A mathematical model of mucilage expansion in myxospermous seeds of Capsella bursa-pastoris (shepherd's purse)

Background and Aims

Myxospermy is a term which describes the ability of a seed to produce mucilage upon hydration. The mucilage is mainly comprised of plant cell-wall polysaccharides which are deposited during development of those cells that comprise the seed coat (testa). Myxospermy is more prevalent among those plant species adapted to surviving on arid sandy soils, though its significance in determining the ecological fitness of plants is unclear. In this study, the first mathematical model of myxospermous seed mucilage expansion is presented based on seeds of the model plant species Capsella bursa-pastoris (shepherd''s purse).

Methods

The structures underpinning the expansion process were described using light, electron and time-lapse confocal micrographs. The data and experimental observations were used to create a mathematical model of myxospermous seed mucilage expansion based on diffusion equations.

Key Results

The mucilage expansion was rapid, taking 5 s, during which the cell mucilage volume increased 75-fold. At the level of the seed, this represented a 6-fold increase in seed volume and a 2·5-fold increase in seed surface area. These increases were shown to be a function of water uptake (16 g water g⁻¹ mucilage dry weight), and relaxation of the polymers which comprised the mucilage. In addition, the osmotic pressure of the seed mucilage, estimated by assessing the mucilage expansion of seeds hydrated in solutions of varying osmotic pressure, was –0·54 MPa (equivalent to 0·11 m or 6·6 g L⁻¹ NaCl).

Conclusions

The results showed that the mucilage may be characterized as hydrogel and seed-mucilage expansion may be modelled using the diffusion equation described. The potential of myxospermous seeds to affect the ecological services provided by soil is discussed briefly.     

Co-adaptation of seed dormancy and flowering time in the arable weed Capsella bursa-pastoris (shepherd's purse)

Background and Aims

The duration of the plant life cycle is an important attribute that determines fitness and coexistence of weeds in arable fields. It depends on the timing of two key life-history traits: time from seed dispersal to germination and time from germination to flowering. These traits are components of the time to reproduction. Dormancy results in reduced and delayed germination, thus increasing time to reproduction. Genotypes in the arable seedbank predominantly have short time to flowering. Synergy between reduced seed dormancy and reduced flowering time would create stronger contrasts between genotypes, offering greater adaptation in-field. Therefore, we studied differences in seed dormancy between in-field flowering time genotypes of shepherd''s purse.

Methods

Genotypes with early, intermediate or late flowering time were grown in a glasshouse to provide seed stock for germination tests. Secondary dormancy was assessed by comparing germination before and after dark-incubation. Dormancy was characterized separately for seed myxospermy heteromorphs, observed in each genotype. Seed carbon and nitrogen content and seed mass were determined as indicators of seed filling and resource partitioning associated with dormancy.

Key Results

Although no differences were observed in primary dormancy, secondary dormancy was weaker among the seeds of early-flowering genotypes. On average, myxospermous seeds showed stronger secondary dormancy than non-myxospermous seeds in all genotypes. Seed filling was similar between the genotypes, but nitrogen partitioning was higher in early-flowering genotypes and in non-myxospermous seeds.

Conclusions

In shepherd''s purse, early flowering and reduced seed dormancy coincide and appear to be linked. The seed heteromorphism contributes to variation in dormancy. Three functional groups of seed dormancy were identified, varying in dormancy depth and nitrate response. One of these groups (FG-III) was distinct for early-flowering genotypes. The weaker secondary dormancy of early-flowering genotypes confers a selective advantage in arable fields.     

Spatial distribution of mRNA during pre-fertilization ovule development in Capsella bursa-pastoris

Summary In situ hybridization of sections of the ovary and ovule of Capsella bursa-pastoris with ³H-polyuridylic acid [³H-poly(U)] showed the presence of polyadenylic acid-containing RNA [poly(A) + RNA] in the cells of the placenta and nucellus. During megasporogenesis there was a decrease in ³H-poly(U) binding activity of the nucellar cells concomitant with the appearance of poly(A) + RNA in the integuments. As the typical eight-nucleate embryo sac was formed, ³H-poly(U) binding was not apparent around the quartet of nuclei at the chalazal end, while it persisted at the micropylar end. Both the egg and synergids as well as the chalazal proliferating tissue showed high concentrations of poly(A) + RNA in their cytoplasm. The results suggest a role for transient localizations of poly(A) + RNA during female sporogenesis and gametogenesis in C. bursa-pastoris.     

Production and genetic analysis of partial hybrids in intertribal crosses between Brassica species (B. rapa, B. napus) and Capsella bursa-pastoris

Capsella bursa-pastoris (L.) Medic (2n = 4x = 32) is a natural double-low (erucic acid < 1%, glucosinolates < 30 micromol/g) germplasm and shows high degree of resistance to Sclerotinia sclerotiorum. Hybridizations were carried out between two Brassica species viz. B. rapa (2n = 20) and B. napus (2n = 38) as female and C. bursa-pastoris as male parent to introduce these desirable traits into cultivated Brassica species. Majority of F(1) plants resembled female parents in morphology and only a few expressed some characters of male parent, including the white petals. Based on cytological observation of somatic cells, the F(1) plants were classified into five types: two types from the cross with B. rapa, type I had 2n = 27-29; type II had 2n = 20; three types from the crosses with B. napus, type III was haploids with 2n = 19; type IV had 2n = 29; type V had 2n = 38. One to two chromosomes of C. bursa-pastoris were detected in pollen mother cells (PMCs) of type I plant by genomic in situ hybridization (GISH), together with chromosomal segments in ovary cells and PMCs of some F1 plants. Amplified fragment length polymorphism (AFLP) bands specific for the male parent, novel for two parents and absent bands in Brassica parents were generated in majority of F1 plants, even in Brassica-types and haploids, indicating the introgressions at various levels from C. bursa-pastoris and genomic alterations following hybridization. Some Brassica-type progeny plants had reduced contents of erucic acid and glucosinolates associated with improved resistance to S. sclerotiorum. The cytological and molecular mechanisms behind these results are discussed.     

Somatic embryogenesis from cell suspension and protoplast cultures ofCapsella bursa-pastoris (L.) Medic

Summary Rapidly growing cell suspension cultures of shepherd’s purse (Capsella bursa-pastoris L. Medic.) were established from leaf-derived calli. These suspensions remained unorganized in the presence of 2,4-D, but underwent extensive root organogenesis in a growth regulator-free liquid medium. Attempts to induce direct embryogenesis in liquid cultures were unsuccessful, but numerous embryos were obtained from cells plated onto growth-regulator-free solid medium. These embryos were frequently abnormal, and secondary embryogenesis was problematic for plant recovery but fertile plants were recovered. Viable protoplasts could readily be isolated from these cell suspensions. After 1 wk of culture, protoplast viability was 62%, and 7% of the cells had divided. Embryogenesis was observed from protoplast-derived microcolonies, plated on growth-regulator-free medium. Although these somatic embryos were difficult to root, plants were recovered. New cell suspensions were more recently established, which were only 4 to 6 mo. old when plant regeneration was attempted. Numerous shoots were obtained when these cells were plated onto growth-regulator-free solid media. However, these shoots differed from the embryos previously obtained in that they readily rooted and rapidly developed into plantlets. This system may allow the use of shepherd’s purse as a gene source for introgression of agronomically interesting traits intoBrassica crop species through protoplast manipulation and somatic hybridization.     

An antimicrobial peptide of the earthworm Pheretima tschiliensis: cDNA cloning, expression and immunolocalization

A cDNA encoding a putative antimicrobial peptide (named PP-1) was obtained using a rapid amplification of cDNA ends from the Asian earthworm, Pheretima tschiliensis. PP-1 showed 77.6% homology with the antimicrobial peptide lumbricin I isolated from the earthworm Lumbricus rubellus. PP-1 lacked an obvious signal peptide sequence. RT-PCR analysis demonstrated that this gene was expressed mainly in the body wall. PP-1 was expressed in Escherichia coli as a fusion protein with a maltoze-binding protein. A polyclonal antiserum was raised in mice using this recombinant fusion protein as antigen. Immunohistochemical studies showed that PP-1 was only in the mucus of the epidermis.     

Antibacterial activities of peptides designed as hybrids of antimicrobial peptides

Hybrid peptides (HP-MA, HP-ME), each of 20 residues and incorporating 2–9 residues of Helicobacter pylori ribosomal protein L1 (HP) and 1–12 residues of magainin 2 and melittin, were designed. The antibiotic activities of these peptides were evaluated using bacterial, tumor and human erythrocyte cells. HP-MA had a stronger antibacterial activity against Gram-positive bacteria and Gram-negative bacteria than HP (2-20) and magainin 2, and HP-ME was similar to melittin. None of the hybrids had anti-tumor or hemolytic activity. These peptides were further investigated using an artificial liposomal vesicle and 1,6-diphenyl-1,3,5-hexatriene as a membrane probe, and confirmed to have similar antibacterial activities. The antibacterial effect of these hybrids is probably caused by their ability to damage the bacterial plasma membrane. Additional circular dichroism spectra suggested that the -helical structure of these peptides plays an important role in their antibiotic effect but that -helical property is less connected with the enhanced antibiotic activity.     

Capsella as a model system to study the evolutionary relevance of floral homeotic mutants

Several lines of evidence suggest that homeotic changes played a considerable role during the evolution of flowers. This, however, is difficult to reconcile with the predominant evolutionary theory which rejects any drastic, saltational change of the phenotype as reasonable mode of evolution due to its assumed negative impact on the fitness of the affected organism. A better understanding of the evolutionary potential of homeotic transitions requires a study of the performance of respective mutant varieties in the wild. Here we introduce ``Stamenoid petals' (Spe), a variety of Capsella bursa-pastoris (shepherd's purse), as a suitable model to study the evolutionary potential of floral homeotic mutants. In the flowers of the Spe variety all petals are transformed into stamens, while all other floral organs are unaffected. In contrast to most other homeotic mutants the Spe variety occurs on several locations in relatively large and stable populations in the wild. Due to its close relationship to the model plant Arabidopsis thaliana, the Spe variety of C. bursa-pastoris can be rigorously studied, from the molecular genetic basis of the phenotype to its consequences on the fitness in wild habitats. Investigations on Spe may thus help to clarify whether homeotic transformations have the potential to contribute to macroevolution.     

L-lactate dehydrogenase from leaves of Capsella bursa-pastoris (L.) Med.

By ammonium sulfate fractionation and gel filtration an enzyme preparation which catalyzed NAD⁺-dependent L-lactate oxidation (10^-4 kat kg^-1 protein), as well as NADH-dependent pyruvate reduction (10^-3 kat kg^-1 protein), was obtained from leaves of Capsella bursa-pastoris. This lactate dehydrogenase activity was not due to an unspecific activity of either glycolate oxidase, glycolate dehydrogenase, hydroxypyruvate reductase, alcohol dehydrogenase, or a malate oxidizing enzyme. These enzymes could be separated from the protein displaying lactate dehydrogenase activity by gel filtration and electrophoresis and distinguished from it by their known properties. The enzyme under consideration does not oxidize D-lactate, and reduces pyruvate to L-lactate (the configuration of which was determined using highly specific animal L-lactate dehydrogenase). Based on these results the studied Capsella leaf enzyme is classified as L-lactate dehydrogenase (EC 1.1.1.27). It has a K_m value of 0.25 mmol l^-1 (pH 7.0, 0.3 mmol l^-1 NADH) for pyruvate and of 13 mmol l^-1 (pH 7.8, 3 mmol l^-1 NAD⁺) for L-lactate. Lactate dehydrogenase activity was also detected in the leaves of several other plants.Abbreviation FMN flavin adenine mononucleotide     

Halocyntin and papillosin,two new antimicrobial peptides isolated from hemocytes of the solitary tunicate,Halocynthia papillosa

We report here the screening of five marine invertebrate species from two taxa (tunicates and echinoderms) for the presence of cationic antimicrobial peptides (AMP) in defence cells (hemocytes). Antimicrobial activities were detected only in the two tunicates Microcosmus sabatieri and Halocynthia papillosa. In addition, we report the isolation and characterization of two novel peptides from H. papillosa hemocytes. These molecules display antibacterial activity against Gram‐positive and Gram‐negative bacteria. Complete peptide characterization was obtained by a combination of Edman degradation and mass spectrometry. The mature molecules, named halocyntin and papillosin, comprise 26 and 34 amino acid residues, respectively. Their primary structure display no significant similarities with previously described AMP. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Characterization of two crustin antimicrobial peptides from the freshwater crayfish Pacifastacus leniusculus

The two bacteria-induced crustin genes, Plcrustin1 and Plcrustin2, previously found in the hemocyte cDNA library of Pacifastacus leniusculus, contain the open reading frames of 357 bp encoding a putative protein of 118 amino acid residues and 330 bp encoding a putative protein of 109 amino acid residues, respectively. The carboxyl-terminal part of the two crustins possesses, respectively, 7 and 8 conserved cysteine residues representation of a WAP domain that is found in carcinins and crustins in other several crustaceans. The amino acid sequences of Plcrustin1 and Plcrustin2 show that they belong to type I crustins. In order to characterize their properties and biological activities, the two recombinant crustin proteins were produced in the Escherichia coli expression system. Antimicrobial assays showed that the growth of only one Gram-positive bacterium, Micrococcus luteus M1 11, was inhibited by the recombinant Plcrustin1 and Plcrustin2 with MIC of about 0.07-0.27 μM and 3.5-8 μM, respectively. In addition, the study of inhibition mechanism revealed that the antimicrobial activity of the two recombinant crustin proteins was a result of bactericidal effect. However, the two crustins did not exhibit the inhibitory activities against trypsin, chymotrypsin, elastase and subtilisin A.     

Characterization of antimicrobial peptides isolated from the skin of the Chinese frog, Rana dybowskii

The skins of amphibians secrete small antimicrobial peptides that fight infection and are being explored as potential alternatives to conventional antibiotics. In this study we combined mass spectrometry with cDNA sequencing to examine antimicrobial peptides in skin secretions from the Chinese frog Rana dybowskii. Thirteen peptides having precursor sequences that resemble known antimicrobial peptides from this genus were identified, ten of which were members of previously described peptide families based on their primary structures; i.e., brevinin-1, Japonicin-1, brevinin-2 and temporin. The other three peptides from R. dybowskii, which were named dybowskin-1CDYa, dybowskin-2 CDYa and dybowskin-2CDYb, had different amino acid compositions and little sequence similarity to known antimicrobial peptides. The carboxyl terminus of dybowskin-1CDY lacked amidation and is therefore clearly distinct from temporin peptides, whereas dybowskin-2CDYa and dybowskin-2CDYb consisted of 18 amino acids and were rich in Arg residues. Chemically synthesized peptides corresponding to mature dybowskin-1CDYa and dybowskin-2CDYa had strong antimicrobial activity and caused little hemolysis of human erythrocytes, suggesting they may serve as interesting templates for the development of novel antibiotics.     

Molecular cloning and characterization of antimicrobial peptides from skin of the broad-folded frog, Hylarana latouchii

Seven cDNA sequences encoding antimicrobial peptide (AMP) precursors were cloned by screening the skin-derived cDNA library of the broad-folded frog, Hylarana latouchii. Seven of the deduced peptides are highly similar to AMPs in five families of brevinin-2 (brevinin-2LTa, brevinin-2LTb, and brevinin-2LTc), esculentin-1 (esculentin-1LTa), esculentin-2 (esculentin-2LTa), palustrin-2 (palustrin-2LTa), and temporin (temporin-LTe). The actual sequences and characteristics of mature AMPs were analyzed by RP-HPLC and LC–MS/MS-based proteomics approaches in combination of four different protein digestion processes and by LTQ XL in combination of gas-phase fractionation (GPF) analysis. Moreover, most of the peptides found in this study hardly display hemolytic activity in vitro, suggesting they are promising antimicrobial drug candidates.     

Purification and cloning of a novel antimicrobial peptide from salivary glands of the hard tick, Ixodes sinensis

A novel antimicrobial peptide named as ixosin-B was isolated from the salivary glands of the hard tick, Ixodes sinensis, by gel filtration, ion exchange chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Its amino acid sequence was determined as QLKVDLWGTRSGIQPEQHSSGKSDVRRWRSRY by Edman degradation. The cDNA encoding ixosin-B was cloned by cDNA library screening. The predicted protein from the cDNA sequence composed of 89 amino acids including mature ixosin-B. Purified ixosin-B exerted its antimicrobial activities against bacteria and fungi. No similarity was found by BLAST search to any database entries and, thus, our findings describe a novel antimicrobial peptide. It is also the fourth family of antimicrobial peptide from hard ticks.