Characterization of five new low-molecular-mass trypsin inhibitors from white mustard (Sinapis alba L.) seed.

Five new low-molecular-mass trypsin inhibitors belonging to the RTI/MTI-2 family were identified from white mustard (Sinapis alba L. ; MTI-2) seed. Purified MTI-2 consisted of a peptide mixture, displaying Ile or Arg at position 43, Trp or kynurenine (Kyn) at position 44, and C-terminal ragged ends. The occurrence of Ile or Arg at position 43 suggested that MTI-2 inhibitors originated from different genes. The presence of 5-oxo-proline (pyroglutamic acid; 5-oxoPro1) and Kyn44 reflected post-translational processing of the serine proteinase inhibitor. MTI-2 showed approximately 70% amino-acid identity with low-molecular-mass trypsin inhibitors isolated from oil rape (Brassica napus var. oleifera; RTI-III) seed and with serine proteinase inhibitors mapped in Arabidopsis thaliana chromosome II (ATTI). Furthermore, MTI-2 was homologous to brazzein, the sweet-tasting protein from Pentadiplandra brazzeana Baillon fruit ( approximately 30% amino-acid identity). Although snake-venom toxins showed a low amino-acid identity (< 20%) with MTI-2, RTI-III, and ATTI, some structurally relevant residues were conserved. The disulfide bridge pattern of MTI-2 (Cys5-Cys27, Cys18-Cys31, Cys42-Cys52, and Cys54-Cys57) corresponded to that of RTI-III and of snake-venom toxins, being different from that of brazzein. Therefore, protein similarity might be attributable to the three-dimensional arrangement rather than to the amino-acid sequence. Values of Ka for MTI-2 binding to bovine beta-trypsin (trypsin) and bovine alpha-chymotrypsin were 6.3 x 109 M-1 and 2.0 x 106 M-1, respectively, at pH 8.0 and 21.0 degrees C. Moreover, values of kon for MTI-2 binding to trypsin and of koff for the dissociation of the serine proteinase:inhibitor complex were 5.6 x 105 M-1.s-1 and 8.9 x 10-5 M-1.s-1, respectively, at pH 8.0 and 21.0 degrees C. Despite the heterogeneity of the purified inhibitor peptide mixture, the inhibition properties of the different MTI-2 inhibitors were indistinguishable.     

Regeneration capacity of selected genotypes of white mustard (Sinapis alba L.)

Traditional breeding methods based on inbreeding are difficult to implement in the case of Sinapis alba (white mustard) because this plant displays high levels of self-incompatibility. More rapid progress in breeding could be possible if biotechnological methods and in vitro cultures were used. However, white mustard is not readily amenable to biotechnological treatment. Seeds of traditional S. alba cultivars (e.g., Nakielska) are characterized by high levels of glucosinolates and erucic acid. However, a new Polish variety of white mustard (Bamberka) possesses low erucic acid content in the oil. The main goal of the study was elaboration of a plant regeneration system via in vitro culture of hypocotyl and cotyledon explants from low and high erucic acid-containing white mustard cultivars. In these experiments, a simple system for in vitro regeneration of white mustard was developed, with the aim to promote maximum formation of shoots within a short period of time. Traditional and improved cultivars of S. alba showed comparable capacity for shoot development from hypocotyl-derived and cotyledon-derived explants. The two types of cultivars were characterized by essentially equivalent shoot regeneration responses, being slightly higher in hypocotyl than the cotyledonary explants. A greater influence on shoot regeneration from hypocotyl explants was observed on medium supplemented with 4.4 μmol 6-benzylaminopurine, 0.57 μmol indole-3-acetic acid, and a low concentration of kinetin (4.6 μmol). This technique will allow for rapid generation of sufficient plant material for further use in a variety of white mustard breeding projects.     

Analysis of light-controlled accumulation of carotenoids in mustard (Sinapis alba L.) seedlings

Carotenoid accumulation in the cotyledons of the mustard seedling (Sinapis alba L.) is controlled by light. Besides the stimulatory function of phytochrome in carotenogenesis the experiments reveal the significance of chlorophyll accumulation for the accumulation of larger amounts of acrotenoids. A specific blue light effect was not found. The data suggest that light exerts its control over carotenoid biogenesis through two separate mechanisms: A phytochrome regulation of enzyme levels before a postulated pool of free carotenoids, and a regulation by chlorophyll draining the pool by complex-formation.Abbreviations Chl chlorophyll(s) - PChl protochlorophyll(ide) - HIR high irradiance reaction (of phytochrome) - P_fr far-red absorbing, physiologically active form of phytochrome - P_r red absorbing, physiologically inactive form of phytochrome - P_fof total phytochrome, i.e. [P_r]+[P_fr] - [P_fr]/[P_fof], wavelength dependent photoequilibrium of the phytochrome system - red red light - fr far-red light     

Phytochrome-mediated development of mitochondria in the cotyledons of mustard (Sinapis alba L.) seedlings

Summary The development of mitochondria from promitochondria is regulated by phytochrome. This conclusion is based on four lines of evidence: 1. The activity of representative mitochondrial marker enzymes (fumarase, EC 4.2.1.2; succinate dehydrogenase, EC 1.3.99.1; cytochrome oxidase, EC 1.9.3.1) is increased by continuous far-red light and (in 2 of the 3 enzymes) by brief red pulses, the effect of which is reversible by brief far-red pulses. These effects do not merely represent a general growth or proliferation of mitochondria already present but specific responses of individual enzymes. Inhibitors of protein synthesis but not of RNA synthesis suppress the increase of these enzyme activities. 2. Continuous far-red light changes some structural properties of the mitochondrial membranes, detectable by an increased requirement of detergent (Triton X-100) for the solubilization of cytochrome oxidase and a more efficient retainment of the matrix enzyme fumarase during isolation of mitochondria. Continuous far-red light increases the apparent buoyant density of mitochondria on a sucrose density gradient. 3. Continuous far-red light has a strong effect on the morphology of the inner mitochondrial membrane system. Electron micrographs from dark-grown cotyledons show arrays of parallel, plate-like cristae while typical plant mitochondria with irregularly oriented sacculi are formed in the light. These responses indicate the involvement of mitochondria in cytophotomorphogenesis during the transition of the cotyledons from dissimilatory to assimilatory metabolism.Abbreviations DCPIP 2.6-dichlorophenole indophenole - EDTA Na₂-ethylenediaminetetraacetate - HEPES 2-[4-(2-hydroxyethyl)-piperazine-(1)ethanesulfonic acid - PMS phenazine methosulfate     

Agrobacterium-mediated transformation of white mustard (Sinapis alba L.) and regeneration of transgenic plants

Summary A procedure for the regeneration of fertile transgenic white mustard (Sinapis alba L.) is presented. The protocol is based on infection of stem explants of 7–9 day old plants with an Agrobacterium tumefaciens strain harboring a disarmed binary vector with chimeric genes encoding neomycin phosphotransferase and -glucuronidase. Shoots are regenerated from callus-forming explants within 3–4 weeks. Under selection, 10% of the explants with transgenic embryonic callus develop into fertile transgenic plants. Rooting shoots transferred to soil yield seeds within 14–16 weeks following transformation. Integration and expression of the T-DNA encoded marker genes was confirmed by histochemical glucuronidase assays and Southern-DNA hybridization using primary transformants and S1-progeny. The analysis showed stable integration and Mendelian inheritance of trans-genes in transformed Sinapis lines.Abbreviations BAP 6-benzylaminopurine - CaMV cauliflower mosaic virus - GUS -glucuronidase - IBA indole-3-butyric acid - IM infection medium - NAA 1-naphthalene acetic acid - neo gene encoding NPTII - NPTII neomycin phosphotransferase - RIM root-inducing medium - SEM shoot-elongation medium - SIM shoot-inducing medium - t-nos polyadenylation site of the nopaline synthase gene - uidA gene encoding GUS - WM wash medium - X-Gluc 5-bromo-4-chloro-3-indolyl -D-glucuronide     

Phytochrome control of plastid mRNA in mustard (Sinapis alba L.)

The steady-state levels of plastid RNA sequences in dark-grown and light-grown mustard (Sinapis alba L.) seedlings have been compared. Total cellular RNAs were labeled in vitro with ³²P and hybridized to separated restriction fragments of plastid DNA. Cloned DNA fragments which encode the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxylase (dimerizing), EC 4.1.1.39] and a 35,000 plastid polypeptide were used as probes to assess the levels of these two plastid mRNAs. The 1.22-kilobase-pair mRNA for the 35,000 polypeptide is almost undetectable in dark-grown seedlings, but is a major plastid mRNA in light-grown seedlings. The hybridization analysis of RNA from seedlings which were irradiated with red and far-red light indicates that the level of this mRNA, but not of LS mRNA, is controlled by phytochrome.Abbreviations LS large subunit - RuBP ribulose-1,5-bisphosphate - ptDNA plastid DNA     

The role of pigments in germination and dark-growth of white mustard (Sinapis alba L.) seeds

Pigments leached from Sinapis alba L. seeds and extracted from dark-grown seedlings are described and investigated by spectrophotometry and chromatography and their functions examined. Evidence is given that they are quinonoid, and it is suggested that they form part of a complex which absorbs ultraviolet light and utilizes it to produce growth energy at stages before visible light becomes available to the plant, and that this process may be the means by which mutations are introduced into species.     

Hormones are no causal links in phytochrome-mediated adventitious root formation in mustard seedlings (Sinapis alba L.)

The question of whether or not hormones are causal links in the realization of phytochrome control during photomorphogenesis was investigated using the phytochrome-dependent formation of adventitious roots in hypocotyl cuttings excised from mustard seedlings as a test system. Histological examination of regenerating rest seedlings revealed that phytochrome (operationally, continuous far-red light) mediates the de novo formation of root primordia in the pericycle region of the hypocotyl near the cutting surface withing 12–24 h after excision.Auxin (IAA), gibberellin (GA₃), Cytokinin (kinetin), abscisic acid (ABA), and ethylene had no promotive effect on primordium formation in dark-grown or far-red irradiated rest seedlings. Depending on concentration, the application of these hormones was either ineffective or inhibitory in the rooting response. It is concluded that phytochrome does not operate through changes of hormone (auxin, gibberellin, cytokinin, ABA, ethylene) levels.While externally applied ethylene had no specific effect on primordium formation, the number of primordia produced in darkness could be increased to the far-red light level by removing the endogenously formed ethylene. Since the stimulatory effect of light could not be related to a lower ethylene level, it is concluded that ethylene interferes with primordium formation by modulating the susceptibility of this process to phytochrome control. This ethylene effect takes place in a concentration range below the range that can be manipulated by external application of the hormone.Abbreviations ABA abscisic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - P_r P_fr red and far-red absorbing forms of phytochrome     

Binding of the trypsin inhibitor from white mustard (Sinapis alba L.) seeds to bovine beta-trypsin: thermodynamic study

The effect of pH and temperature on the association equilibrium constant (Ka) for the binding of the trypsin inhibitor from white mustard (Sinapis alba L.) seeds (MTI) to bovine beta-trypsin (EC 3.4.21.4) has been investigated. On lowering the pH from 9 to 3, values of Ka for MTI binding to bovine beta-trypsin decrease thus reflecting the acid-pK and -midpoint shifts, upon inhibitor association, of two independent ionizable groups, and of a three-proton transition, respectively. At pH 8.0, values of thermodynamic parameters for MTI binding to bovine beta-trypsin are: Ka = 4.5 X 10(8)M-1, delta G0 = -11.6 kcal/mol, and delta S0 = +53 entropy units (all at 21 degrees C); and delta H0 = +4.1 kcal/mol (temperature independent between 5 degrees C and 45 degrees C). Binding properties of MTI to bovine beta-trypsin have been analyzed in parallel with those concerning macromolecular inhibitor association to serine (pro)enzymes.     

Glycerolipid synthesis by homogenate and oil bodies from developing mustard (Sinapis alba L.) seed

[1-¹⁴C]Oleic acid and [14-¹⁴C]erucic acid were converted to their acyl-CoA derivatives and incorporated into acyl lipids by a homogenate from developing mustard (Sinapis alba L.) seed and oil bodies, as well as supernatant isolated by centrifugation at 20000 g. In both homogenate and oil bodies, the oleoyl moieties from exogenous [1-¹⁴C]oleoyl-CoA were most extensively incorporated into phosphatidic acids, but very little into phosphatidylcholines. The pattern of labelling of acyl lipids by oleoyl versus erucoyl moieties from either of the corresponding fatty acids, added individually or as a mixed substrate, indicates that oleoyl-CoA directly acylates sn-glycerol-3-phosphate to yield lysophosphatidic acids and phosphatidic acids that are subsequently converted to mono- and diacylglycerols. In contrast, erucoyl-CoA predominantly acylates preformed mono-and diacylglycerols containing oleoyl moieties to yield triacylglycerols containing erucoyl moieties.     

Effect of light on the development of glyoxysomal functions in the cotyledons of mustard (Sinapis alba L.) seedlings

The degradation of storage fat in the cotyledons of mustard seedlings is unaffected by phytochrome and photosynthesis (irradiation with continuous red or far-red light from sowing of the seeds) although light imposes a strong constraint on the translocation of organic matter from the cotyledons into the seedling axis. Likewise, the development and disappearance of glyoxysomal enzyme activities (isocitrate lyase, malate synthase, citrate synthase) takes place independently of light. It is concluded that the mobilization of storage fat (fatcarbohydrate transformation) is independent of photomorphogenesis. The surplus of carbohydrate produced from fat in the light seems to be converted to starch grains in the plastids, which function as a secondary storage pool in the cotyledons.Abbreviations CS citrate synthase - ICL isocitrate lyase - MS malate synthase     

Leaf growth of white mustard (Sinapis alba) in different environments

Summary The numbers of cells and area of fully expanded leaves were determined on successive leaves of Sinapis alba grown either in 8 hr. photoperiod (vegetative plants) or 16 hr. photoperiod (flowering plants) at a constant temperature of 20°C. In the 8 hr. photoperiod leaf 9 had the greatest area but leaf 12 had most cells. In 16 hr. photperiod leaf 5 had the greatest area but leaf 9 had most cells. The relationship between area and cell number of successive leaves on the main stem fell into 3 distinct phases: in phase (1), cell number increased at a greater rate than leaf area; in phase (2), leaf area decreased while cell number increased; in phase (3), cell number and leaf area decreased proportionally. For an increase in unit area, cell number increased more in 8 hr. than in 16 hr. photoperiod.Using final area and final cell number of successive leaves, by extrapolation the cell number of unit area of primordium has been deduced. Cell number per unit area increased in successive primordia up to a certain node after which it remained constant at succeeding nodes. It was found that in plants grown under different conditions the cell number per unit area in successive primordia increased at a constant logarithmic rate. That is, cells became progressively smaller. It is concluded that changes in cell size of successive primordia are not influenced by the environment but are under internal control.