Biosynthesis and insecticidal properties of plant cyclotides: The cyclic knotted proteins from Oldenlandia affinis

Several members of the Rubiaceae and Violaceae families produce a series of cycloticles or macrocyclic peptides of 29-31 amino acids with an embedded cystine knot. We aim to understand the mechanism of synthesis of cyclic peptides in plants and have isolated a cDNA clone that encodes the cyclotide kalata Ell as well as three other clones for related cycloticles from the African plant Olden-landia affinis. The cDNA clones encode prepropeptides with a 20-aa signal sequence, an N-terminal prosequence of 46-68 amino acids and one, two, or three cyclotide domains separated by regions of about 25 aa. The corresponding cycloticles have been isolated from plant material, indicating that the cyclotide domains are excised and cyclized from all four predicted precursor proteins. The exact processing site is likely to lie on the N-terminal side of the strongly conserved GlyLeuPro or SerLeuPro sequence that flanks both sides of the cyclotide domain. Cyclotides have previously been assigned an antimicrobial function; here we describe a potent inhibitory effect on the growth and development of larvae from the Lepidopteran species Helicoverpa punctigera.

Smoke derived from burnt vegetation stimulates germination of arable weeds

A commercially available smoke-water solution (Seed Starter (R)) stimulated the germination of caryopses and intact florets of Avena fatua L. The solution was most effective when diluted (5-50%) and presented to intact or dehulled grain that had received a short period of dry after-ripening. It was less effective when applied at full strength or to grains that had been freshly harvested. The same stimulatory effect was observed in par-fly after-ripened caryopses of nine different wild oat biotypes obtained from three different cropping regions of the world. When freshly harvested caryopses were re-tested with the commercial solution (100%) for just 7 days prior to placement on to distilled water, a much higher germination percentage was possible than seen with continuous smoke-water incubation. The stimulatory ability of smoke water was more closely matched to that of gibberellic acid than to potassium nitrate, which had little or no effect on freshly harvested caryopses. The smoke-water solution (5-100%) was tested on the germination of 18 other cool temperate arable weed species. All monocotyledonous species tested (viz. Avena sterilis ssp. ludoviciana L., Alopecurus myosuroides, Sorghum halepense, Phalaris paradoxa) responded positively, while those of the dicotyledonous species were either strongly stimulated (greater than or equal to 40% stimulation Malva neglecta), moderately stimulated (greater than or equal to 20% stimulation Galium aparine, Veronica persica), slightly stimulated (Polygonum persicaria, P pennsylvanicum, Fallopia convolvulus), unaffected (P. aviculare, Sinapis arvensis, Heracleum sphondylium, Angelica sylvestris, Mercurialis annua, Veronica hederifolia) or inhibited (Lamium purpureum). The optimal concentrations required to stimulate germination of the monocotyledonous species were similar to those observed for A. fatua (5-10%). However, for the dicotyledonous species slightly stronger solutions were required (10-20%). When the unaffected species were retested using a 10-day pre-chilling treatment, smoke water showed a small promotive response in three (S. arvensis, P. aviculare and V hederifolia) of the six species. When four different smoke-water solutions (Seed Starter (R), Regen 2000 (R), charred-wood solution and wheat-straw solution) were tested on two representative species (A. fatua and M. neglecta), three formulations were effective in promoting the germination of both species, while the fourth (charred-wood solution) was only active on A. fatua. The active concentrations were different for the four solutions. Three solutions were active in the 2-20% dilution range, while the fourth (Regen 2000 (R)) was only active in the 1-2% dilution range and was inhibitory at higher concentrations. These observations are discussed in the context that smoke may play an important ecological role in the management and control of introduced weeds in native and arable communities.

The plant isoflavenoid genistein activates p53 and Chk2 in an ATM-dependent manner

Genistein is an isoflavenoid that is abundant in soy beans. Genistein has been reported to have a wide range of biological activities and to play a role in the diminished incidence of breast cancer in populations that consume a soy-rich diet. Genistein was originally identified as an inhibitor of tyrosine kinases; however, it also inhibits topoisomerase II by stabilizing the covalent DNA cleavage complex, an event predicted to cause DNA damage. The topoisomerase II inhibitor etoposide acts in a similar manner. Here we show that genistein induces the up-regulation of p53 protein, phosphorylation of p53 at serine 15, activation of the sequence-specific DNA binding properties of p53, and phosphorylation of the hCds1/Chk2 protein kinase at threonine 68. Phosphorylation and activation of p53 and phosphorylation of Chk2 were not observed in ATM-deficient cells. In contrast, the topoisomerase II inhibitor etoposide induced phosphorylation of p53 and Chk2 in ATM-positive and ATM-deficient cells. In addition, genistein-treated ATM-deficient cells were significantly more susceptible to genistein-induced killing than were ATM-positive cells. Together our data suggest that ATM is required for activation of a DNA damage-induced pathway that activates p53 and Chk2 in response to genistein.