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.     

Leaf extract of neem (Azadirachta indica) alleviates adverse effects of drought in quinoa (Chenopodium quinoa Willd.) plants through alterations in biochemical attributes and antioxidants

The influence of varying concentrations (0, 1, 3, 4, 5, 6, 8, and 10 % v/v) of neem (Azadirachta indica) leaf extract on drought stressed (40 % field capacity) quinoa (Chenopodium quinoa Willd.) plants was assessed. During the current study two cultivars of quinoa (V7 and V9) were used. This study revealed that water stress adversely affects the fresh and dry weight of shoots and roots as well as chlorophyll pigments (a and b) of both quinoa cultivars. In contrast, drought stress enhanced glycinebetaine (GB), free proline, phenolic content, hydrogen peroxide (H₂O₂), activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) enzymes, and relative membrane permeability (RMP). However, application of neem leaf extract improved the accumulation of key osmoprotectants like proline, GB and activities of enzymatic antioxidants. Our findings showed 5 % neem leaf extract is an effective treatment in counteracting the oxidative damage caused by water stress, thereby improving overall plant growth. Of both cultivars of quinoa, the response of cv. V9 to stress as well as foliar applied neem was relatively more promising.     

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.     

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.     

Molecular and cytological characterization of ribosomal RNA genes in Chenopodium quinoa and Chenopodium berlandieri.

The nucleolus organizer region (NOR) and 5S ribosomal RNA (rRNA) genes are valuable as chromosome landmarks and in evolutionary studies. The NOR intergenic spacers (IGS) and 5S rRNA nontranscribed spacers (NTS) were PCR-amplified and sequenced from 5 cultivars of the Andean grain crop quinoa (Chenopodium quinoa Willd., 2n = 4x = 36) and a related wild ancestor (C. berlandieri Moq. subsp. zschackei (Murr) A. Zobel, 2n = 4x = 36). Length heterogeneity observed in the IGS resulted from copy number difference in subrepeat elements, small re arrangements, and species-specific indels, though the general sequence composition of the 2 species was highly similar. Fifteen of the 41 sequence polymorphisms identified among the C. quinoa lines were synapomorphic and clearly differentiated the highland and lowland ecotypes. Analysis of the NTS sequences revealed 2 basic NTS sequence classes that likely originated from the 2 allopolyploid subgenomes of C. quinoa. Fluorescence in situ hybridization (FISH) analysis showed that C. quinoa possesses an interstitial and a terminal pair of 5S rRNA loci and only 1 pair of NOR, suggesting a reduction in the number of rRNA loci during the evolution of this species. C. berlandieri exhibited variation in both NOR and 5S rRNA loci without changes in ploidy.     

Micropropagation protocol for coastal quinoa

Plant Cell, Tissue and Organ Culture (PCTOC) - Quinoa is a model halophyte plant, with seeds rich in proteins, fatty acids and lacking prolamines. There are two types of quinoa cultivars: highland...     

Non-destructive evaluation of chlorophyll content in quinoa and amaranth leaves by simple and multiple regression analysis of RGB image components

Leaf chlorophyll content provides valuable information about physiological status of plants; it is directly linked to photosynthetic potential and primary production. In vitro assessment by wet chemical extraction is the standard method for leaf chlorophyll determination. This measurement is expensive, laborious, and time consuming. Over the years alternative methods, rapid and non-destructive, have been explored. The aim of this work was to evaluate the applicability of a fast and non-invasive field method for estimation of chlorophyll content in quinoa and amaranth leaves based on RGB components analysis of digital images acquired with a standard SLR camera. Digital images of leaves from different genotypes of quinoa and amaranth were acquired directly in the field. Mean values of each RGB component were evaluated via image analysis software and correlated to leaf chlorophyll provided by standard laboratory procedure. Single and multiple regression models using RGB color components as independent variables have been tested and validated. The performance of the proposed method was compared to that of the widely used non-destructive SPAD method. Sensitivity of the best regression models for different genotypes of quinoa and amaranth was also checked. Color data acquisition of the leaves in the field with a digital camera was quick, more effective, and lower cost than SPAD. The proposed RGB models provided better correlation (highest R ²) and prediction (lowest RMSEP) of the true value of foliar chlorophyll content and had a lower amount of noise in the whole range of chlorophyll studied compared with SPAD and other leaf image processing based models when applied to quinoa and amaranth.     

A genetic linkage map of quinoa (<Emphasis Type="Italic">Chenopodium quinoa</Emphasis>) based on AFLP,RAPD, and SSR markers

Quinoa (Chenopodium quinoa Willd.) is an important seed crop for human consumption in the Andean region of South America. It is the primary staple in areas too arid or saline for the major cereal crops. The objective of this project was to build the first genetic linkage map of quinoa. Selection of the mapping population was based on a preliminary genetic similarity analysis of four potential mapping parents. Breeding lines Ku-2 and 0654, a Chilean lowland type and a Peruvian Altiplano type, respectively, showed a low similarity coefficient of 0.31 and were selected to form an F₂ mapping population. The genetic map is based on 80 F₂ individuals from this population and consists of 230 amplified length polymorphism (AFLP), 19 simple-sequence repeat (SSR), and six randomly amplified polymorphic DNA markers. The map spans 1,020 cM and contains 35 linkage groups with an average marker density of 4.0 cM per marker. Clustering of AFLP markers was not observed. Additionally, we report the primer sequences and map locations for 19 SSR markers that will be valuable tools for future quinoa genome analysis. This map provides a key starting point for genetic dissection of agronomically important characteristics of quinoa, including seed saponin content, grain yield, maturity, and resistance to disease, frost, and drought. Current efforts are geared towards the generation of more than 200 mapped SSR markers and the development of several recombinant-inbred mapping populations.     

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.     

A dopamine-based biosynthetic pathway produces decarboxylated betalains in Chenopodium quinoa

Betalains are the nitrogenous pigments that replace anthocyanins in the plant order Caryophyllales. Here, we describe unconventional decarboxylated betalains in quinoa (Chenopodium quinoa) grains. Decarboxylated betalains are derived from a previously unconsidered activity of the 4,5-DOPA-extradiol-dioxygenase enzyme (DODA), which has been identified as the key enzymatic step in the established biosynthetic pathway of betalains. Here, dopamine is fully characterized as an alternative substrate of the DODA enzyme able to yield an intermediate and structural unit of plant pigments: 6-decarboxy-betalamic acid, which is proposed and described. To characterize this activity, quinoa grains of different colors were analyzed in depth by chromatography, time-of-flight mass spectrometry, and reactions were performed in enzymatic assays and bioreactors. The enzymatic-chemical scheme proposed leads to an uncharacterized family of 6-decarboxylated betalains produced by a hitherto unknown enzymatic activity. All intermediate compounds as well as the final products of the dopamine-based biosynthetic pathway of pigments have been unambiguously determined and the reactions have been characterized from the enzymatic and functional perspectives. Results evidence a palette of molecules in quinoa grains of physiological relevance and which explain minor betalains described in plants of the Caryophyllales order. An entire family of betalains is anticipated.

A biosynthetic pathway produces unconventional plant pigments betalains derived from dopamine in quinoa.     

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.     

Effects of Photoperiod, Temperature and Radiation on the Rate of Leaf Appearance in Quinoa (Chenopodium quinoa Willd.) under Field Conditions

Effects of radiation on the phyllochron of nine quinoa (Chenopodiumquinoa Willd.) cultivars growing under field conditions werequantified. Rate of change of photoperiod had no effect on phyllochron.Radiation responses differed markedly between cultivars, rangingfrom insensitive in the most tropical cultivars (Nariñoand Ecu-621) to 0.55 °Cd mol PPFD m^-2 d^-1in the Peruviancultivar Amarilla de Maranganí. Cultivars reported ashaving the highest photoperiod sensitivity and longest phyllochronwere insensitive to radiation, while cultivars insensitive tophotoperiod from the Bolivian and Peruvian altiplano (Kanckolla,Blanca de Julí and Sajama) or moderately sensitive tophotoperiod from central Chile (Faro and Baer) were affectedby radiation. It is suggested that variation in phyllochronsensitivity to radiation could be associated with differencesin photoassimilate demand per unit time to sustain leaf growth.A model based on phyllochron responses to radiation and temperatureproduced an important improvement in the quality of predictionswhen compared with a model based on responses to photoperiodand temperature for seven of the nine cultivars evaluated. Thisis the first instance in which a quantitative relationship betweenincident radiation and the phyllochron in natural environmentshas been established, allowing the inclusion of this factorin predictions of the rate of leaf appearance in the field.Copyright 2001 Annals of Botany Company Chenopodium quinoa, quinoa, phyllochron, radiation, photoperiod, rate of change of photoperiod     

Soil drenching of paclobutrazol: An efficient way to improve quinoa performance under salinity

Salinity extent and severity is rising because of poor management practices on agricultural lands, possibility lies to grow salt‐tolerant crops with better management techniques. Therefore, a highly nutritive salt‐tolerant crop quinoa with immense potential to contribute for future food security was selected for this investigation. Soil drenching of paclobutrazol (PBZ; 20 mg l^?1) was used to understand the ionic relations, gaseous exchange characteristics, oxidative defense system and yield under saline conditions (400 mM NaCl) including normal (0 mM NaCl) and no PBZ (0 mg l^?1) as controls. The results revealed that salinity stress reduced the growth and yield of quinoa through perturbing ionic homeostasis with the consequences of overproduction of reactive oxygen species (ROS), oxidative damages and reduced photosynthesis. PBZ improved the quinoa performance through regulation of ionic homeostasis by decreasing Na⁺, Cl^?, while improving K⁺, Mg²⁺ and Ca²⁺ concentration. It also enhanced the antioxidative system including ascorbic acid, phenylalanine ammonia‐lyase, polyphenol oxidase and glutathione peroxidase, which scavenged the ROS (H₂O₂ and O₂^?‐) and lowered the oxidative damages (malondialdehyde level) under salinity in roots and more specifically in leaf tissues. The photosynthetic rate and stomatal conductance consequently improved (16 and 21%, respectively) in salt‐stressed quinoa PBZ‐treated compared to the non‐treated ones and contributed to the improvement of panicle length (33%), 100‐grain weight (8%) and grain yield (38%). Therefore, PBZ can be opted as a shotgun approach to improve quinoa performance and other crops under high saline conditions.     

Central Role of Salicylic Acid in Resistance of Wheat Against <Emphasis Type="Italic">Fusarium graminearum</Emphasis>

Head blight caused by Fusarium graminearum (F. graminearum) is one of the major threats to wheat and barley around the world. The importance of this disease is due to a reduction in both grain yield and quality in infected plants. Currently, there is limited knowledge about the physiological mechanisms involved in plant resistance against this pathogen. To reveal the physiological mechanisms underlying the resistance to F. graminearum, spikes of resistant (Sumai3) and susceptible (Falat) wheat cultivars were analyzed 4 days after inoculation, as the first symptoms of pathogen infection appeared. F. graminearum inoculation resulted in a greater induction level and activity of salicylic acid (SA), callose, phenolic compounds, peroxidase, phenylalanine ammonia lyase (PAL), and polyphenol oxidase in resistant versus susceptible cultivars. Soil drench application to spikes of SA, 24 h before inoculation with F. graminearum alleviated Fusarium head blight symptoms in both resistant and susceptible cultivars. SA treated plants showed a significant increment in hydrogen peroxide (H₂O₂) production, lipid peroxidation, SA, and callose content. SA-induced H₂O₂ level seems to be related to increased superoxide dismutase and decreased catalase activities. In addition, real-time quantitative PCR analysis showed that SA pretreatment induced expression of PAL genes in both infected and non-infected head tissues of the susceptible and resistant cultivars. Our data showed that soil drench application of SA activates antioxidant defense responses and may subsequently induce systemic acquired resistance, which may contribute to the resistance against F. graminearum. These results provide novel insights about the physiological and molecular role of SA in plant resistance against hemi-biotrophic pathogen infection.     

大小粒型藜麦籽粒表型、灌浆特性及淀粉合成酶活性的差异分析

【目的】籽粒大小是影响藜麦产量、商品性和加工特性的重要因素,考察灌浆期大小粒型藜麦籽粒表型、灌浆特性和淀粉合成酶活性的差异,为大粒型藜麦品种的选育提供理论指导。【方法】选择千粒重大于5.0 g和小于3.0 g的藜麦材料各两份,在青海省农林科学院种质资源创新试验基地进行田间试验,比较自灌浆期始7 d、14 d、21 d和28 d籽粒表型、灌浆特性和淀粉合成酶活性等在大小粒型藜麦间的差异。【结果】（1）大小粒型藜麦籽粒面积、周长、直径、粒长、粒宽表型性状随着生育时期均极显著增大,且粒型间存在显著差异,并以籽粒面积和周长差异最大,大粒型藜麦显著高于小粒型藜麦9.12%~11.54%和21.49~23.92%。（2）灌浆期间大粒型藜麦百粒干重始终显著高于同期小粒型藜麦,平均增幅在21.23%～31.04%;大小粒型藜麦灌浆速率随生育期均先上升后下降,均符合“慢-快-慢”的变化规律,但达到峰值时间和峰高明显不同,大粒型峰值出现早而高,小粒型则低而迟。（3）淀粉分支酶（SBE）、蔗糖合成酶（SS）、可溶性淀粉合成酶（SSS）和ADPG焦磷酸化酶（AGP）在大小粒型藜麦籽粒灌浆期呈现不同的变化趋势,SBE和SS活性表现为小粒型藜麦强于大粒型藜麦,而SSS和AGP活性则表现为大粒型藜麦强于小粒型藜麦。【结论】藜麦籽粒灌浆期间4种淀粉合成酶活性的差异,致使淀粉合成积累量和灌浆速率峰值的不同,进而形成籽粒表型性状的差异,而SSS和AGPase是影响藜麦籽粒大小形成的关键酶。     

Induction of somatic embryogenesis in cultured cells of <Emphasis Type="Italic">Chenopodium quinoa</Emphasis>

A major limiting factor for quinoa cultivation as a grain crop on a large scale are virus diseases, in particularly seed borne diseases. Therefore, a somatic embryogenesis protocol is a necessary tool to produce virus free plants. Somatic embryogenesis offers the possibility of mass production of transgenic plants and therefore can be used easily to study the effect on plants resulting from breeding processes. An in vitro protocol has been developed for somatic embryogensis from calluses and cell cultures of Chenopodium quinoa. Callus was induced from hypocotyl explants within 2 weeks of culture on a modified Murashige and Skoog (MS) medium supplemented with 0.45 M 2,4-D. Calluses were cultured on solid or liquid MS medium and later the development of somatic embryos was observed on both employing the same MS medium without 2,4-D. To our knowledge this is the first report of somatic embryogenesis in Chenopodium quinoa.     

Genotypic difference in salinity tolerance in quinoa is determined by differential control of xylem Na loading and stomatal density

Quinoa is regarded as a highly salt tolerant halophyte crop, of great potential for cultivation on saline areas around the world. Fourteen quinoa genotypes of different geographical origin, differing in salinity tolerance, were grown under greenhouse conditions. Salinity treatment started on 10 day old seedlings. Six weeks after the treatment commenced, leaf sap Na and K content and osmolality, stomatal density, chlorophyll fluorescence characteristics, and xylem sap Na and K composition were measured. Responses to salinity differed greatly among the varieties. All cultivars had substantially increased K⁺ concentrations in the leaf sap, but the most tolerant cultivars had lower xylem Na⁺ content at the time of sampling. Most tolerant cultivars had lowest leaf sap osmolality. All varieties reduced stomata density when grown under saline conditions. All varieties clustered into two groups (includers and excluders) depending on their strategy of handling Na⁺ under saline conditions. Under control (non-saline) conditions, a strong positive correlation was observed between salinity tolerance and plants ability to accumulate Na⁺ in the shoot. Increased leaf sap K⁺, controlled Na⁺ loading to the xylem, and reduced stomata density are important physiological traits contributing to genotypic differences in salinity tolerance in quinoa, a halophyte species from Chenopodium family.