Pea (Pisum sativum L.) seed isolectins 1 and 2 and pea root lectin result from carboxypeptidase-like processing of a single gene product

The complete amino acid sequences of the -subunits of pea (Pisum sativum L.) seed and root lectin, the C-terminal amino acids of the -subunits of pea seed lectin, and most of the sequence of the -subunit of pea root lectin were determined. In contrast to earlier reports it was shown that the -subunits of both seed isolectins end at Asn-181. The 1 subunits end at Gln-241 (major fraction) or Lys-240 (minor fraction), whereas the 2 subunits end at Ser-239, Ser-238, Ser-237 or Thr-236. psl cDNA clones from seed are identical to psl cDNA clones from root, and root PSL is identical to seed PSL2, ending at Ser-239, Ser-238 or Ser-237. It seems that the presence of Lys-240 is the sole determinant of the charge difference between pea isolectins. PSL1 can be converted into PSL2 by carboxypeptidase P from Penicillium janthinellum. These results confirm that PSL from roots is encoded by the same gene as PSL from seeds. Thus, it seems that, next to an Asn-X specific protease responsible for the processing at positions 181/182 and 187/188, a carboxypeptidase is responsible for the conversion of PSL1 into PSL2, which is probably the final processing product.     

Flow-cytometric cell counting and DNA estimation for the study of plant cell population dynamics

A one-step procedure is presented for simultaneous measurement of cell number and DNA content in cultured plant cells by flow cytometry. In order to obtain nuclei representative of the growth stadium of the culture and of all phases of the cell cycle, cells were carefully sampled and immediately fixed. Next, nuclei were isolated by enzymatic and mechanical maceration, and stained with a DNA-specific fluorescent dye. In the resultant preparation, cells can be counted at relative ease by means of a fluorescence microscope. However, flow-cytometric counting appeared to be superior to manual counting since the time needed for flow-cytometric counting was one-fourth that for manual counting and the variance between counts of the samples was significantly less. In addition, from the same routine, accurate DNA distributions were obtained as a second important parameter of the population dynamics.     

Light-dependent expression of two catalase subunits in leaves of barley seedlings

In young barley seedlings high levels of a 57 kDa catalase subunit were found in the dark, while increased levels of a 53 kDa subunit were detected in the leaves after growth in the light. A catalase-deficient mutant fails to upregulate expression of the 53 kDa subunit after transfer to a high light intensity.     

Rhizobium induces marked root hair curling by redirection of tip growth: a computer simulation

Computer simulation shows that Rhizobium can induce marked curling in legume root hairs by growth induction. Essential elements are: a) the attachment of one inducing principle (e.g. one bacterium or a group of bacteria), preferably within the growth area of the root hair; b) translocation of the inductor along the growing root hari tip; and c) redirection of the original plant-driven tip growth. Also other root hair deformations, for example root hair branching and infection thread growth, can be explained with the proposed model.     

Arachidonic acid can induce leukotriene C4 formation by purified human eosinophils in the absence of other stimuli

Stimulation of purified human eosinophils with 50 microM arachidonic acid leads to the production of leukotriene C4, 15-hydroxy-eicosatetraenoic acid and 15-series leukotrienes. The ratio of the amounts of leukotriene C4 and 15-lipoxygenase products was found to be strongly dependent on the arachidonic acid concentration, being relatively large at low arachidonic acid concentrations and very small at high arachidonic acid concentrations. In the presence of 1 microM platelet-activating factor a significant elevation of leukotriene C4 formation is observed, whereas the formation of 15-lipoxygenase products remains unaltered. As arachidonic acid was found to be capable of inducing a fast, transient rise in the cytosolic free Ca2+ concentration, this explains at least partly its ability to induce the Ca2+-dependent formation of leukotriene C4.     

The excretion of leukotriene E4 into urine following inhalation of leukotriene D4 by human individuals

Healthy volunteers underwent bronchial challenge with increasing doses of nebulized leukotriene D4 (0.007 - 200 nmol) at 15 min intervals. Total amounts of 200 nmol (females) and 400 nmol (males) were inhaled, corresponding to approximately 100 nmol and 200 nmol deposited in the lung, respectively. Of the latter amounts 3 +/- 1% (mean +/- S.E.M., n = 5) was found to be excreted as leukotriene E4 into the urine within 12 h. No further excretion after this period was observed. Approximately 50% of the total urinary leukotriene E4 was excreted during the first 2 h. These results suggest that a possible formation of sulfidopeptide leukotrienes in the lung in vivo can be monitored by measuring leukotriene E4 excretion into the urine.     

Vitrification and a heat-shock treatment improve cryopreservation of tobacco cell suspensions compared to two-step freezing

Vitrification and two-step freezing were comparatively tested for cryopreservation of tobacco cell suspensions. The optimal growth phase of the culture and the optimal length of the protecting preculture period were determined. With both methods, late-exponential cells showed higher survival rates compared to early exponential and growth-limited cells. Under optimal conditions vitrification yielded higher survival rates than two-step freezing (55% and 36%, respectively). Using two-step freezing a preculture period of 72 h in medium supplemented with 0.3 M mannitol was necessary to obtain maximal survival, whereas for vitrification 24 h of preculture sufficed. Heat shock treatment prior to the cryopreservation procedure could improve survival when mannitol precultured cells in a non-optimal growth phase were used. Heat-shocked cells, which were not precultured with mannitol, did not survive vitrification. Vitrification is the method recommended for cryopreservation of tobacco cell suspensions, in view of the shorter preculture period and higher survival rates resulting in quicker regrowth.     

Mutational analysis of the sugar-binding site of pea lectin

Comparison of x-ray crystal structures of several legume lectins, co-crystallized with sugar molecules, showed a strong conservation of amino acid residues directly involved in ligand binding. For pea (Pisum sativum) lectin (PSL), these conserved amino acids can be classified into three groups: (I) D81 and N125, present in all legume lectins studied so far; (II) G99 and G216, conserved in almost all legume lectins; and (III) A217 and E218, which are only found in Vicieae lectins and are possibly determinants of sugar-binding specificity. Each of these amino acids in PSL was changed by site-directed mutagenesis, resulting in PSL molecules with single substitutions: for group I D81A, D81N, N125A; for group II G99R, G216L; and for group III A217L, E218Q, respectively. PSL double mutant Y124R; A126S was included as a control. The modified PSL molecules appeared not to be affected in their ability to form dimeric proteins, whereas the sugar-binding activity of each of the PSL mutants, with the exception of the control mutant (as shown by haemagglutination assays), was completely eliminated. These results confirm the model of the sugar-binding site of Vicieae lectins as deduced from X-ray analysis.     

Molecular mechanisms of attachment of Rhizobium bacteria to plant roots

Attachment of bacteria to plant cells is one of the earliest steps in many plant-bacterium interactions. This review covers the current knowledge on one of the best-studied examples of bacterium-plant attachment, namely the molecular mechanism by which Rhizobium bacteria adhere to plant roots. Despite differences in several studies with regard to growth conditions of bacteria and plants and to methods used for measuring attachment, an overall consensus can be drawn from the available data. Rhizobial attachment to plant root hairs appears to be a two-step process. A bacterial Ca(2+)-binding protein, designated as rhicadhesin, is involved in direct attachment of bacteria to the surface of the root hair cell. Besides this step, there is another step which results mainly in accumulation and anchoring of the bacteria to the surface of the root hair. This leads to so-called firm attachment. Depending on the growth conditions of the bacteria, the latter step is mediated by plant lectins and/or by bacterial appendages such as cellulose fibrils and fimbriae. The possible role of these adhesions in root nodule formation is discussed.