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Questions: Do long-term observations in permanent plots confirm the conceptual model of Metrosideros polymorpha cohort dynamics as postulated in 1987? Do regeneration patterns occur independently of substrate age, i.e. of direct volcanic disturbance impact? Location: The windward mountain slopes of the younger Mauna Loa and the older Mauna Kea volcanoes (island of Hawaii, USA). Methods: After widespread forest decline (dieback), permanent plots were established in 1976 in 13 dieback and 13 non-dieback patches to monitor the population structure of M. polymorpha at ca. 5-yr intervals. Within each plot of 20 × 20 m, all trees with DBH >2.5 cm were individually tagged, measured and tree vigour assessed; regeneration was quantified in 16 systematically placed subplots of 3 × 5 m. Data collected in the subplots included the total number of M. polymorpha seedlings and saplings (five stem height classes). Here we analyse monitoring data from six time steps from 1976 to 2003 using repeated measures ANOVA to test specific predictions derived from the 1987 conceptual model. Results: Regeneration was significantly different between dieback and non-dieback plots. In dieback plots, the collapse in the 1970s was followed by a 'sapling wave' that by 2003 led to new cohort stands of M. polymorpha. In non-dieback stands, seedling emergence did not result in sapling waves over the same period. Instead, a 'sapling gap' (i.e. very few or no M. polymorpha saplings) prevailed as typical for mature stands. Canopy dieback in 1976, degree of recovery by 2003 and the number of living trees in 2003 were unrelated to substrate age. Conclusions: Population development of M. polymorpha supports the cohort dynamics model, which predicts rebuilding of the forest with the same canopy species after dieback. The lack of association with substrate age suggests that the long-term maintenance of cohort structure in M. polymorpha does not depend on volcanic disturbance but may be related to other environmental mechanisms, such as climate anomalies.

The oxidative burst, the generation of reactive oxygen species (ROS) in response to microbial pathogen attack, is a ubiquitous early part of the resistance mechanisms of plant cells. It has also become apparent from the study of a number of plant–pathogen interactions and those modelled by elicitor treatment of cultured cells that there may be more than one mechanism operating. However, one mechanism may be dominant in any given species. NADPH oxidases have been implicated in a number of systems and have been cloned and characterized. However, the enzyme system which is the major source of ROS in French bean (Phaseolus vulgaris) cells treated with a cell wall elicitor from Colletotrichum lindemuthianum, appears to be dependent on an exocellular peroxidase. The second component, the extracellular alkalinization, occurs as a result of the Ca2+ and proton influxes and the K+ efflux common to most elicitation systems as one of the earliest responses. The third component, the actual reductant/substrate, has remained elusive. The low molecular weight compound composition of apoplastic fluid was compared before and after elicitation. The substrate only becomes available some min after elicitation and can be extracted, so that by comparing the profiles by LC‐MS it has been possible to identify possible substrates. The mechanism has proved to be complex and may involve a number of low molecular weight components. Stimulation of H2O2 production was observed with saturated fatty acids such as palmitate and stearate without concomitant oxylipin production. This biochemical evidence is supported by immunolocalization studies on papillae forming at bacterial infection sites that show the peroxidase isoform present at sites of H2O2 production revealed by cerium chloride staining together with the cross‐linked wall proteins and callose and callose synthase. The peroxidase has been cloned and expressed in Pichia pastoris and has been shown to catalyse the oxidation reaction with the same kinetics as the purified enzyme. Furthermore, Arabidopsis plants transformed heterologously using the French bean peroxidase in antisense orientation have proved to be highly susceptible to bacterial and fungal pathogens. Thus it is possible that Arabidopsis is another species with the potential to mount an apoplastic oxidative burst and these transformed plant lines may be useful to identify the peroxidase that is responsible.

• Modulators of cAMP, calcium and G proteins were used to treat bean (Phaseolus vulgaris) cells before addition of an elicitor from Colletotrichum lindemuthianum in order to elucidate the early steps of signal transduction leading to the production of the apoplastic oxidative burst. • Hydrogen peroxide production by elicited bean cells was monitored with luminolor xylenol-orange-based assays. • Pretreatment with forskolin, dibutyryl cAMP or the Ca2+ ionophore A23187 enhanced the production of reactive oxygen species (ROS). The Ca2+ channel blocker, verapamil, and the calmodulin antagonist W7 led to a decreased oxidative burst and cancelled the dibutyryl cAMP effect. The production of ROS was increased by cholera toxin (CTX), an activator of G proteins. • Thus, an increase of cytosolic calcium ([ Ca2+] cyt) mediated through an increased level of cAMP is required for ROS production. The data support a role for G proteins and cAMP in extracellular alkalinization and Ca2+ influx, possibly in the provision of a reductant, which with the extracellular peroxidase, are required for the apoplastic oxidative burst.

Cultured cells of rose (Rosa damascena) treated with an elicitor derived from Phytophthora spp. and suspension-cultured cells of French bean (Phaseolus vulgaris) treated with an elicitor derived from the cell walls of Colletotrichum lindemuthianum both produced H2O2. It has been hypothesized that in rose cells H2O2 is produced by a plasma membrane NAD(P)H oxidase (superoxide synthase), whereas in bean cells H2O2 is derived directly from cell wall peroxidases following extracellular alkalinization and the appearance of a reductant. In the rose/Phytophthora spp. system treated with N,N-diethyldithiocarbamate, superoxide was detected by a N,N'-dimethyl-9,9'-biacridium dinitrate-dependent chemiluminescence; in contrast, in the bean/C. lindemuthianum system, no superoxide was detected, with or without N,N-diethyldithiocarbamate. When rose cells were washed free of medium (containing cell wall peroxidase) and then treated with Phytophthora spp. elicitor, they accumulated a higher maximum concentration of H2O2 than when treated without the washing procedure. In contrast, a washing treatment reduced the H2O2 accumulated by French bean cells treated with C. lindemuthianum elicitor. Rose cells produced reductant capable of stimulating horseradish (Armoracia lapathifolia) peroxidase to form H2O2 but did not have a peroxidase capable of forming H2O2 in the presence of reductant. Rose and French bean cells thus appear to be responding by different mechanisms to generate the oxidative burst

Changes in protein kinase activity have been investigated during the early response of suspension cultured cells of French bean to fungal elicitor. One of the kinases activated has a known target, phenylalanine ammonia-lyase (PAL), which has an important role in plant defence responses, and was purified. Kinase acivity during purification was monitored for both the PAL-derived peptide and syntide-2, which it also phosphorylated. The kinase had an Mr of 55,000 on the basis of gel migration, 45Ca2+ binding, autophosphorylation and phosphorylation of various substrates using in-gel assays. The kinase has been characterised with respect to kinetics and other properties in vitro and appears to be a CDPK. In-gel assays were also used to show that this kinase and a number of other CDPKs of similar Mr showed complex changes in elicitor-treated suspension-cultured cells of French bean. An activation was observed within 10 min and was maintained for up to 4 h. The time course of activation was different from MAP kinase and casein kinase assayed in the same extracts. However, at 5 min after addition of elicitor there is a transient inactivation of the CDPKs before activation. This inactivation can be mimicked by adding forskolin to the cells 30 min before elicitation, which brings about changes in the cellular pH. Forskolin potentiates the oxidative burst when elicitor is subsequently added while the CDPK cannot be activated by elicitor upon forskolin treatment. In contrast, intracellular acidification brought about by forskolin brings about slight activation of MAPkinase.