Cutin:cutin-acid endo-transacylase (CCT), a cuticle-remodelling enzyme activity in the plant epidermis

Cutin is a polyester matrix mainly composed of hydroxy-fatty acids that occurs in the cuticles of shoots and root-caps. The cuticle, of which cutin is a major component, protects the plant from biotic and abiotic stresses, and cutin has been postulated to constrain organ expansion. We propose that, to allow cutin restructuring, ester bonds in this net-like polymer can be transiently cleaved and then re-formed (transacylation). Here, using pea epicotyl epidermis as the main model, we first detected a cutin:cutin-fatty acid endo-transacylase (CCT) activity. In-situ assays used endogenous cutin as the donor substrate for endogenous enzymes; the exogenous acceptor substrate was a radiolabelled monomeric cutin-acid, 16-hydroxy-[3H]hexadecanoic acid (HHA). High-molecular-weight cutin became ester-bonded to intact [3H]HHA molecules, which thereby became unextractable except by ester-hydrolysing alkalis. In-situ CCT activity correlated with growth rate in Hylotelephium leaves and tomato fruits, suggesting a role in loosening the outer epidermal wall during organ growth. The only well-defined cutin transacylase in the apoplast, CUS1 (a tomato cutin synthase), when produced in transgenic tobacco, lacked CCT activity. This finding provides a reference for future CCT protein identification, which can adopt our sensitive enzyme assay to screen other CUS1-related enzymes.

Dehydrocostus lactone, a naturally occurring polar auxin transport inhibitor, inhibits epicotyl growth by interacting with auxin in etiolated Pisum sativum seedlings

We have isolated germacranolide-type sesquiterpene lactones with an α-methylene-γ-lactone moiety, dehydrocostus lactone (DHCL), costunolide, santamarine, and a novel compound denoted artabolide [3-hydroxy-4,6,7(H)-germacra-1(10),11(13)-dien-6,12-olide] from oriental medicinal Asteraceae plants as novel naturally occurring inhibitors of polar auxin transport detected by the radish hypocotyl bioassay. To investigate the mode of action of natural sesquiterpene lactones on the inhibition of polar auxin transport as well as its relation to the growth of seedlings, the function of DHCL on growth and auxin dynamics in etiolated pea seedlings was studied intensively. DHCL reduced polar auxin transport in a dose-dependent manner together with the inhibition of the accumulation of mRNA of <em>PsAUX1</em> and <em>PsPIN1</em> genes encoding influx and efflux carrier proteins of auxin, respectively. DHCL applied to the apical hook region as a lanolin paste substantially inhibited elongation growth in the subapical region of epicotyls in intact etiolated pea seedlings, coupled with a significant reduction of endogenous levels of indole-3-acetic acid (IAA). DHCL also revealed the inhibition of IAA-induced cell elongation in etiolated pea epicotyl segments by affecting IAA-induced changes in the mechanical properties of cell walls. These facts suggest that germacranolide-type sesquiterpene lactones with an α-methylene-γ-lactone moiety affect the expression of <em>PsAUX1</em> and <em>PsPINs</em> genes, and then inhibit polar auxin transport and reduce endogenous levels of IAA necessary for stem growth in etiolated pea seedlings. These compounds are also suggested to show the inhibitory effects on auxin action in pea stem growth.

Influence of lead on auxin-induced cell elongation

The influence of lead chloride on plant tissue growth is described. Lead reduced elongation of etiolated wheat coleoptile segments, green pea epicotyl fragments and etiolated and green sunflower hypocotyls. Green tissues were more susceptible to lead than etiolated ones. PbCl₂ in a 10^-4 M concentration significantly reduced plastic and elastic extensibility of the wheat coleoptile cell walls and diminished the hydration of sunflower hypocotyl segments. Auxin (indolyl-3-acetic acid - IAA) applied in concentration optimal for growth of the particular tissues partly attenuated the inhibitory action of lead on elongation, plastic and elastic extensibility and water absorption. Auxin applied in supraoptimal concentrations did not abolish the inhibitory action of lead on tissue growth.

Species-Specificity of an Elicitor-Active Oligosaccharide, LN-3, to Leguminous Plants

LN-3, a linear pyridylaminated hepta-β-glucoside previously found to show elicitor activity in alfalfa cotyledons, was examined for phytoalexin-inducing activity in pea epicotyl and bean cotyledon assays. LN-3 did not show (+)-pisatin-inducing activity in pea epicotyls. In the bean cotyledon assay, however, the (± )-kievitone content gradually increased with increasing LN- 3 concentration, and reached a maxim um (ca. 17 |ig/g fresh wt) at 100 μ/ml. Half-maximal elicitor activity was seen at ca. 16 μm . After three legumes, alfalfa, pea and bean, were treated with LN-3, the recovery of the rem aining LN-3 or its fragments was examined. Almost 100% of LN-3 or its fragments was recovered from the pea test solution; in contrast, recoveries from alfalfa and bean were only 63.8 and 38.1% , respectively. HPLC and LC-MS analyses of the recovered samples indicated that LN-3 was hydrolyzed to give mono- and/or diglucoside( s) in the alfalfa and the bean solutions, while in the pea experiment a small portion of LN-3 was hydrolyzed to give sugar fragments with different degrees of polymerization < 7.