Signalling responses in the bark and foliage of canker-susceptible and -resistant cypress clones inoculated with Seiridium cardinale.

The necrotrophic fungus Seiridium cardinale is the main responsible for Cypress Canker Disease (CCD), a pandemic affecting many Cupressaceae worldwide. The present study aims to elucidate the signalling of the early responses in the bark and foliage of CCD-susceptible and -resistant C. sempervirens clones to S. cardinale inoculation (SI and RI, respectively). In the bark of SI, a peaking production of ethylene (Et) and jasmonic acid (JA) occurred at 3 and 4 days post inoculation (dpi), respectively, suggesting an attempted plant response to the pathogen. A response that, however, was ineffective, as confirmed by the severe accumulation of malondialdehyde by-products at 13 dpi (i.e., lipid peroxidation). Differently, Et emission peaked in RI bark at 3 and 13 dpi, whereas abscisic acid (ABA) accumulated at 1, 4 and 13 dpi, resulting in a lower MDA accumulation (and unchanged levels of antioxidant capacity). In the foliage of SI, Et was produced at 1 and 9 dpi, whereas JA and salicylic acid (SA) accumulated at 1 and 3 dpi. Conversely, an increase of ABA and SA occurred at 1 dpi in the RI foliage. This outcome indicates that some of the observed metabolic alterations, mainly occurring as local defence mechanisms, might be able to gradually shift to a systemic resistance, although an accumulation of MDA was observed in both SI and RI foliage (but with an increased antioxidant capacity reported only in the resistant clone). We believe that the results reported here will be useful for the selection of clones able to limit the spread and damage of CCD.

When "thirsty" means "less able to activate the signalling wave trigged by a pulse of ozone": A case of study in two Mediterranean deciduous oak species with different drought sensitivity.

There is a lack of knowledge about the possibility that plants facing abiotic stressors, such as drought, have an altered perception of a pulse of O3 and incur in alterations of their signalling network. This poses some concerns as to whether defensive strategy to cope episodic O3 peaks in healthy plants may fail under stress. In this study, a set of saplings of two Mediterranean deciduous species, Quercus cerris and Q. pubescens, was subjected to water withholding (20% of daily evapotranspiration for 15 days) while another set was kept well-watered. Saplings were then subjected to a pulse of O3 (200 nl l-1 for 5 h) or maintained in filtered air. Q. pubescens had a more severe decline of photosynthesis and leaf PDΨw (about -65% and 5-fold lower than in well-watered ones) and events of cell death were observed under drought when compared to Q. cerris, which is supportive for a higher sensitivity to drought exhibited by this species. When O3 was applied after drought, patterns of signalling compounds were altered in both species. Only in Q. pubescens, the typical O3-induced accumulation of apoplastic reactive oxygen species, which is the first necessary step for the activation of signalling cascade, was completely lost. In Q. cerris the most frequent changes encompassed the weakening of peaks of key signalling molecules (ethylene and salicylic acid), whereas in Q. pubescens both delayed (salicylic and jasmonic acid) or weakened (ethylene and salicylic acid) peaks were observed. This is translated to a higher ability of Q. cerris to maintain a prompt activation of defensive reaction to counteract oxidative damage due to the pollutant. Our results reveal the complexity of the signalling network in plants facing multiple stresses and highlight the need to further investigate possible alteration of defensive mechanism of tree species to predict their behavior.

What about the detoxification mechanisms underlying ozone sensitivity in Liriodendron tulipifera?

Liriodendron tulipifera (known as the tulip tree) is a woody species that has been previously classified as sensitive to ozone (O3) in terms of visible leaf injuries and photosynthetic primary reactions. The objective of this work is to give a thorough description of the detoxification mechanisms that are at the basis of O3 sensitivity. Biochemical and molecular markers were used to characterize the response of 1-year-old saplings exposed to O3 (120 ppb, 5 h day-1, for 45 consecutive days) under controlled conditions. O3 effects resulted in a less efficient metabolism of Halliwell-Asada cycle as confirmed by the diminished capacity to convert the oxidized forms of ascorbate and glutathione in the reduced ones (AsA and GSH, respectively). The reduced activity of AsA and GSH regenerating enzymes indicates that de novo AsA biosynthesis occurred. This compound could be a cofactor of several plant-specific enzymes that are involved in the early part of the phenylpropanoid and flavonoid biosynthesis pathway, as confirmed by the significant rise of PAL activity (+75%). The induction of the defence-related secondary metabolites (in particular, rutin and caffeic acid were about threefold higher) and the concomitant increase in transcript levels of PAL and CHS genes (+120 and 30%, respectively) suggest that L. tulipifera utilized this route in order to partially counteract the O3-induced oxidative damage.

Losing the Warning Signal: Drought Compromises the Cross-Talk of Signaling Molecules in Quercus ilex Exposed to Ozone.

Understanding the interactions between drought and acute ozone (O3) stress in terms of signaling molecules and cell death would improve the predictions of plant responses to climate change. The aim was to investigate whether drought stress influences the responses of plants to acute episodes of O3 exposure. In this study, the behavior of 84 Mediterranean evergreen Quercus ilex plants was evaluated in terms of cross-talk responses among signaling molecules. Half of the sample was subjected to drought (20% of the effective daily evapotranspiration, for 15 days) and was later exposed to an acute O3 exposure (200 nL L-1 for 5 h). First, our results indicate that in well-water conditions, O3 induced a signaling pathway specific to O3-sensitive behavior. Second, different trends and consequently different roles of phytohormones and signaling molecules (ethylene, ET; abscisic acid, ABA; salycilic acid, SA and jasmonic acid, JA) were observed in relation to water stress and O3. A spatial and functional correlation between these signaling molecules was observed in modulating O3-induced responses in well-watered plants. In contrast, in drought-stressed plants, these compounds were not involved either in O3-induced signaling mechanisms or in leaf senescence (a response observed in water-stressed plants before the O3-exposure). Third, these differences were ascribable to the fact that in drought conditions, most defense processes induced by O3 were compromised and/or altered. Our results highlight how Q. ilex plants suffering from water deprivation respond differently to an acute O3 episode compared to well-watered plants, and suggest new effect to be considered in plant responses to environmental changes. This poses the serious question as to whether or not multiple high-magnitude O3 events (as predicted) can change these cross-talk responses, thus opening it up possible further investigations.

Can Ozone Alter the Terpenoid Composition and Membrane Integrity of in vitro Melissa officinalis Shoots?

Ozone affects volatile organic compounds that protect plants from biotic and abiotic stress. In vitro Melissa officinalis shoots were exposed to ozone (200 ppb, 3 h) in controlled environmental conditions: leaf pigments, membrane integrity and headspace composition were assayed during fumigation and after the recovery period (3 h from the beginning of the exposure, FBE). At the end of the exposure, no injury was observed in untreated and treated shoots, although an evident increase in lipid peroxidation was reported (+38.5 and +37.2% of TBARS levels in comparison with controls, respectively after 1 and 3 h FBE). The levels of total carotenoids significantly rose as a normal response mechanism to oxidative stress. SPME-GS-MS analysis showed that, as a consequence of the fumigation, the trends in non-terpenoid compounds increased after 1 and 3 h FBE. This suggests that the concentration and the duration of the treatment were enough to cause a breakdown of cells (as evidenced by increased TBARS levels) and involves an association between volatile products of the lipoxygenase pathway (LOX products) and membrane degradation.

Role of nitric oxide in actin depolymerization and programmed cell death induced by fusicoccin in sycamore (Acer pseudoplatanus) cultured cells.

Programmed cell death (PCD) plays a vital role in plant development and is involved in defence mechanisms against biotic and abiotic stresses. Different forms of PCD have been described in plants on the basis of the cell organelle first involved. In sycamore (Acer pseudoplatanus L.) cultured cells, the phytotoxin fusicoccin (FC) induces cell death. However, only a fraction of the dead cells shows the typical hallmarks of animal apoptosis, including cell shrinkage, chromatin condensation, DNA fragmentation and release of cytochrome c from the mitochondrion. In this work, we show that the scavenging of nitric oxide (NO), produced in the presence of FC, by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and rutin inhibits cell death without affecting DNA fragmentation and cytochrome c release. In addition, we show that FC induces a massive depolymerization of actin filaments that is prevented by the NO scavengers. Finally, the addition of actin-depolymerizing drugs induces PCD in control cells and overcomes the inhibiting effect of cPTIO on FC-induced cell death. Vice versa, the addition of actin-stabilizing drugs to FC-treated cells partially inhibits the phytotoxin-induced PCD. These results suggest that besides an apoptotic-like form of PCD involving the release of cytochrome c, FC induces at least another form of cell death, likely mediated by NO and independent of cytochrome c release, and they make it tempting to speculate that changes in actin cytoskeleton are involved in this form of PCD.

Budding yeast PAK kinases regulate mitotic exit by two different mechanisms.

We report the characterization of the dominant-negative CLA4t allele of the budding yeast CLA4 gene, encoding a member of the p21-activated kinase (PAK) family of protein kinases, which, together with its homologue STE20, plays an essential role in promoting budding and cytokinesis. Overproduction of the Cla4t protein likely inhibits both endogenous Cla4 and Ste20 and causes a delay in the onset of anaphase that correlates with inactivation of Cdc20/anaphase-promoting complex (APC)-dependent proteolysis of both the cyclinB Clb2 and securin. Although the precise mechanism of APC inhibition by Cla4t remains to be elucidated, our results suggest that Cla4 and Ste20 may regulate the first wave of cyclinB proteolysis mediated by Cdc20/APC, which has been shown to be crucial for activation of the mitotic exit network (MEN). We show that the Cdk1-inhibitory kinase Swe1 is required for the Cla4t-dependent delay in cell cycle progression, suggesting that it might be required to prevent full Cdc20/APC and MEN activation. In addition, inhibition of PAK kinases by Cla4t prevents mitotic exit also by a Swe1-independent mechanism impinging directly on the MEN activator Tem1.