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Three basic proteins, M1, M2A and M2B, that are substrates for plant Ca(2+)-dependent protein kinase (CDPK) were purified from seeds of yellow mustard (Sinapis alba L.) by a protocol involving batchwise chromatography on carboxymethylcellulose (CM52), cation-exchange HPLC on an SP5PW column and reversed-phase HPLC on a C18 column. The complete amino-acid sequences of these proteins have been determined employing Edman sequencing and electrospray ionization mass spectrometry (ESMS) applied to the proteins and their tryptic and chymotryptic fragments. M1 (observed mass 5676.8 +/- 1.0 Da; calculated mass 5677.57 Da), M2A (observed mass 5704.8 +/- 0.8 Da; calculated mass 5704.60 Da) and M2B (observed mass 5839.5 +/- 1.2 Da; calculated mass 5838.78 Da) have been identified as gamma-thionins, which are potent antifungal proteins. M1, M2A and M2B are phosphorylated by plant CDPK on Ser residues, the site of phosphorylation on M2A being S8 as directly confirmed by Edman sequencing and mass spectrometry of the chymotryptically generated phosphopeptide CQRPS(HPO3)GTW11. M1 and M2A have apparent calmodulin (CaM) antagonist activity with IC50 values of 4.8 +/- 1.3 microM and 5.5 +/- 1.5 microM, respectively, for inhibition of CaM-dependent myosin light chain kinase (MLCK). M2A and/or M2B interacts with dansyl-CaM in both the presence and absence of calcium.

Bioassay-guided analysis of an EtOH extract of Moringa oleifera leaves showing hypotensive activity led to the isolation of two nitrile glycosides, niazirin [1] and niazirinin [2], and three mustard oil glycosides, 4-[(4'-O-acetyl-alpha-L-rhamnosyloxy)benzyl]isothiocyanate [4], niaziminin A, and niaziminin B. Glycoside 2 is a new compound. Niaziminins A and B have previously been obtained from the left extract as a mixture, while compound 4 is new from this source. Structural determination was accomplished by means of spectroscopic methods including appropriate 2D nmr experiments and chemical reactions. This is the first report of the isolation of nitriles, an isothiocyanate, and thiocarbamates from the same plant species. Isothiocyanate 4 and the thiocarbamate glycosides niaziminin A and B showed hypotensive activity while nitrile glycosides 1 and 2 were found to be inactive in this regard.

In a pot experiment the soil application of different levels of Cd2+ (0, 10, 20, 30, 40 and 60 micrograms g-1 soil) affected the lipid components of mustard seeds (Brassica juncea L. Cv. RH-30) markedly. Total lipids declined with the Cd2+ levels regularly while phospho and glycolipids increased only at higher levels. Fatty acids profile of total, neutral and polar lipid fractions were affected considerably. Erucic acid in total and neutral lipids was observed to increase while it decreased in polar lipids with Cd2+ as compared to control. On the other hand palmitic, oleic and linoleic acids had reverse trend. Cadmium concentration increased consistently with increasing levels of Cd2+. Plant dry weight was also decreased significantly with Cd2+ levels.

To investigate physiological processes generated by endogenous circadian rhythms on the molecular level, we have identified clock-controlled genes in the long-day plant Sinapis alba L. A cDNA library was differentially screened using cDNA probes representing transcripts expressed at either the middle of the light period or the middle of the dark period. Two closely related groups of transcripts, Sagrp1 and Sagrp2, controlled by a circadian rhythm have been isolated. RNA blot analysis verified that transcript levels oscillate in plants grown in light/dark cycles with maxima between 'Zeitgeber' time (zt)8 and zt12 (8-12 h after onset of illumination) and minima around zt20. Steady-state mRNA levels continue to oscillate in plants shifted from light/dark cycles to constant light. No synchronous mRNA oscillations are found in plants grown from seed in constant light at constant temperature, suggesting that the clock has to be entrained initially. In contrast, when plants grown in constant light are exposed to rhythmic temperature shifts oscillations of steady-state Sagrp mRNA levels are induced, indicating that temperature acts as an alternative external stimulus (zeitgeber) other than light to entrain the oscillator. In situ hybridization reveals that both transcript groups are expressed predominantly in meristematic and growing tissue. Strong expression is observed in the leaf primordia of the shoot apex, the procambial strands, cambium and in all cell layers of young leaves around zt12. In contrast, little or no signal is found on tissue sections isolated at zt20. This indicates that the oscillator(s) regulating Sagrp transcript fluctuations operate(s) synchronously in different organs. For both transcript groups cDNAs were isolated corresponding to unspliced pre-mRNAs or to transcripts generated by the use of a second 5' splice site. The cDNAs corresponding to the fully spliced transcripts contain open reading frames for polypeptides of 16 kDa, each containing a putative N-terminal RNA recognition motif and a C-terminal region rich in glycine. The predicted proteins show strong homology to an ABA-inducible glycine-rich protein from maize embryos and to the mammalian RNA-binding protein A1 of the heterogeneous nuclear ribonucleoprotein complex involved in pre-mRNA splicing. The SaGRP protein fluctuates with a very low amplitude over light/dark cycles. Immunogold labeling demonstrates the presence of the SaGRP protein within the nucleus of the investigated meristematic cells of young leaves.

A protein with inhibitory activity toward trypsin has been isolated from Sinapis arvensis L (charlock). It has a molecular weight of 15,500 and consists of two chains connected by disulfide bonds. The amino acid sequence was determined and showed that it belongs to the napin family of storage proteins. CD studies showed an alpha-helix content of 12% and a beta-structure of about 50%.

A new serine proteinase inhibitor, rapeseed trypsin inhibitor (RTI), has been isolated from rapeseed (Brassica napus var. oleifera) seed. The protein inhibits the catalytic activity of bovine beta-trypsin and bovine alpha-chymotrypsin with apparent dissociation constants of 3.0 x 10(-10) M and 4.1 x 10(-7) M, at pH 8.0 and 21 degrees C, respectively. The stoichiometry of both proteinase-inhibitor complexes is 1:1. The amino acid sequence of RTI consists of 60 amino acid residues, corresponding to an M(r) of about 6.7 kDa. The P1-P1' reactive site bond has been tentatively identified at position Arg20-Ile21. RTI shows no similarity to other serine proteinase inhibitors except the low molecular weight mustard trypsin inhibitor (MTI-2). RTI and MTI-2 could be members of a new class of plant serine proteinase inhibitors.

Three proteins resembling bacterial sigma factors were previously isolated from mustard chloroplasts (K. Tiller, A. Eisermann and G. Link, Eur J Biochem 198: 93-99, 1991). These sigma-like factors (SLFs) confer DNA-binding and transcription specificity to a system consisting of Escherichia coli core RNA polymerase and cloned DNA regions that carry a chloroplast promoter. Sigma-like activity was now isolated also from etioplasts and could be assigned to three polypeptides of M(r) 67,000 (SLF67), 52,000 (SLF52) and 29,000 (SLF29), i.e. the same sizes as for the chloroplast SLFs. The purification scheme for the factors from either plastid type included an initial heparin-Sepharose and a final gel filtration step. For the etioplast factors, however, an additional phosphocellulose step was required to release these polypeptides from the RNA polymerase. The etioplast SLFs have similar, but not identical, salt requirements for DNA binding as compared to their chloroplast counterparts. Under conditions of maximum binding activity there is overall preference of etioplast SLFs for the psbA promoter over the trnQ and rps16 promoters.

A new serine proteinase inhibitor, mustard trypsin inhibitor 2 (MTI-2), has been isolated from white mustard (Sinapis alba L.) seed by affinity chromatography and reverse phase HPLC. The protein inhibits the catalytic activity of bovine beta-trypsin and bovine alpha-chymotrypsin, with dissociation constants (Kd) of 1.6 x 10(-10) M and 5.0 x 10(-7) M, respectively, at pH 8.0 and 21 degrees C, the stoichiometry of both proteinase-inhibitor complexes being 1:1. The amino acid sequence of MTI-2, which was determined following S-pyridylethylation, is comprised of 63 residues, corresponding to a molecular weight of about 7 kDa, and shows only extremely limited homology to other serine proteinase inhibitors.

The low molecular weight seed storage protein of Brassica campestris has been isolated and its amino acid composition determined. Antibody raised against this low molecular weight protein has been used to compare the antigenic similarity between the low molecular weight storage proteins of different Cruciferae seeds by immunoprecipitation and Western blotting. These studies revealed the existence of antigenically homologous proteins of identical molecular weights in seeds of other Cruciferae but absent in some other dicots like mung bean and tobacco seeds.