Growth Dynamics of Salmonella enterica Strains on Alfalfa Sprouts and in Waste Seed Irrigation Water

Alfalfa sprouts and other seed sprouts have been implicated in numerous outbreaks of salmonellosis. The source of these epidemics appears to have been low-level contamination of seeds by Salmonella bacteria that developed into clinically significant populations during the seed germination process. To test the possibility that Salmonella enterica strains carry host range determinants that allow them to grow on alfalfa, strains isolated from alfalfa or other sources were surveyed for their ability to grow on germinating alfalfa seeds. An S. enterica serovar Cubana strain originally isolated from contaminated alfalfa sprouts multiplied most rapidly during the initial 24 h of the seed germination process. Germinating alfalfa seeds supported the multiplication of S. enterica cells prior to the emergence of the root radicle at 72 h. Thereafter, much lower rates of multiplication were apparent. The ability of S. enterica to grow on germinating alfalfa seeds was independent of the serovar, isolation source, or virulence of the strain. Isolates obtained from alfalfa attained population levels similar to those observed for strains isolated from contaminated meat products or stools. Each of the strains could be detected in the waste irrigation water, with populations being strongly correlated with those detected on the germinating alfalfa seeds. The S. enterica strains were capable of utilizing the waste irrigation water as a sole carbon and nitrogen source. S. enterica strains thus appear to grow saprophytically on soluble organics released from seeds during early phases of germination. The ability to detect S. enterica in the waste irrigation water early in the germination process indicates that this method may be used as a simple way to monitor the contamination of sprouts during commercial operations.     

A convenient method for the synthesis of methyl 2-acetamido-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-α-d-glucopyranoside

In the seeds of Tropaeolum and Tamarindus, which have been found to have the highest content of amyloids, these polysaccharides are associated with sucrose and O-d-galactosylsucroses of the raffinose-stachyose series. Balsamine seeds have a low content of amyloid and do not contain the aforementioned galactosides, which are otherwise widely distributed in plant seeds. They contain sucrose and a mono-O-D-galactosylsucrose that has been crystallized and identified as planteose. Planteose has previously been isolated from some Sympetalous plants. As far as we know, this is the first report of its occurrence in an Archichlamydeaeous plant seed.     

Imidazole,a new natural product from the leguminosae

Imidazole was isolated from Lens culinaris seeds as its dansyl derivative and shown to be identical to a synthetic standard by co-chromatography, ¹H NMR and EIMS. Chromatographic evidence indicated that it was also present in the seeds of 11 other legumes, 30% of those tested.     

Defense peptides from barnyard grass (Echinochloa crusgalli L.) seeds

A number of defense polypeptides from latent seeds of weed cereal barnyard grass (Echinochloa crusgalli L.) has been isolated and characterized using an acidic extraction and high performance liquid chromatography methods in combination with MALDI-TOF mass spectrometry and Edman sequencing. Members of three antimicrobial peptide families and two protease inhibitor families were found to be localized in barnyard grass seeds. Their biological activity concerning to Gram-Positive and Gram-Negative phytopathogenic bacteria, as well as oomycete Phytophthora infestans, has been investigated. Diversity of barnyard grass defense peptides is a significant factor that provides a resistance of E. crusgalli seeds to germination and latent phases.     

2-Aminoimidazole in seeds of Mundulea sericea

An amine present as ca 0.9 % of the fr. wt of the seeds of the legume Mundulea sericea has been isolated and characterised as 2-aminoimidazole     

2-amino-4-acetylaminobutyric acid, 2,4-diaminobutyric acid and 2-amino-6N-oxalylureidopropionic acid (oxalylalbizziine) in seeds of acacia angustissima

A new amino acid previously detected in 17 species of Acacia has been isolated from seeds of Acacia angustissima and identified as oxalylalbizziine. These seeds also contain more than 6% dry weight of 2-amino-4-acetylaminobutyric acid, which has not been reported previously in a legume, and lower concentrations of 2,4-diaminobutyric acid.     

Different effects on triacylglycerol packaging to oil bodies in transgenic rice seeds by specifically eliminating one of their two oleosin isoforms

Expression of OLE16 and OLE18, two oleosin isoforms in oil bodies of rice seeds, was suppressed by RNA interference. Electron microscopy revealed a few large, irregular oil clusters in 35S::ole16i transgenic seed cells, whereas accumulated oil bodies in 35S::ole18i transgenic seed cells were comparable to or slightly larger than those in wild-type seed cells. Large and irregular oil clusters were observed in cells of double mutant seeds. These unexpected differences observed in oil bodies of 35S::ole16i and 35S::ole18i transgenic seeds were further analyzed. In comparison to wild-type plants, OLE18 levels were reduced to approximately 40% when OLE16 was completely eliminated in 35S::ole16i transgenic plants. In contrast, OLE16 was reduced to only 80% of wild-type levels when OLE18 was completely eliminated in 35S::ole18i transgenic plants. While the triacylglycerol content of crude seed extracts of 35S::ole16i and 35S::ole18i transgenic seeds was reduced to approximately 60% and 80%, respectively, triacylglycerol in isolated oil bodies was respectively reduced to 45% and 80% in accordance with the reduction of their oleosin contents. Oil bodies isolated from both 35S::ole16i and 35S::ole18i transgenic seeds were found to be of comparable size and stability to those isolated from wild-type rice seeds, although they were merely sheltered by a single oleosin isoform. The drastic difference between the triacylglycerol contents of crude seed extracts and isolated oil bodies from 35S::ole16i transgenic plants could be attributed to the presence of large, unstable oil clusters that were sheltered by insufficient amounts of oleosin and therefore could not be isolated together with stable oil bodies.     

Asparagine Enhances Starch Accumulation in Developing and Germinating Lupin Seeds

Regulation of starch accumulation in yellow (Lupinus luteus L.), white (L. albus L.), and Andean lupin (L. mutabilis Sweet) developing and germinating seeds was investigated. Research was conducted on cotyledons isolated from developing seeds as well as on organs of germinating seeds, that is, isolated embryo axes, excised cotyledons, and seedling axes and cotyledons. All organs were cultured in vitro for 96 h in different carbon (60 mM sucrose) and nitrogen (35 mM asparagine or 35 mM nitrate) conditions. Ultrastructure observation showed one common pattern of changes in the number and size of starch granules caused by sucrose, asparagine, and nitrate in both developing and germinating seeds. Sucrose increased the number and size of starch granules. Asparagine additionally increased starch accumulation (irrespective of sucrose nutrition) but nitrate had no effect on starch accumulation. Asparagine treatment resulted in a significant decrease in soluble sugar level in all organs of germinating lupin seeds of the three species investigated. The above-mentioned changes were most clearly visible in white lupin organs. In white lupin, starch granules were visible even in cells of sucrose-starved isolated embryo axes where advanced autophagy occurs. The importance of asparagine-increased starch content in the creation of a strong source–sink gradient in developing and germinating lupin seeds is discussed.     

Isolimonic acid,a new citrus limonoid

Isolimonic acid was isolated as its methyl ester from seeds of Citrus aurantium, C. reticulata and C. paradisi, and its structure was determined by ¹H and ¹³C NMR. The isomeric limonoid ichangin was also isolated from C. aurantium and C. paradisi.     

Role of indehiscent pericarp in formation of soil seed bank in five cold desert Brassicaceae species

The dispersal and germination unit of some Brassicaceae species is the fruit, and we hypothesized that it could affect germination phenology and promote formation of a soil seed bank. We determined the effects of the indehiscent pericarp on germination and longevity of buried seeds of five Brassicaceae species native to cold deserts of central Asia. Germination phenology (seedling emergence) was monitored for intact dispersal units and isolated seeds of Chorispora sibirica, Goldbachia laevigata, Spirorrhynchus sabulosus, Tauscheria lasiocarpa (annuals), and Sterigmostemum fuhaiense (perennial) at natural temperatures in watered and non-watered (natural precipitation) soil. Intact dispersal units and isolated seeds were buried under natural conditions and exhumed at regular intervals for 35 months to monitor germination, viability and moisture content of isolated seeds, seeds in dispersal units, and seeds removed from dispersal units after burial. Isolated seeds of Goldbachia, Spirorrhynchus, and Tauscheria germinated only the first autumn and those of Chorispora and Sterigmostemum the first autumn and first spring, with higher germination percentages in all species in watered than in non-watered soil. A high percentage of seeds in buried dispersal units of Chorispora, Goldbachia, and Sterigmostemum was viable after 35 months, and seeds exhibited a 6-month dormancy cycle, being non-dormant only in autumn and spring. Seeds in buried dispersal units of Spirorrhynchus and Tauscheria germinated when exhumed in the first spring, but all non-germinated seeds were dead after 1 year. Thus, the presence of the pericarp allows Chorispora, Goldbachia, and Sterigmostemum to form a persistent seed bank but not Spirorrhynchus and Tauscheria.     

24β-Ethyl-31-norlanosta-8,25(27)-dien-3β-ol and 24β-ethyl-25(27)-dehydrolophenol in seeds of three cucurbitaceae species

The structures of two 4α-methylsterols is isolated from Cucumis sativus(Cucurbitaceae) seeds were determined based mainly on their ¹³CNMR spectra as 24β-ethyl-31-norlanosta-8,25(27)-dien-3β-ol and 24β-ethyl-25(27)- dehydrolophenol, respectively, of which the former is a new sterol from natural sources. These two 4α-methylsterols were identified in the seeds of two other Cucurbitaceae species, Lagenaria leucantha var. Gourda and Citrullus battich. The probable biogenetic significance of the two 4α-methylsterols is discussed. Other 4α-methylsterols identified in the seeds of the three Cucurbitaceae species were obtusifoliol, cycloeucalenol and gramisterol.     

A reappraisal of the free amino acids in seeds of Crotalaria Juncea (Leguminosae)

The seeds of Crotalaria juncea have been reported at different times to contain β-hydroxy-N-methyl-dl-norvaline and δ-hydroxynorleucine, which are isomers. We detected only one non-protein amino acid in seeds obtained from various sources; it has been isolated its identity as δ-hydroxynorleucine confirmed.     

In vitro symbiotic seed germination of South Indian endemic orchid Coelogyne nervosa

Study on the dependence of orchids on fungi for seed germination and seedling development provides a mean for understanding the role of fungi in the orchid development process. The epiphytic orchid Coelogyne nervosa endemic to south India is exploited in an unsustainable manner for its therapeutic value. So a protocol for symbiotic seed germination was established for C. nervosa. We isolated a fungus by plating mycorrhizal root discs of the terrestrial orchid Eulophia epidendreae and identified it as Epulorhiza sp., by sequencing the internal transcribed spacer (ITS) regions of the ribosomal RNA gene. Germination of C. nervosa seeds was higher when inoculated with Epulorhiza sp. Uninoculated seeds of C. nervosa ceased to develop soon after the initiation of germination, and the embryo failed to rupture the seed testa. The isolated fungal hyphae entered the germinating seeds either through the pores in-between the integuments, or through the rhizoids. After the fungal establishment (peloton formation) in embryonic cells, the embryo transformed into a protocorm and after 45 days, 66% of the germinated seeds were transformed into protocorms. Nevertheless, promeristem formation occurred only after fungal association. Sixty-three percent of the protocorms developed their first leaf by 90 days and 62% of these produced a second leaf by 120 days after fungal inoculation. All the seedlings in green leaf stage produced roots and contained fungal pelotons. Our results suggest that the Epulorhiza sp. could be successfully used in the in vitro production of C. nervosa for their reintroduction into its natural environment.     

Seed deposition patterns and the survival of seeds and seedlings of the palm Euterpe edulis

The seed deposition pattern created by a seed disperser is one of the components of the efficiency of a species as seed disperser, and ultimately may influence the recruitment of a plant species. In this study, we used the seeds of a bird-dispersed forest palm, Euterpe edulis, to investigate the effects of two distinct seed deposition patterns created by birds that defecate (clumped pattern) and regurgitate seeds (loose-clumped pattern) on the survival of seeds experimentally set in an E. edulis-rich site, and of seedlings grown under shade-house conditions. The study was conducted in the lowland forest of Parque Estadual Intervales, SE Brazil. Clumped and loose-clumped seeds were equally preyed upon by rodents and insects. Although clumped and isolated seedlings had the same root weight after 1 year, the isolated seedlings survived better and presented more developed shoots, suggesting intraspecific competition among clumped seedlings. Our results indicate that animals that deposit E. edulis seeds in faecal clumps (e.g. cracids, tapirs) are less efficient seed dispersers than those that regurgitate seeds individually (e.g. trogons, toucans). Intraspecific competition among seedlings growing from faecal clumps is a likely process preventing the occurrence of clumps of adult palms.     

Profiling of phenylpropanoids in transgenic low-sinapine oilseed rape (Brassica napus)

A dsRNAi approach silencing a key enzyme of sinapate ester biosynthesis (UDP-glucose:sinapate glucosyltransferase, encoded by the UGT84A9 gene) in oilseed rape (Brassica napus) seeds was performed to reduce the anti-nutritive properties of the seeds by lowering the content of the major seed component sinapine (sinapoylcholine) and various minor sinapate esters. The transgenic seeds have been produced so far to the T6 generation and revealed a steady suppression of sinapate ester accumulation. HPLC analysis of the wild-type and transgenic seeds revealed, as in the previous generations, marked alterations of the sinapate ester pattern of the transformed seeds. Besides strong reduction of the amount of the known sinapate esters, HPLC analysis revealed unexpectedly the appearance of several minor hitherto unknown rapeseed constituents. These compounds were isolated and identified by mass spectrometric and NMR spectroscopic analyses. Structures of 11 components were elucidated to be 4-O-glucosides of syringate, caffeyl alcohol and its 7,8-dihydro derivative as well as of sinapate and sinapine, along with sinapoylated kaempferol glycosides, a hexoside of a cyclic spermidine alkaloid and a sinapine derivative with an ether-bridge to a C₆-C₃-unit. These results indicate a strong impact of the transgenic approach on the metabolic network of phenylpropanoids in B. napus seeds. Silencing of UGT84A9 gene expression disrupt the metabolic flow through sinapoylglucose and alters the amounts and nature of the phenylpropanoid endproducts.     

Cissogenin,a pregnane genin from Marsdenia tenacissima

A new polyhydroxy pregnane designated as cissogenin has been isolated from the seeds of Marsdenia tenacissima. Its structure has been established as 3β,11α,12β,14β,20 S-pentahydroxy-pregn-5-ene.     

Biologically active gibberellins in immature seeds of Pyrus serotin

Biologically active gibberellins were isolated from immature seeds of Japanese pear (Pyrus serotina). The major one was tentatively identified as GA₄ by selected ion monitoring. Evidence for the possible presence of GA₇ in the seeds was inconclusive but GA₃ was not detected by either the rice seedling or barley half-seed bioassay.     

The lipoxygenases in developing soybean seeds, their characterization and synthesis in vitro

A number of lipoxygenase isoenzymes were identified in developing soybean (Glycine max L. Merrill cv Provar) seeds and two have been partially characterized. In a study of lipoxygenase level in developing soybean seeds, the enzyme content increased markedly during development. Comparisons of the lipoxygenases from mature soybean seeds and immature seeds by isoelectric focusing, chromatofocusing, sodium dodecyl sulfate polyacrylamide gel electrophoresis and peptide mapping identified two categories of isoenzyme. The isoenzymes from immature seeds were found by electron paramagnetic resonance spectroscopy to be isolated at least in part as the high spin iron(III) or active form of the enzyme in contrast to lipoxygenases from mature seeds which were isolated as electron paramagnetic resonance silent, high spin iron(II) species. The discovery of increased levels of lipoxygenases during seed development and their isolation in an active form suggests that the enzyme may play a physiological role during the maturation process. The incorporation of iron-59 from the nutrient medium into lipoxygenase during culture of immature seeds was indicative of de novo synthesis of the enzyme. The efficiency of the iron uptake was high, as indicated by the level of radioactivity found in the enzyme (one gram atom of iron per mole of lipoxygenase).     

Isolation and Activity Analysis of a Seed-Abundant soyAP1 Gene Promoter from Soybean

The soybean aspartic proteinase gene soyAP1 has previously been shown to be expressed specifically in soybean seeds. To investigate the expression pattern and active cis-elements of the soyAP1 promoter, the 1,650-bp 5??-upstream genomic DNA fragment named PS-552 was isolated by PCR walking. Sequence analysis revealed that this fragment contains a series of motifs related to seed-specific promoters and some pollen-expressed elements. Stable expression in transgenic Arabidopsis thaliana showed that the PS-552 promoter can regulate beta-glucuronidase gene accumulation in mature seeds at much higher levels than other tissues, especially vegetative tissues, and exhibits similar activity to the 35S promoter in mature seeds. These results show that the PS-552 promoter is a highly active promoter controlling downstream gene expression, mainly in mature seeds. The 5??-end deletion studies of PS-552 showed that the cis-elements of CAAACAC, AACA, E-box, and CCAA play a role in increasing the seed-specific activity. The proportion of mature seed activity and flower activity was increased as the deletion fragment lengthened, indicating that seed cis-elements possibly lessen or suppress the effect of pollen-expressed elements, increasing the activity of PS-552 in mature seeds.