Responses of Scots pine to nickel and copper exposure and herbivory

The goal of this thesis was to examine the ecophysiological responses of Scots pine (Pinus sylvestris L.), with an emphasis on the oxidative enzyme peroxidase and plant phenolics to environmental stresses like elevated levels of nickel (Ni) and copper (Cu), and herbivory. The effects of Ni and Cu were studied in a gradient survey at a sulphur dioxide contaminated site in the Kola Peninsula, and with experiments in which seedlings were exposed to Ni mist or to Ni and Cu amended into the soil. In addition, experimental Ni exposure was combined with disturbance of the natural lichen cover of the forest ground layer. Pine sawfly attack was simulated in the early season defoliation experiment, in which mature Scots pine were defoliated (100 %) during two successive years in a dry, nutrient-poor Scots pine stand. In addition, the effect of previous defoliation on the growth of sawfly (Diprion pini L.) larvae was studied. Apoplastic peroxidase activity was elevated in the needles of pine in a Ni- , Cu- and SO2- polluted environment, which indicated an increased oxidative stress. Increased foliar peroxidase activity due to Ni contamination was shown in the experiment, in which Ni was added as mist. No such response was found in peroxidase acitivity of the roots exposed to elevated Ni and/or Cu in the soil. Elevated Ni in the soil increased the concentration of foliar condensed tannins, which are able to bind heavy metals in the cells. Addition of low levels of Ni in the soil appeared to benefit pine seedlings, which was seen as promoted shoot growth and better condition of the roots. Wet Ni deposition of 2000 mg m-2 reduced growth and survival of pine seedlings, whereas deposition levels 200 mg m-2 or 20 mg m-2 caused no effects in a 2-y lasting experiment. The lichen mat on the forest floor did not act as an effective buffer against the adverse impacts of heavy metals on pine seedlings. However, some evidence was found indicating that soil microbes profited from the lichen mat. Artificial defoliation increased peroxidase activity in the Scots pine needles. In addition, defoliation decreased nitrogen, diamine putrescine and glucose concentrations in the needles and increased the concentrations of several phenolic compounds, starch and sucrose. Previous artificial defoliation led to poor growth of sawfly larvae reared on the pines, suggesting delayed induced resistance in Scots pine. However, there was no consistent relationship between inducibility (proportional increase in a compound following defoliation) and adverse effects on the growth of pine sawfly larvae. The observed inducible responses in needle phenolics due to previous defoliation thus appear to represent non-specific responses against sawflies.

Resistance Mechanisms of Non-Hodgkin Lymphomas against Rituximab Treatment

Rituximab, a monoclonal antibody against B-cell specific CD20 antigen, is used for the treatment of non-Hodgkin lymphomas (NHL) and chronic lymphatic leukemia. In combination with chemotherapeutics rituximab has remarkably improved the outcome of NHL patients, but a vast variation in the lengths of remissions remains and the outcome of individual patients is difficult to predict. This thesis has searched for an explanation for this by studying the effector mechanisms of rituximab and by comparing gene expression in lymphoma tissue samples of patients with long- and short-term survival. This work demonstrated that activation of complement (C) system is in vitro more efficient effector mechanism of rituximab than cellular mechanisms or apoptosis. Activation of the C system was also shown in vivo during rituximab treatment. However, intravenously administered rituximab could not enter the cerebrospinal fluid, and neither C activation nor removal of lymphoma cells was observed in central nervous system. In vitro cytotoxicity assays showed that rituximab-induced cell killing could be markedly improved with simultaneous neutralization of the C regulatory proteins CD46 (Membrane cofactor protein), CD55 (Decay-accelerating factor), and CD59 (protectin). In a retrospective study of follicular lymphoma (FL) patients, low lymphoma tissue mRNA expressions of CD59 and CD55 were associated with a good prognosis and in a progressive flow cytometry study high expression of CD20 relative to CD55 was correlated to a longer progression free survival. Gene expression profile analysis revealed that expression of certain often cell cycle, signal transduction or immune response related genes correlate with clinical outcome of FL patients. Emphasizing the role of tumor microenvironment the best differentiating genes Smad1 and EphA1 were demonstrated to be mainly expressed in the non-malignant cells of tumors. In conclusion, this thesis shows that activation of the C system is a clinically important effector mechanism of rituximab and that microenvironment factor in tumors and expression of C regulatory proteins affect markedly the efficacy of immunochemotherapy. This data can be used to identify more accurately the patients for whom immunochemotherapy is given. It may also be beneficial in development of rituximab-containing and other monoclonal antibody therapies against cancer.

Pathogen-Induced Defense Signaling and Signal Crosstalk in Arabidopsis

Erwinia carotovora subsp. carotovora is a bacterial phytopathogen that causes soft rot in various agronomically important crop plants. A genetically specified resistance to E. carotovora has not been defined, and plant resistance to this pathogen is established through nonspecific activation of basal defense responses. This, together with the broad host range, makes this pathogen a good model for studying the activation of plant defenses. Production and secretion of plant cell wall-degrading enzymes (PCWDE) are central to the virulence of E. carotovora. It also possesses the type III secretion system (TTSS) utilized by many Gram-negative bacteria to secrete virulence- promoting effector proteins to plant cells. This study elucidated the role of E. carotovora HrpN (HrpNEcc), an effector protein secreted through TTSS, and the contribution of this protein in the virulence of E. carotovora. Treatment of plants with HrpNEcc was demonstrated to induce a hypersensitive response (HR) as well as resistance to E. carotovora. Resistance induced by HrpNEcc required both salicylic acid (SA)- and jasmonate/ethylene (JA/ET)-dependent defense signaling in Arabidopsis. Simultaneous treatment of Arabidopsis with HrpNEcc and PCWDE polygalacturonase PehA elicited accelerated and enhanced induction of defense genes but also increased production of superoxide and lesion formation. This demonstrates mutual amplification of defense signaling by these two virulence factors of E. carotovora. Identification of genes that are rapidly induced in response to a pathogen can provide novel information about the early events occurring in the plant defense response. CHLOROPHYLLASE 1 (AtCLH1) and EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15) are both rapidly triggered by E. carotovora in Arabidopsis. Characterization of AtCLH1 encoding chlorophyll-degrading enzyme chlorophyllase indicated that it might have a role in chlorophyll degradation during plant tissue damage. Silencing of this gene resulted in increased accumulation of reactive oxygen species (ROS) in response to pathogen infection in a light-dependent manner. This led to enhanced SA-dependent defenses and resistance to E. carotovora. Moreover, crosstalk between different defense signaling pathways was observed; JA-dependent defenses and resistance to fungal pathogen Alternaria brassicicola were impaired, indicating antagonism between SA- and JA-dependent signaling. Characterization of ERD15 suggested that it is a novel, negative regulator of abscisic acid (ABA) signaling in Arabidopsis. Overexpression of ERD15 resulted in insensitivity to ABA and reduced tolerance of the plants to dehydration stress. However, simultaneously, the resistance of the plants to E. carotovora was enhanced. Silencing of ERD15 improved freezing and drought tolerance of transgenic plants. This, together with the reducing effect of ABA on seed germination, indicated hypersensitivity to this phytohormone. ERD15 was hypothesized to act as a capacitor that controls the appropriate activation of ABA responses in Arabidopsis.

Novel Molecular Mechanisms of Arabidopsis Disease Resistance

Plants are capable of recognizing phytopathogens through the perception of pathogen-derived molecules or plant cell-wall degradation products due to the activities of pathogen-secreted enzymes. Such elicitor recognition events trigger an array of inducible defense responses involving signal transduction networks and massive transcriptional re-programming. The outcome of a pathogen infection relies on the balance between different signaling pathways, which are integrated by regulatory proteins. This thesis characterized two key regulatory components: a damage control enzyme, chlorophyllase 1 (AtCHL1), and a transcription factor, WRKY70. Their roles in defense signaling were then investigated. The Erwinia-derived elicitors rapidly activated the expression of AtCLH1 and WRKY70 through different signaling pathways. The expression of the AtCHL1 gene was up-regulated by jasmonic acid (JA) but down-regulated by salicylic acid (SA), whereas WRKY70 was activated by SA and repressed by JA. In order to elucidate the functions of AtCLH1 and WRKY70 in plant defense, stable transgenic lines were produced where these genes were overexpressed or silenced. Additionally, independent knockout lines were also characterized. Bacterial and fungal pathogens were then used to assess the contribution of these genes to the Arabidopsis disease resistance. The transcriptional modulation of AtCLH1 by either the constitutive over-expression or RNAi silencing caused alterations in the chlorophyll-to-chlorophyllide ratio, supporting the claim that chlorophyllase 1 has a role in the chlorophyll degradation pathway. Silencing of this gene led to light-dependent over-accumulation of the reactive oxygen species (ROS) in response to infection by Erwinia carotovora subsp. carotovora SCC1. This was followed by an enhanced induction of SA-dependent defense genes and an increased resistance to this pathogen. Interestingly, little effect on the pathogen-induced SA accumulation at the early infection was observed, suggesting that action of ROS might potentiate SA signaling. In contrast, the pathogen-induced JA production was significantly reduced in the RNAi silenced plants. Moreover, JA signaling and resistance to Alternaria brassicicola were impaired. These observations provide support for the argument that the ROS generated in chloroplasts might have a negative impact on JA signaling. The over-expression of WRKY70 resulted in an enhanced resistance to E. carotovora subsp. carotovora SCC1, Pseudomonas syringae pv. tomato DC3000 and Erysiphe cichoracearum UCSC1, whilst an antisense suppression or an insertional inactivation of WRKY70 led to a compromised resistance to E. carotovora subsp. carotovora SCC1 and to E. cichoracearum UCSC1 but not to P. syringae pv. tomato DC3000. Gene expression analysis revealed that WRKY70 activated many known defense-related genes associated with the SAR response but suppressed a subset of the JA-responsive genes. In particular, I was able to show that both the basal and the induced expression of AtCLH1 was enhanced by the antisense silencing or the insertional inactivation of WRKY70, whereas a reduction in AtCLH1 expression was observed in the WRKY70 over-expressors following an MeJA application or an A. brassicicola infection. Moreover, the SA-induced suppression of AtCLH1 was relieved in wrky70 mutants. These results indicate that WRKY70 down-regulates AtCLH1. An epistasis analysis suggested that WRKY70 functions downstream of the NPR1 in an SA-dependent signaling pathway. When challenged with A. brassicicola, WRKY70 over-expressing plants exhibited a compromised disease resistance while wrky70 mutants had the opposite effect. These results confirmed the WRKY70-mediated inhibitory effects on JA signaling. Furthermore, the WRKY70-controlled suppression of A. brassicicola resistance was mainly through an NPR1-dependent mechanism. Taking all the data together, I suggest that the pathogen-responsive transcription factor WRKY70 is a common component in both SA- and JA-dependent pathways and plays a crucial role in the SA-mediated suppression of JA signaling.