New Insights into the Antimicrobial Properties of Hydrolysates and Peptide Fractions Derived from Chia Seed (Salvia hispanica L.)

Bioactive peptides derived from chia (Salvia hispanica) seed with antioxidant, antihypertensive, and anti-inflammatory activities have been well documented; however, few studies describe the antimicrobial properties of these peptides, which is of great interest not only in the prevention of food-borne diseases but also food spoilage. The aim of this study was to generate chia seed peptides using microwave-assisted hydrolysis with sequential (alcalase + flavourzyme) enzymes (AF-MW), fractionate them into 3–10 and < 3 kDa fractions, and evaluate their potential antimicrobial activity towards Escherichia coli, Salmonella enterica, and Listeria monocytogenes. Overall, the peptide fraction < 3 kDa showed higher antimicrobial activity than both chia seed hydrolysate and peptide fraction 3–10 kDa. Furthermore, the < 3 kDa fraction showed remarkable increase in membrane permeability of E. coli (71.49% crystal violet uptake) and L. monocytogenes (80.10% crystal violet uptake). These peptides caused a significant extension in the lag phase, decreases in the maximum growth, and growth rate in the bacteria and promoted multiple indentations (transmembrane tunnels), membrane wrinkling, and pronounced deformations in the integrity of the bacterial cell membranes. Finally, a select group of peptides in the AF-MW < 3 kDa fraction contained 16 sequences with cationic and hydrophobic character, with seven of them sharing the exact same sequence (GDVIAIR) and eight of them having the amino acid K as either N- or C-terminal or both. In conclusion, our results indicate that bioactive peptides obtained from chia seed proteins by microwave and enzymatic hydrolysis could be employed as antimicrobial agents in foods and therapeutic applications.

     

Genetics of qualitative traits in domesticated chia (Salvia hispanica L.)

In Salvia hispanica L., several changes in qualitative characters, including seed coat color, stem pigmentation, and shattering, have evolved with cultivation and domestication. Three F(2) segregating generations from crosses between wild and domesticated parents were scored for three qualitative traits. A single recessive gene, designated scc, was found to govern the white seed characteristic. A single dominant gene, designated SSP, was found to control striated stem pigmentation. A complete dominance of open calyx over closed calyx was observed in F(1) generations and small numbers of plants with closed calyxes were observed in F(2) generations, not conforming to Mendelian ratios. For this non-shattering trait, a complementation test was conducted between two lines representative of geographically and morphologically divergent domesticated varieties. Complementary gene action was not observed in any F(1) plants, and all F(2) plants were homogeneous with respect to the trait, suggesting the same genetic control for non-shattering among domesticated varieties. An analysis of limited data for linkage of SSP and scc indicated that the two loci segregate independently.     

Fatty acid and nutritive quality of chia (Salvia hispanica L.) seeds and plant during growth

The fatty acid (FA) profile, chemical composition, gross energy and organic matter digestibility of chia (Salvia hispanica L.) have been determined in the seed and in the plant collected at five progressive morphological stages from early vegetative to budding stage. The FA analyses disclosed quantitative differences between the plant stages that were characterised by a high percentage of polyunsaturated fatty acids (PUFA), which made up from 752 to 623 g/kg of the total FA of the plant during the growth cycle. The α-linolenic acid (ALA, C_18:3n–3) decreased from 649 g/kg, at the early vegetative stage, to 499 g/kg of the total FA, at the budding stage, while all the other FAs increased with increasing growth stage. The chia seed FAs were also highly unsaturated, with their main components being ALA (641 g/kg of the total FA) and linoleic acid (LA, C_18:2n–6; 188 g/kg of the total FA).The evolution of the quality of chia is closely related to the ageing of the plant. The chia plant provides a forage with a good nutritive value when harvested at a stage before the shooting period. After this, the nutritional quality of the plant considerably decreases with an increase in the fibrous fractions and a dramatical decrease of the crude protein content.     

Ethnobotany of chia,Salvia hispanica L. (Lamiaceae)

Salvia hispanica L, was an important staple Mesoamerican food and medicinal plant in pre-Columbian times. Unlike other Mesoamerican pseudocereal crops such asAmaranthus andChenopodium, it has received comparatively little research attention. An ethnobotanical review of this Mesoamerican crop plantSalvia hispanica has been undertaken to examine changes in use accompanying Spanish colonization. A comparative analysis of accounts of use from the 16th century codices of Mexico and subsequent publications has revealed subtle changes in medicinal, culinary, artistic, and religious uses. Several hypotheses surrounding changes in use through time and the original use(s) that led to domestication are developed and tested through collection of ethnobotanical data in the highlands of western Mexico and Guatemala. A general decline in ethnobotanical knowledge associated with wild populations coupled with a loss of habitat in some locations has degraded important germplasm and knowledge resources for a species with great economic potential.     

蓝粒小麦花青素生物合成途径中的二氢黄酮醇4-还原酶基因的分子克隆

蓝色色素在蓝粒小麦种子糊粉层中的生物合成途径的分子生物学机制至今仍不清楚。应用RT—PCR和RACE方法从蓝粒小麦正在发育的种子中克隆到一个编码二氢黄酮醇4-还原酶的基因(DFR)。推测其为花青素生物合成途径中的一个关键基因,且与蓝粒小麦中蓝色色素形成密切相关;其开放阅读框编码一个包含354个氨基酸残基的多肽,与一些从其他植物中已克隆到的DFR有很高的同源性：大麦(94％)、水稻(83％)、玉米(84％)。从长穗偃麦草(2n=70)、蓝粒小麦、浅蓝粒小麦自交产生的白粒后代小麦以及中国春的基因组中分别分离到一个全长DFR序列。经聚类分析表明DFR cDNA核甘酸序列与从中国春基因组中克隆的DFR具有100％的同源性,且与长穗偃麦草、蓝粒小麦、白粒小麦基因组中分离的DFR均有很高的同源性。4个DFR基因组DNA均含有3个内含子,且它们之间的差异主要在内含子区,表明该基因在进化上很保守。经Southern杂交分析,DFR小麦中至少有3-5个拷贝,不同小麦材料间未见明显差异,但与长穗偃麦草有明显差异,属于一个DFR超基因家族。Northern分析表明该DFR在蓝粒和白粒种子的不同发育时期的表达存在明显差异,都在开花后大约18d表达最强,在同一时期的蓝白种子中,DFR蓝粒种子中的表达量高于白粒。DFR转录本在小麦和长穗偃麦草的幼叶中积累多,但在芽鞘中的表达显著低于幼叶中;在小麦的根和长穗偃麦草的发育种子中均未检测到DFR的表达。推测蓝粒小麦中可能存在调控DFR在蓝粒小麦中表达的调控基因,类似于玉米花青素合成途径中的调节基因。     

蓝粒小麦花青素生物合成途径中的二氢黄酮醇4-还原酶基因的分子克隆

蓝色色素在蓝粒小麦种子糊粉层中的生物合成途径的分子生物学机制至今仍不清楚.应用RT-PCR和RACE方法从蓝粒小麦正在发育的种子中克隆到一个编码二氢黄酮醇4-还原酶的基因(DFR).推测其为花青素生物合成途径中的一个关键基因,且与蓝粒小麦中蓝色色素形成密切相关;其开放阅读框编码一个包含354个氨基酸残基的多肽,与一些从其他植物中已克隆到的DFR有很高的同源性:大麦(94%)、水稻(83%)、玉米(84%).从长穗偃麦草(2n=70)、蓝粒小麦、浅蓝粒小麦自交产生的白粒后代小麦以及中国春的基因组中分别分离到一个全长DFR序列.经聚类分析表明DFR cDNA核甘酸序列与从中国春基因组中克隆的DFR具有100%的同源性,且与长穗偃麦草、蓝粒小麦、白粒小麦基因组中分离的DFR均有很高的同源性.4个DFR基因组DNA均含有3个内含子,且它们之间的差异主要在内含子区,表明该基因在进化上很保守.经Southern杂交分析,DFR在小麦中至少有3～5个拷贝,不同小麦材料间未见明显差异,但与长穗偃麦草有明显差异,属于一个DFR超基因家族.Northern分析表明该DFR在蓝粒和白粒种子的不同发育时期的表达存在明显差异,都在开花后大约18 d表达最强,在同一时期的蓝白种子中,DFR在蓝粒种子中的表达量高于白粒.DFR转录本在小麦和长穗偃麦草的幼叶中积累多,但在芽鞘中的表达显著低于幼叶中;在小麦的根和长穗偃麦草的发育种子中均未检测到DFR的表达.推测蓝粒小麦中可能存在调控DFR在蓝粒小麦中表达的调控基因,类似于玉米花青素合成途径中的调节基因.     

Cell-free Synthesis of Globulin by Developing Oat (Avena sativa L.) Seeds

The primary storage protein synthesized during oat (Avena sativa L.) groat development is a globulin. Polysomes were isolated from oat groats 12 days after anthesis. These polysomes directed the incorporation of radioactive amino acids into protein in a cell-free protein synthesis system containing wheat germ supernatant. The Mg(2+) optimum was 4 mm, the pH optimum was 6-8, and the amount of amino acid incorporation depended on polysome concentration. Incorporation of amino acids was linear for about 10 min and approached a maximum after 20 min. Using the initiation inhibitor, T-2 toxin, it was determined that about 36% of the amino acid incorporation was due to the initiation of new polypeptide chains. The in vitro product co-electrophoresed with authentic oat groat globulin on polyacrylamide-sodium dodecyl sulfate (SDS) gels. The cyanogen bromide peptides of the in vitro product partially corresponded with those from authentic globulin when electrophoresed on polyacrylamide-SDS gels. These data suggest that the in vitro product is primarily oat globulin. The polysome population was separated into membrane-bound and free polysomes. Membrane-bound polysomes synthesized about twice the amount of protein as did free polysomes. Products synthesized in vitro on both types of polysomes were essentially the same.     

Changes in Polysomes and RNA in Developing Seeds of Chickpea (Cicer arietinum L.)

In the present investigation changes in polyribosomes and RNAs in the developing seeds of chickpea (Cicer arietinum L.) have been studied. The total polysome yield was higher in the early stages of development and declined at the later stages. The maximum level of polyribosomes was obtained at 18 days after flowering and a drastic decrease was noticed at maturity. The total RNA yield correlated with the polysomal yield. Northern hybridization with a heterologous probe (pea legumin cDNA) gave distinct hybridization with the mRNA coding for legumin proteins at different stages of seed development. Hybridization showed a direct relation between mRNA levels and seed weight accumulation.     

On the Compartmentation of Triacylglycerol Synthesis in Developing Seeds of Brassica napus

Compartmentation of storage lipid biosynthesis in developing erucate-rich rapeseeds during the period of rapid triacylglycerol accumulation has been investigated by labelling acyl residues and the glycerol backbone in endomembrane lipids of isolated embryos with radioactive precursors, either before (“in vivo”) or after (“in vitro”) subcellular fractionation. In contrast to the low light environment within the pod under normal environmental conditions, the photosynthetic and lipid synthesizing capacities of the embryos were significantly stimulated by their illumination in the isolated state. Both ways of demonstrating “de novo” synthesis of triacylglycerols and erucic acid in endomembrane vesicles show their significantly higher accumulation in oil bodies than in microsomal fractions, where membrane lipids predominate. The increased diacylglycerol acylation in erucate-rich rape embryos appears to be coupled to an alternative elongation mechanism for oleic acid, with another immediate acyl donor than 18:1-CoA. The present results are interpreted as a spatial separation of triacylglycerol formation, with very long-chain fatty acids obtained from residual lipid synthesis and fatty acid elongating capacity located on the endoplasmic reticulum.     

Site of Synthesis of the Enzymes of the Pyrimidine Biosynthetic Pathway in Oat (Avena sativa L.) Leaves

Heat-bleached oat (Avena sativa L. cv Porter) leaves lacking 70S chloroplast ribosomes have been used to demonstrate that four chloroplast-localized enzymes of pyrimidine nucleotide biosynthesis: aspartate carbamoyl-transferase, dihydroorotase, orotidine phosphoribosyl-transferase, and orotidine-5′-phosphate decarboxylase, are synthesized on cytoplasmic ribosomes. Two other chloroplast enzymes, carbamoyl phosphate synthetase, involved in both pyrimidine and arginine biosynthesis, and ornithine carbamoyltransferase, an enzyme of arginine biosynthesis, were also shown to be made on 80S ribosomes.     

Carbon Dioxide Fixation in the Carbon Economy of Developing Seeds of Lupinus albus (L.)

The effects of CO₂ concentration and illumination on net gas exchange and the pathway of ¹⁴CO₂ fixation in detached seeds from developing fruits of Lupinus albus (L.) have been studied.

Increasing the CO₂ concentration in the surrounding atmosphere (from 0.03 to 3.0% [v/v] in air) decreased CO₂ efflux by detached seeds either exposed to the light flux equivalent to that transmitted by the pod wall (500 to 600 micro-Einsteins per square meter per second) in full sunlight or held in darkness. Above 1% CO₂ detached seeds made a net gain of CO₂ in the light (up to 0.4 milligrams of CO₂ fixed per gram fresh weight per hour) but ¹⁴CO₂ injected into the gas space of intact fruits (containing around 1.5% CO₂ naturally) was fixed mainly by the pod and little by the seeds.

Throughout development seeds contained ribulose-1,5-bisphosphate carboxylase activity (EC 4.1.1.39), especially in the embryo (up to 99 micromoles of CO₂ fixed per gram fresh weight per hour) and phosphoenolpyruvate carboxylase (EC 4.1.1.31) in both testa (up to 280 micromoles of CO₂ fixed per gram fresh weight per hour) and embryo (up to 355 micromoles of CO₂ fixed per gram fresh weight per hour).

In kinetic experiments the most significant early formed product of ¹⁴CO₂ fixation in both light and dark was malate but in the light phosphoglyceric acid and sugar phosphates were also rapidly labeled. ¹⁴CO₂ fixation in the light was linked to the synthesis of sugars and amino acids but in the dark labeled sugars were not formed.

     

A beta-Galactosidase from Radish (Raphanus sativus L.) Seeds

A basic β-galactosidase (β-Galase) has been purified 281-fold from imbibed radish (Raphanus sativus L.) seeds by conventional purification procedures. The purified enzyme is an electrophoretically homogeneous protein consisting of a single polypeptide with an apparent molecular mass of 45 kilodaltons and pl values of 8.6 to 8.8. The enzyme was maximally active at pH 4.0 on p-nitrophenyl β-d-galactoside and β-1,3-linked galactobiose. The enzyme activity was inhibited strongly by Hg²⁺ and 4-chloromercuribenzoate. d-Galactono-(1→4)-lactone and d-galactal acted as potent competitive inhibitors. Using galactooligosaccharides differing in the types of linkage as the substrates, it was demonstrated that radish seed β-Galase specifically split off β-1,3- and β-1,6-linked d-galactosyl residues from the nonreducing ends, and their rates of hydrolysis increased with increasing chain lengths. Radish seed and leaf arabino-3,6-galactan-proteins were resistant to the β-galase alone but could be partially degraded by the enzyme after the treatment with a fungal α-l-arabinofuranosidase leaving some oligosaccharides consisting of d-galactose, uronic acid, l-arabinose, and other minor sugar components besides d-galactose as the main product.     

Desiccation Tolerance and Potential Longevity of Developing Seeds of Rice (Oryza sativa L.)

The longevity and desiccation tolerance of samples of seedsof a japonica rice (Oryza sativa L.) harvested serially duringdevelopment from plants grown in two temperature regimes, viz28/20 °C and 32/24 °C (12/12 h) were determined. Massmaturity (defined as the end of the seed-filling phase) occurred19·7 and 18·3 d after 50% anthesis, respectively.Longevity (determined at 40 °C with 15% moisture contentand quantified by the value of the constant K_i of the seed viabilityequation) improved during seed development and maturation until17 and 14 d after mass maturity in the cooler and warmer regimes,respectively, but declined thereafter. Changes in K_i with timewere similar in the two environments until mass maturity, butthe increase in K_i values after mass maturity was much greaterin the cooler regime. Tolerance of desiccation to low (4%) moisturecontents improved until 22 and 14 d after mass maturity in thecooler and warmer regimes, respectively, when maturation dryinghad reduced seed moisture contents naturally to 24 and 32% moisturecontent, respectively. Further delays to seed harvest reduceddesiccation tolerance, particularly in the warmer environment.Comparison among 15 samples of seeds harvested at differenttimes in the two environments showed a strong correlation (r= 0·947, P < 0·01) between longevity (K_i) anddesiccation tolerance (to 4% moisture content). Hence, it issuggested that the regulation of desiccation tolerance to lowmoisture contents and potential air-dry longevity during seeddevelopment and maturation determined here may have a commoncause.Copyright 1994, 1999 Academic Press Oryza sativa L., rice, desiccation tolerance, genebanks, seed development, seed longevity, temperature     

Verticillium wilt of sage (Salvia officinalis L.)

Verticillium dahliae Kleb. was established as the cause of wilt in sage. The disease causes leaf yellowing and necrosis from the base of the plant upwards, followed by defoliation. Microsclerotia found in the bark and sometimes the pith of severely affected stems are regarded as a diagnostic feature. Surveys confirmed the widespread occurrence of the disease in the Vale of Evesham and showed that it was most severe on heavy clay soils and in poorly drained areas but was not related to soil pH, phosphate or potash content. The rate of disease spread and the distribution of affected plants suggested that infected debris was the main source of disease dispersal, particularly as microsclerotia were abundant in many fallen leaves. It was shown that the disease could be disseminated in propagating material, and field evidence suggested that the pathogen could survive in the field in the absence of sage for at least 10 years. Replanted sage in affected areas generally contracted the disease within 1 year. Control measures suggested include careful stock selection, weed control and improved general hygiene.     

Contribution of the Pentose Phosphate Pathway and Glycolytic Pathway to Dormancy Breakage and Germination of Peanut (Arachis hypogaea L.) Seeds

Levels of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADPH oxidoreductase, E.C. 1.1.1.49 [EC] ) 6-phosphogluconate dehydrogenase(6-phospho-D-gluconate : NADP⁺ oxidoreduc tase, E.C. 1.1.1.44 [EC] )and aldolase (fructose 1, 6-diphosphate, D-glyceraldehyde, 3-phosphatelyase, E.C. 4.1.2.13 [EC] ) were assayed in the seeds of geneticallydormant and non-dormant pure lines of groundnut. In dormantlines cotyledons showed increased levels of activity of G-6-PDHand 6-PGDH during dry storage after-ripening. While the embryonicaxis did not exhibit detectable levels of enzyme activitiesimmediately after harvest, the activity started after a lapseof time during dry storage. When seeds of dormant lines wereincubated with kinetin (6-furfurylaminopurine) a distinct increasein the levels of both the enzymes was observed. The levels ofaldolase activity gradually decreased in the cotyledons andincreased in the embryonic axis of both control and kinetintreated seeds during the period of after-ripening. Comparedto control, kinetin treatment increased the aldolase activityin the embryonic axis and decreased it in the cotyledons. In non-dormant lines the activity of both the enzymes of PPpathway increased sharply both in the cotyledons and embryonicaxis while aldolase activity decreased in the cotyledons andincreased in the embryonic axis during germination i.e., from24 h to 96 h of germination. Abscisic acid caused inhibitionof enzyme activities to a large extent. Key words: PP pathway, dormancy breakage, germination, peanut     

Quantitative Analysis of Photosynthate Unloading in Developing Seeds of Phaseolus vulgaris L. : II. Pathway and Turgor Sensitivity

Phloem import and unloading in perfused bean (Phaseolus vulgaris L.) seed coats were investigated using steady-state labeling. Though photosynthate import and unloading were significantly reduced by perfusion, measurements of photosynthate fluxes in perfused seed coats proved useful for the study of unloading mechanisms in vivo. Phloem import was stimulated by lowered seed coat cell turgor, as demonstrated by an increase in tracer and sucrose import to seed coats perfused with high concentrations of an osmoticum. The partitioning of photosynthates between retention in the seed coat and release to the perfusion solution also was turgor sensitive; increases in seed coat cell turgor stimulated photosynthate release to the apoplast at the expense of photosynthate retention within the seed coat. There was no evidence of a turgor-sensitive sucrose uptake mechanism in perfused seed coats. Thus, the turgor sensitivity of photosynthate partitioning within perfused seed coats was consistent with a turgor-sensitive efflux control mechanism. Measurements of tracer equilibration and sugar partitioning in perfused seed coats provided strong evidence for symplastic phloem unloading in seed coats.     

Neo-clerodane Diterpenoids with Hypoglycemic Effects in Vivo from the Aerial Parts of Salvia hispanica L.

A new neo-clerodane diterpenoid, salvihispin H ( 1 ), and six known ones ( 2 – 7 ) were identified from the aerial parts of Salvia hispanica L. The structure and absolute configuration of 1 were elucidated by extensive analysis of spectroscopic (¹H, ¹³C, and 2D NMR, and HR-ESI-MS) and single-crystal X-ray diffraction data. The anti-diabetic effects of salvihispin H ( 1 ) and salvifaricin ( 2 ) were evaluated in diabetic db/db mice. The data showed that 1 and 2 could significantly reduce fasting blood glucose level and improve insulin resistance, and compound 1 exerted glucose-lowering effect more quickly than metformin. In addition, 1 and 2 could also reduce serum TG level in db/db mice. These results demonstrated that compounds 1 and 2 could be considered as potent candidates for the therapy of type 2 diabetes mellitus (T2DM).