Metabolome and water status phenotyping of Arabidopsis under abiotic stress cues reveals new insight into ESK1 function

Metabolomic investigation of the freezing-tolerant Arabidopsis mutant esk1 revealed large alterations in polar metabolite content in roots and shoots. Stress metabolic markers were found to be among the most significant metabolic markers associated with the mutation, but also compounds related to growth regulation or nutrition. The metabolic phenotype of esk1 was also compared to that of wild type (WT) under various environmental constraints, namely cold, salinity and dehydration. The mutant was shown to express constitutively a subset of metabolic responses which fits with the core of stress metabolic responses in the WT. But remarkably, the most specific metabolic responses to cold acclimation were not phenocopied by esk1 mutation and remained fully inducible in the mutant at low temperature. Under salt stress, esk1 accumulated lower amounts of Na⁺ in leaves than the WT, and under dehydration stress its metabolic profile and osmotic potential were only slightly impacted. These phenotypes are consistent with the hypothesis of an altered water status in esk1 , which actually exhibited basic lower water content (WC) and transpiration rate (TR) than the WT. Taken together, the results suggest that ESK1 does not function as a specific cold acclimation gene, but could rather be involved in water homeostasis.     

The abundance,distribution and structural characteristics of tree‐holes in Nothofagus forest,New Zealand

Tree‐holes provide an important microhabitat that is used for feeding, roosting and breeding by numerous species around the world. Yet despite their ecological importance for many of New Zealand's endangered species, few studies have investigated the abundance or distribution of tree‐holes in native forests. We used complementary ground and climbed tree surveys to determine the abundance, distribution and characteristics of tree‐holes in undisturbed Nothofagus forest in the Lewis Pass, New Zealand. We found that hole‐bearing trees were surprisingly abundant compared with many other studies, including Australian Eucalyptus species and American beech. In fact, we estimated as many as 3906 tree‐holes per hectare, of which 963 holes per hectare were potentially large enough to provide roost sites for hole‐nesting bats in New Zealand, while only eight holes per hectare were potentially suitable for specialist hole‐nesting birds. This was of great interest as primary cavity‐excavating animals are absent from New Zealand forests, compared with North America and Australia. Moreover, tree‐hole formation in New Zealand is likely to be dominated by abiotic processes, such as branch breakage from windstorms and snow damage. As has been found in many other studies, tree‐holes were not uniformly distributed throughout the forest. Tree‐holes were significantly more abundant on the least abundant tree species, Nothofagus fusca, than on either N. menziesii or N. solandri. In addition to tree species, tree size was also an important factor influencing the structural characteristics of tree‐holes and their abundance in this forest. Moreover, these trends were not fully evident without climbed tree surveys. Our results revealed that ground‐based surveys consistently underestimated the number of tree‐holes present on Nothofagus trees, and illustrate the importance of using climbed inspections where possible in tree‐hole surveys. We compare our results with other studies overseas and discuss how these are linked to the biotic and abiotic processes involved in tree‐hole formation. We consider the potential implications of our findings for New Zealand's hole‐dwelling fauna and how stand dynamics and past and future forest management practices will influence the structural characteristics of tree‐holes and their abundance in remnant forest throughout New Zealand.     

Growth and Dormancy Cycles in Citrus Bud Cultures and Their Hormonal Control

An in vitro bud culture method was devised in order to better understand the control mechanism of Citrus bud development. This technique offers a new approach to the study of hormonal control of growth, dormancy and flowering cycles in perennial plants. Buds were excised from orchard trees throughout the year, cultured on defined media for prolonged periods, and their vegetative growth responses to various growth hormones were determined. The buds proceeded with their vegetative development in vitro and achieved sprouting on a basal medium. The various growth regulators affected both the time required for sprouting (TRS) and the type of growth. In summer buds, IAA delayed sprouting, while GA enhanced it and caused shoot elongation. Cytokinins specifically induced the formation of numerous adventitious buds, whereas ABA completely inhibited sprouting; this inhibition, however, was reversible. A marked decrease in total protein and in the rate of its synthesis was evident during the first 20 days of sprouting induction and early bud growth. The annual growth rhythm was determined in spring buds sampled and cultured throughout the year, and an innate dormancy of citrus buds was revealed. Both the dormancy and the sprouting periods of buds in vitro corresponded to the natural periods occurring under field conditions. The effect of exogenous IAA, GA and cytokinins on the TRS varied at different periods along the season, suggesting the concept of “critical levels” in the endogenous balance of hormones.     

Interrelationship of Abscisic Acid and Gibberellic Acid in the Promotion of Callus Formation in the Abscission Zone of Citrus Bud Cultures

The mechanism of ABA-induced callus formation was studied in sterile bud cultures of Citrus [Citrus sinensis (L.) Osbeck] on defined media. ABA was found to promote callus formation in the abscission zone between the petiole and the branch while inhibiting bud growth. The promoting effect of ABA was dependent on the physiological state of the shoot from which buds were excised, and on the size of the explant. Callus formation was highest in autumn and summer (i.e. younger) buds, and lowest in older buds excised from previous summer flush. GA was only slightly active in promoting callus formation when applied separately, but showed a highly synergistic effect when applied with ABA: maximal callus formation was attained at a combination of 10^?5M ABA and 10^?6 MGA in the medium. Subcultures of ABA-induced callus revealed that ABA inhibited the growth of isolated subcultured callus, while IAA and kinetin, and especially GA, promoted its rapid proliferation. A general decrease in protein synthesis was found in the abscission zone during the first 5 days of induction, while total protein content changed only slightly. The results suggest that ABA-induced callus formation in Citrus bud explants is a multiphasic phenomenon involving, at least, two stages: (1) activation of certain cells in the abscission zone by ABA, resulting in the formation of callus layers, and (2) subsequent proliferation of the callus tissue, which is dependent on the hormonal balance in the explant. This growth-promoting effect of ABA seems to be a general phenomenon in explants exposing a cut-surface.     

Effects of landscape features and demographic history on the genetic structure of Testudo marginata populations in the southern Peloponnese and Sardinia

Testudo marginata, the largest European land tortoise, is suffering habitat degradation and destruction. Some populations, in markedly degraded habitats, are characterized by divergent morphotypes. However, the evolutionary significance of these morphotypes is of debate. Using 11 polymorphic microsatellites, we studied: (1) marginated tortoises from Sardinia that display a divergent morphotype – this population was potentially introduced from Greece; and (2) an area in the southern Peloponnese that includes a small and degraded zone in which marginated tortoises are dwarf. Genetic analyses run without any a priori assignment clearly acknowledge the specimens sampled in the territory of the dwarf form as a single group whilst Sardinian specimens are clustered with other specimens from the northern part of the area sampled in Greece. Demographic analyses suggest that Sardinian tortoises originated recently from some of the populations sampled in the northern part of the area sampled in Greece. Over locations sampled in Greece, a landscape‐genetic analysis allowed us to detect potential landscape features that may reduce gene flow between the dwarf form territory and surrounding areas. Our results suggest that the territory of the dwarf form is particularly propitious for marginated tortoises and that conservation regulations in Greece should be reinforced to protect this area from increasing impact of human activities changing from traditional agriculture to mechanization and extensive use of chemicals. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 591–606.     

CANNABINOID ENANTIOMER ACTION ON THE CYTOARCHITECTURE

The negative and positive enantiomers of 7-hydroxy- Δ⁶-tetrahydrocannabinol-dimethylheptyl (designated HU-210 and HU-211 respectively) differentially affect undifferentiated and differentiating cultured pheochromocytoma cells (PC-12 cells). In general, cell viability and cell proliferation were suppressed to a much greater extent with HU-210 than with HU-211 in differentiating cells. The effects of these synthetic cannabinoids on the cytoskeleton of PC-12 cells were examined by epifluorescence and confocal microscopy. In both undifferentiated and differentiating PC-12 cells, HU-211 has little effect on the cytoarchitecture whereas HU-210 disrupts the distribution of microtubules and microfilaments. Vacuoles (2–4 μm) were evident in the cytoplasm of HU-210-treated cells but not in the cytoplasm of HU-211-treated cells or in vehicle controls. Tubulin and actin mRNA levels were reduced to 5 and 40 %, respectively (relative to untreated controls) in 10 μmHU-210-treated cells whereas the same concentration of HU-211 reduced tubulin and actin mRNA levels to 90 and 95 %, respectively. A comparison of the effects of the paired enantiomers and Δ¹-THC on the cellular parameters studied reveals that in differentiating cells the action of Δ¹-THC is intermediate between that of HU-210 and HU-211. This study demonstrates that compared to HU-210 and Δ¹-THC the positive enantiomer HU-211 has little cellular activity.