hybrids. This high incidence of interspecific gene flow from crop to weed appears to be the result of asymmetric pollen flow to free-living plants from high-density cultivated populations. The observed level of crop/weed hybridization, combined with heterosis and partial fertility of F₁ crop/weed hybrids, suggests that repeated annual cycles ofC. quinoa cultivation within the North American range ofC. berlandieri could produce introgressive change among sympatric wild populations. In terms of risk assessment for biotechnology, these results suggest that the breeding system may not provide an accurate indication of the potential for genetic interaction among predominately self-pollinating grain crops and their free-living relatives.     

A recessive allele inhibiting saponin synthesis in two lines of Bolivian quinoa (Chenopodium quinoa Willd.)

Quinoa cultivars currently grown in North America and Europe require removal of bitter-tasting saponins from the grain prior to human consumption. This need for postharvest processing is a barrier to expanding production of the crop outside its Andean area of origin. Grain saponin content in quinoa shows continuous variation and is considered to be a quantitative trait. However, segregation for the presence or absence of grain saponin in F2 generations derived from crosses between high- and low-saponin parents indicates a major gene effect, with plants homozygous for a recessive allele spl having no detectable grain saponin. Variation in saponin levels among F2 plants with detectable grain saponin was consistent with polygenic inheritance. It appears that grain saponin level in quinoa is both qualitatively and quantitatively controlled, with saponin production requiring at least one dominant allele at the Sp locus and the amount of grain saponin being determined by an unknown number of additional quantitative loci. Introgression of sp1 into day-neutral lines will facilitate the development of short-season "sweet" quinoa cultivars which do not require postharvest processing to remove grain saponin.     

Genetic structure in cultivated quinoa (Chenopodium quinoa Willd.), a reflection of landscape structure in Northwest Argentina

Quinoa (Chenopodium quinoa Willd.), one of the main crops domesticated in the Andean highlands 1,000 of years ago, played an important role as a protein source. 35 germplasm accessions collected along the Northwest Argentina (NWA) region were studied using 22 microsatellite (SSR) markers. Results showed a great level of genetic diversity, differing from previous reports about the geographical distribution of quinoa variability. All SSR loci analysed were highly polymorphic detecting a total of 354 alleles among all populations, with an average of 16 alleles per locus. Cluster analyses grouped the accessions into four main clusters at the average genetic distance level (0.80), each of which represented a different environment of the NWA region: Puna (UHe?=?0.42, ±0.07 SE), Dry Valleys (UHe?=?0.27, ±0.05 SE), Eastern Humid Valleys (UHe?=?0.16, ±0.04 SE) and a transition area with high altitudes between the last two environments (UHe?=?0.25, ±0.03 SE). An eastward decreasing genetic diversity gradient was found. AMOVA analyses showed a strong genetic structure: a high population subdivision relative to the grouping by region (Fsr?=?0.47) together with a high genetic differentiation among populations (Fst?=?0.58) and a heterozygous defect (Fis?=?0.63) in each of them. The variability structure, a reflection of the structure of the NWA landscapes, is discussed in connection with environmental variables.     

Chromosomal localization of two novel repetitive sequences isolated from the Chenopodium quinoa Willd. genome

The chromosomal organization of two novel repetitive DNA sequences isolated from the Chenopodium quinoa Willd. genome was analyzed across the genomes of selected Chenopodium species. Fluorescence in situ hybridization (FISH) analysis with the repetitive DNA clone 18-24J in the closely related allotetraploids C. quinoa and Chenopodium berlandieri Moq. (2n = 4x = 36) evidenced hybridization signals that were mainly present on 18 chromosomes; however, in the allohexaploid Chenopodium album L. (2n = 6x = 54), cross-hybridization was observed on all of the chromosomes. In situ hybridization with rRNA gene probes indicated that during the evolution of polyploidy, the chenopods lost some of their rDNA loci. Reprobing with rDNA indicated that in the subgenome labeled with 18-24J, one 35S rRNA locus and at least half of the 5S rDNA loci were present. A second analyzed sequence, 12-13P, localized exclusively in pericentromeric regions of each chromosome of C. quinoa and related species. The intensity of the FISH signals differed considerably among chromosomes. The pattern observed on C. quinoa chromosomes after FISH with 12-13P was very similar to GISH results, suggesting that the 12-13P sequence constitutes a major part of the repetitive DNA of C. quinoa.     

Assessment of genetic diversity patterns in Chilean quinoa (Chenopodium quinoa Willd.) germplasm using multiplex fluorescent microsatellite markers

Quinoa (Chenopodium quinoa Willd.) is a staple seed crop in the Andean region of South America. Improving quinoa productivity is a primary food-security issue for this region, and has been part of the impetus for the establishment of several new quinoa breeding programs throughout the Andean region. Chilean quinoa has been characterized as morphologically diverse and bifurcated into coastal and highland ecotypes. The success of emerging breeding programs will rely heavily on the development of core germplasm collections and germplasm evaluation—especially of the coastal quinoa ecotypes that are often neglected in traditional breeding programs. Thus, the objective of this study was to characterize and quantify the genetic diversity within 28 Altiplano and 31 coastal Chilean accessions of quinoa using microsatellite markers. To facilitate the analysis, we also report the development of seven sets of fluorescent multiplexed microsatellite PCR reactions that result in genetic information for 20 highly polymorphic microsatellite loci. A total of 150 alleles were detected among the quinoa accession, ranging from 2 to 20 alleles per locus and an average 7.5 allele/locus. Both cluster (UPGMA) and principal component analyses separated the accessions into two discrete groups. The first group contained quinoa accessions from the north (Andean highlands) and the second group consisted of accessions from the south (lowland or coastal). Three accessions from Europe were classified into the southern quinoa group. The data obtained in the diversity analyses highlights the relationships within and among northern and southern Chilean quinoa accessions and provides the quinoa scientific community with a new set of easy to use and highly informative genetic markers.     

Purification,structural elucidation and in vivo immunity-enhancing activity of polysaccharides from quinoa (Chenopodium quinoa Willd.) seeds

ABSTRACT

Quinoa crude polysaccharides (QPS) were extracted from Chenopodium quinoa Willd. The soluble non-starch polysaccharide fraction (QPS1) was subsequently purified by DEAE-52 cellulose and Sephadex G-50 gel chromatography, using QPS as raw materials. Its chemical structure was identified using FT-IR, NMR, AFM, SEM and Congo red staining. High performance gel permeation chromatography (HPGPC) was used to determine molecular weight, and composition by HPLC. QPS1, with a molecular weight of 34.0 kDa, was mainly composed of mannose, rhamnose, galacturonic acid, glucose, galactose, xylose and arabinose at a molar ratio of 2.63:2.40:1.64:6.28:1.95:2.48:5.01. In addition, we evaluated the ameliorative effects of QPS1 on the improvement of anti-cyclophosphamide (CTX)-induced immunosuppression in ICR mice. The result exhibited significantly immune-enhancing activity: QPS1 successfully improved the content of IFN-γ, IL-6, IFN-ɑ, IgM and lysozyme (LYSO) in serum for three weeks, enhanced the phagocytic function of mononuclear macrophages and ameliorated delayed allergy in mice.     

Oxidative stress protection and stomatal patterning as components of salinity tolerance mechanism in quinoa (Chenopodium quinoa)

Two components of salinity stress are a reduction in water availability to plants and the formation of reactive oxygen species. In this work, we have used quinoa (Chenopodium quinoa), a dicotyledonous C3 halophyte species displaying optimal growth at approximately 150 mM NaCl, to study mechanisms by which halophytes cope with the afore-mentioned components of salt stress. The relative contribution of organic and inorganic osmolytes in leaves of different physiological ages (e.g. positions on the stem) was quantified and linked with the osmoprotective function of organic osmolytes. We show that the extent of the oxidative stress (UV-B irradiation) damage to photosynthetic machinery in young leaves is much less when compared with old leaves, and attribute this difference to the difference in the size of the organic osmolyte pool (1.5-fold difference under control conditions; sixfold difference in plants grown at 400 mM NaCl). Consistent with this, salt-grown plants showed higher Fv/Fm values compared with control plants after UV-B exposure. Exogenous application of physiologically relevant concentrations of glycine betaine substantially mitigated oxidative stress damage to PSII, in a dose-dependent manner. We also show that salt-grown plants showed a significant (approximately 30%) reduction in stomatal density observed in all leaves. It is concluded that accumulation of organic osmolytes plays a dual role providing, in addition to osmotic adjustment, protection of photosynthetic machinery against oxidative stress in developing leaves. It is also suggested that salinity-induced reduction in stomatal density represents a fundamental mechanism by which plants optimize water use efficiency under saline conditions.     

The role of cotyledon metabolism in the establishment of quinoa (Chenopodium quinoa) seedlings growing under salinity

A growth chamber experiment was conducted to assess the effect of salinity on emergence, growth, water status, photosynthetic pigments, osmolyte accumulation, and ionic content of quinoa seedlings (Chenopodium quinoa). The aim was to test the hypothesis that quinoa seedlings are well adapted to grow under salinity due to their ability to adjust the metabolic functionality of their cotyledons. Seedlings were grown for 21 days at 250 mM NaCl from the start of the germination. Germination percentage and cotyledon area were not affected by salt whereas seedling height decreased 15%. FW increased in both control and salt-treated cotyledons, but the increase was higher under salinity. DW only increased in salt-treated cotyledons. The DW/FW ratio did not show significant differences between treatments. Relative water content, chlorophyll, carotenoids, lipids, and proteins were significantly lower under salinity. Total soluble sugars, sucrose and glucose concentrations were higher in salt-treated than in control cotyledons. Ion concentration showed a different distribution pattern. Na⁺ and Cl^? concentrations were higher under salinity, while an inverse result was observed for K⁺ concentration. Proline and glycinebetaine concentrations increased under salinity, but the increase was higher in the former than the latter. The osmoprotective role of proline, glycinebetaine, and soluble sugars is discussed.     

Construction of a quinoa (Chenopodium quinoa Willd.) BAC library and its use in identifying genes encoding seed storage proteins

Quinoa (Chenopodium quinoa Willd.) is adapted to the harsh environments of the Andean Altiplano region. Its seeds have a well-balanced amino acid composition and exceptionally high protein content with respect to human nutrition. Quinoa grain is a staple in the diet of some of the most impoverished people in the world. The plant is an allotetraploid displaying disomic inheritance (2n=4x=36) with a di-haploid genome of 967 Mbp (megabase pair), or 2C=2.01 pg. We constructed two quinoa BAC libraries using BamHI (26,880 clones) and EcoRI (48,000 clones) restriction endonucleases. Cloned inserts in the BamHI library average 113 kb (kilobase) with approximately 2% of the clones lacking inserts, whereas cloned inserts in the EcoRI library average 130 kb and approximately 1% lack inserts. Three plastid genes used as probes of high-density arrayed blots of 73,728 BACs identified approximately 2.8% of the clones as containing plastid DNA inserts. We estimate that the combined quinoa libraries represent at least 9.0 di-haploid nuclear genome equivalents. An average of 12.2 positive clones per probe were identified with 13 quinoa single-copy ESTs as probes of the high-density arrayed blots, suggesting that the estimate of 9.0× coverage of the genome is conservative. Utility of the BAC libraries for gene identification was demonstrated by probing the library with a partial sequence of the 11S globulin seed storage protein gene and identifying multiple positive clones. The presence of the 11S globulin gene in four of the clones was verified by direct comparison with quinoa genomic DNA on a Southern blot. Besides serving as a useful tool for gene identification, the quinoa BAC libraries will be an important resource for physical mapping of the quinoa genome.