Control of the AtMAP65-1 interaction with microtubules through the cell cycle

Cell division depends on the fine control of both microtubule dynamics and microtubule organisation. The microtubule bundling protein MAP65 is a 'midzone MAP' essential for the integrity of the anaphase spindle and cell division. Arabidopsis thaliana MAP65-1 (AtMAP65-1) binds and bundles microtubules by forming 25 nm cross-bridges. Moreover, as AtMAP65-1 bundles microtubules in interphase, anaphase and telophase but does not bind microtubules in prophase or metaphase, its activity through the cell cycle must be under tight control. Here we show that AtMAP65-1 is hyperphosphorylated during prometaphase and metaphase and that CDK and MAPK are involved in this phosphorylation. This phosphorylation inhibits AtMAP65-1 activity. Expression of nonphosphorylatable AtMAP65-1 has a negative effect on mitotic progression resulting in excessive accumulation of microtubules in the metaphase spindle midzone causing a delay in mitosis. We conclude that normal metaphase spindle organisation and the transition to anaphase is dependent on inactivation of AtMAP65-1.

Strategies of actin reorganisation in plant cells

Spatial-temporal flexibility of the actin filament network (F-actin) is essential for all basic cellular functions and is governed by a stochastic dynamic model. In this model, actin filaments that randomly polymerise from a pool of free actin are bundled with other filaments and severed by ADF/cofilin. The fate of the severed fragments is not known. It has been proposed that the fragments are disassembled and the monomeric actin recycled for the polymerisation of new filaments. Here, we have generated tobacco cell lines and Arabidopsis plants expressing the actin marker Lifeact to address the mechanisms of F-actin reorganisation in vivo. We found that F-actin is more dynamic in isotropically expanding cells and that the density of the network changes with a periodicity of 70 seconds. The depolymerisation rate, but not the polymerisation rate, of F-actin increases when microtubules are destabilised. New filaments can be assembled from shorter free cytoplasmic fragments, from the products of F-actin severing and by polymerisation from the ends of extant filaments. Thus, remodelling of F-actin might not require bulk depolymerisation of the entire network, but could occur via severing and end-joining of existing polymers.

The Caspase-Related Protease Separase (EXTRA SPINDLE POLES) Regulates Cell Polarity and Cytokinesis in Arabidopsis

Vesicle trafficking plays an important role in cell division, establishment of cell polarity, and translation of environmental cues to developmental responses. However, the molecular mechanisms regulating vesicle trafficking remain poorly understood. Here, we report that the evolutionarily conserved caspase-related protease separase (EXTRA SPINDLE POLES [ESP]) is required for the establishment of cell polarity and cytokinesis in Arabidopsis thaliana. At the cellular level, separase colocalizes with microtubules and RabA2a (for RAS GENES FROM RAT BRAINA2a) GTPase-positive structures. Separase facilitates polar targeting of the auxin efflux carrier PIN-FORMED2 (PIN2) to the rootward side of the root cortex cells. Plants with the radially swollen4 (rsw4) allele with compromised separase activity, in addition to mitotic failure, display isotropic cell growth, perturbation of auxin gradient formation, slower gravitropic response in roots, and cytokinetic failure. Measurements of the dynamics of vesicle markers on the cell plate revealed an overall reduction of the delivery rates of KNOLLE and RabA2a GTPase in separase-deficient roots. Furthermore, dissociation of the clathrin light chain, a protein that plays major role in the formation of coated vesicles, was slower in rsw4 than in the control. Our results demonstrate that separase is a key regulator of vesicle trafficking, which is indispensable for cytokinesis and the establishment of cell polarity.

In Vitro Evaluation of the Genotoxic Activity and Apoptosis Induction of the Extracts of Roots and Leaves from the Medicinal Plant Coccoloba mollis (Polygonaceae)

Coccoloba mollis (Family Polygonaceae) is a medicinal plant popularly used in cases of memory loss, stress, insomnia, anemia, impaired vision, and sexual impotence, but the scientific literature, to date, lacks studies on the biological effects of this species, particularly with regard to cytotoxicity and induction of DNA damage. The aim of the present study was to assess in vitro (in hepatic HTC cells) ethanolic extracts of the roots and leaves of C. mollis for cytotoxicity, genotoxicity, and induction of apoptosis. For these evaluations the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay, comet assay, micronucleus test with cytokinesis block, and an in situ test for detection of apoptotic cells with acridine orange staining were used. The results showed that the extract obtained from the roots of C. mollis is more cytotoxic than that obtained from the leaves and that the reduction in cell viability observed in the MTT assay was a result, at least in part, from the induction of apoptosis. Both extracts induced DNA damage at a concentration of 20 mu g/mL in the comet assay, but no genotoxicity was detected with any of the treatments carried out in the micronucleus test.

Sexual selection in a tropical fruit fly : role of a plant derived chemical in mate choice

The specific mechanisms by which selective pressures affect individuals are often difficult to resolve. In tephritid fruit flies, males respond strongly and positively to certain plant derived chemicals. Sexual selection by female choice has been hypothesized as the mechanism driving this behaviour in certain species, as females preferentially mate with males that have fed on these chemicals. This hypothesis is, to date, based on studies of only very few species and its generality is largely untested. We tested the hypothesis on different spatial scales (small cage and seminatural field-cage) using the monophagous fruit fly, Bactrocera cacuminata. This species is known to respond to methyl eugenol (ME), a chemical found in many plant species and one upon which previous studies have focused. Contrary to expectation, no obvious female choice was apparent in selecting ME-fed males over unfed males as measured by the number of matings achieved over time, copulation duration, or time of copulation initiation. However, the number of matings achieved by ME-fed males was significantly greater than unfed males 16 and 32 days after exposure to ME in small cages (but not in a field-cage). This delayed advantage suggests that ME may not influence the pheromone system of B. cacuminata but may have other consequences, acting on some other fitness consequence (e.g., enhancement of physiology or survival) of male exposure to these chemicals. We discuss the ecological and evolutionary implications of our findings to explore alternate hypotheses to explain the patterns of response of dacine fruit flies to specific plant-derived chemicals.

Influence of host plant structure and microclimate on the abundance and behaviour of Tephritid fly

Microclimate and host plant architecture significantly influence the abundance and behavior of insects. However, most research in this field has focused at the invertebrate assemblage level, with few studies at the single-species level. Using wild Solanum mauritianum plants, we evaluated the influence of plant structure (number of leaves and branches and height of plant) and microclimate (temperature, relative humidity, and light intensity) on the abundance and behavior of a single insect species, the monophagous tephritid fly Bactrocera cacuminata (Hering). Abundance and oviposition behavior were signficantly influenced by the host structure (density of foliage) and associated microclimate. Resting behavior of both sexes was influenced positively by foliage density, while temperature positively influenced the numbers of resting females. The number of ovipositing females was positively influenced by temperature and negatively by relative humidity. Feeding behavior was rare on the host plant, as was mating. The relatively low explanatory power of the measured variables suggests that, in addition to host plant architecture and associated microclimate, other cues (e.g., olfactory or visual) could affect visitation and use of the larval host plant by adult fruit flies. For 12 plants observed at dusk (the time of fly mating), mating pairs were observed on only one tree. Principal component analyses of the plant and microclimate factors associated with these plants revealed that the plant on which mating was observed had specific characteristics (intermediate light intensity, greater height, and greater quantity of fruit) that may have influenced its selection as a mating site.

Somatic embryogenesis, organogenesis and plant regeneration in taro (Colocasia esculenta var. esculenta)

Callus was initiated in three different ‘‘esculenta’’ taro cultivars by culturing corm slices in the dark on half-strength MS medium supplemented with 2.0 mg/l 2,4- dichlorophenoxyacetic acid (2,4-D) for 20 days followed by subculture of all corm slices to half-strength MS medium containing 1.0 mg/l thidiazuron (TDZ). Depending on the cultivar, 20–30% of corm slices produced compact, yellow, nodular callus on media containing TDZ. Histological studies revealed the presence of typical embryogenic cells which were small, isodiametric with dense cytoplasms. Somatic embryos formed when callus was transferred to hormone-free medium and *72% of the embryos germinated into plantlets on this medium. Simultaneous formation of roots and shoots during germination, and the presence of shoot and root poles revealed by histology, confirmed that these structures were true somatic embryos. Plants derived from somatic embryos appeared phenotypically normal following 2 months growth in a glasshouse. This method is a significant advance on those previously reported for the esculenta cultivars of taro due to its efficiency and reproducibility.

Initiation of embryogenic cell suspensions of taro (Colocasia esculenta var. esculenta) and plant regeneration

Embryogenic callus was initiated by culturing in vitro taro corm slices on agar-solidified half-strength MS medium containing 2.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) for 20 days followed by transfer to 1.0 mg/L thidiazuron (TDZ). Callus was subsequently proliferated on solid medium containing 1.0 mg/L TDZ, 0.5 mg/L 2,4- D and 800 mg/L glutamine before transfer to liquid medium containing the same components but with reduced glutamine (100 mg/L). After 3 months in liquid culture on an orbital shaker, cytoplasmically dense cell aggregates began to form. Somatic embryogenesis was induced by plating suspension cells onto solid media containing reduced levels of hormones (0.1 mg/L TDZ, 0.05 mg/L 2,4-D), high concentrations of sucrose (40–50 g/L) and biotin (1.0 mg/L). Embryo maturation and germination was then induced on media containing 0.05 mg/L benzyladenine (BA) and 0.1 mg/L indole-3-acetic acid (IAA). Histological studies of the developing embryos revealed the presence of typical shoot and root poles suggesting that these structures were true somatic embryos. The rate of somatic embryos formation was 500–3,000 per mL settledcell volume while approximately 60% of the embryos regenerated into plants.

A cost-based optimization of a fiberboard pressing plant using Monte-Carlo simulation (a reliability program)

In this research the reliability and availability of fiberboard pressing plant is assessed and a cost-based optimization of the system using the Monte- Carlo simulation method is performed. The woodchip and pulp or engineered wood industry in Australia and around the world is a lucrative industry. One such industry is hardboard. The pressing system is the main system, as it converts the wet pulp to fiberboard. The assessment identified the pressing system has the highest downtime throughout the plant plus it represents the bottleneck in the process. A survey in the late nineties revealed there are over one thousand plants around the world, with the pressing system being a common system among these plants. No work has been done to assess or estimate the reliability of such a pressing system; therefore this assessment can be used for assessing any plant of this type.