Physiological and Transcriptomic Response of Grey Poplar (Populus ×canescens Aiton Sm.) to Cadmium Stress

(1) Background: Populus ×canescens (Aiton) Sm. is a fast-growing woody plant belonging to the family Salicaceae. Two poplar genotypes characterized by unique phenotypic traits (TP11 and TP20) were chosen to be characterized and tested for a physiological and transcriptomic response to Cd stress. (2) Methods: A comparative analysis of the effects of exposure to high cadmium (Cd) concentrations (10 µM and 100 µM) of TP11 and TP20 was performed. (3) Results: Neither of the tested Cd concentration negatively affected plant growth; however, the chlorophyll content significantly decreased. The potassium (K) content was higher in the shoots than in the roots. The magnesium concentrations were only slightly affected by Cd treatment. The zinc content in the shoots of TP20 was lower than that in the shoots of TP11. Cd accumulation was higher in the roots than in the shoots. After 10 days of exposure, 10 µM Cd resulted in comparable amounts of Cd in the roots and shoots of TP20. The most significant change in transcript amount was observed in endochitinase 2, 12-oxophytodienoate reductase 1 and phi classglutathione S-transferase. (4) Conclusions: Our study provided new insights for effective assessing the ability of different poplar genotypes to tolerate Cd stress and underlying Cd tolerance.

Jasmonic Acid- and Ethylene-Induced Mitochondrial Alternative Oxidase Stimulates Marssonina brunnea Defense in Poplar

Mitochondrial processes are implicated in plant response to biotic stress caused by viruses, actinomyces, bacteria and pests, but their function in defense against fungal invasion remains unclear. Here, we investigated the role and regulation of mitochondrial alternative oxidase (AOX) in response to black spot disease caused by the hemibiotrophic fungus Marssonina brunnea in poplar. M. brunnea inoculation induced the transcription of the AOX1a gene in the mitochondrial electron transport chain and of jasmonic acid (JA) and ethylene (ET) biosynthetic genes, with the accumulation of these phytohormones in poplar leaf, while inhibiting the transcript amount of the mitochondrial cytochrome c oxidase gene (COX6b) and genes related to salicylic acid (SA). Enhanced AOX reduced poplar susceptibility to M. brunnea with a higher ATP/ADP ratio while the repressed AOX caused the reverse effect. Exogenous JA and 1-aminocyclopropane-1-carboxylic acid (ACC, a biosynthetic precursor of ET) inhibited the transcript amount of COX6b and consequently increased the ratio of AOX pathway to total respiration. Furthermore, the transcription of CYS C1 and CYS D1 genes catalyzing cyanide metabolism was induced, while the cysteine (CYS) substrate levels reduced upon M. brunnea inoculation; exogenous JA and ACC mimicked the effect of M. brunnea infection on cysteine. Exogenous SA enhanced, while JA and ACC reduced, poplar susceptibility to M. brunnea. Moreover, inhibiting AOX completely prohibited JA- and ET-increased tolerance to M. brunnea in poplar. These observations indicate that the JA- and ET-induced mitochondrial AOX pathway triggers defense against M. brunnea in poplar. This effect probably involves cyanide. These findings deepen our understanding of plant–pathogenic fungi interactions.

BAALC and WT1 Transcript Amount Discriminates Secondary or Reactive Eosinophilia from Idiopathic Hypereosinophilic Syndrome or Chronic Eosinophilic Leukemia

Idiopathic hypereosinophilic syndromes (HES) or chronic eosinophilic leukemia (CEL) comprise a spectrum of indolent to aggressive diseases characterized by persistent hypereosinophilia. Hypereosinophilia can result from the presence of a defect in the hematopoietic stem cell giving rise to eosinophilia, it can present in many myeloproliferative disorders or alternatively it may be a reactive form, secondary to many clinical conditions. The fusion gene FIP1L1-PDGFR alpha was identified in a subset of patients presenting with HES/CEL. In spite of this, the majority of HES/CEL patients do not present detectable molecular lesions and for many of them the diagnosis is based on exclusion criteria and sometimes it remains doubt. CD34-positive progenitor cells from bone marrow (BM) express BAALC and WT1. Overexpression of BAALC and WT1 were seen in patients with AML and ALL. In a subset of AML it marked poor prognosis, suggesting a role for BAALC or WT1 overexpression in acute leukemia. To explored the possibility to distinguish between HES/CEL and reactive hypereosinophilia based on the measurement of BAALC and WT1 transcript amount. Twenty-two patients with hypereosinophilia were characterized at the molecular level and analyzed for BAALC and WT1 expression. The transcription of FIP1L1-PDGFRalpha fusion gene was detected by nested RT-PCR. The relative transcript amount of BAALC and WT1 were determined by real time PCR analyses. The FIP1L1-PDGFRalpha fusion gene expressed has been identified in bone marrow mononuclear cells of 4 cases. The relative expression level of BAALC and WT1 in these 4 cases with positive FIP1L1-PDGFRalpha fusion gene expression were 2.27(0.27–6.8) and 0.39(0.002–0.90), respectively. Whereas the relative amount of transcripts of BAALC and WT1 in 18 patients with negative FIP1L1-PDGFRalpha fusion gene were 0.069(0.015–0.11) and 0.054(0–0.34) respectively. The relative amount of transcripts of BAALC and WT1 in patients with HES/CEL were 32 times and 7 times than that in those with negative FIP1L1-PDGFRalpha fusion gene, respectively. These results clearly demonstrates that BAALC and WT1 quantitative assessment allows to discriminate between HES/CEL and reactive eosinophilia and represents a useful tool for disease monitoring especially in the patients lacking a marker of clonality.

Prognostic Value of BCR-ABL Transcript Amount for Early Relapse Detection in Chronic Myeloid Leukemia Patients Treated with Allogeneic Hematopoietic Stem Cell Transplantation.

The detection of BCR-ABL mRNA following allogeneic hematopoietic stem cell transplantation (allo-HSCT) is associated with relapse but not absolutely predictive. Early molecular relapse can be detected using real-time quantitative PCR (RQ-PCR) assay, which provides an accurate and reliable method of BCR-ABL expression evaluation. Using this method we analyzed 66 CML patients 3 months to 12 years (median 50 months) after allo-HSCT, who had BCR-ABL transcript measured in peripheral blood or bone marrow samples every 3, 6 and 12 months after allo-HSCT. The patients were analyzed at monthly intervals in the case of rising BCR-ABL transcript level. The median age at diagnosis and median age at time of allo-HSCT were 38 years (range, 16–55). Fifty three patients were in the first chronic phase, and 13 were in the second chronic phase. The median time from diagnosis to allo-HSCT was 8 months (range, 2.5–59). Eighty two percent of patients received allo-HSCT from HLA-identical siblings and 18% from unrelated donors. Transplant conditioning and GvHD prophylaxis were performed according to standard protocols. Quantification of BCR-ABL mRNA transcript was performed according to ‘Europe Against Cancer’ protocol. The median number of assays per patient was 6 (range, 3–16). According to the amount of BCR-ABL transcript after allo-HSCT, patients were allocated into 3 categories, including 35 patients with stable/low level of BCR-ABL transcript (below 0.005%), 11 patients with fluctuating-low level of BCR-ABL (0.005%–0.01%), and 18 patients with high level of BCR-ABL transcript (0.01%–0.1%). We found strong correlation between the amount of BCR-ABL transcript after allo-HSCT and probability of molecular relapse (6% vs. 9% vs. 28%, respectively) as well as relapse-free survival (median time to relapse: 134 months vs. 106 vs. 89, respectively). In the group of patients with high level of BCR-ABL transcript cytogenetic relapse was observed in 75%. In one case BCR-ABL level spontaneously fall down to a low-lewel expression within 6 months. One patient received donor lymphocyte infusion without response, and then was successfully treated with imatinib. RQ-PCR is valuable method to quantitate BCR-ABL expression in CML patients after allo-HSCT, allows monitoring the kinetics of BCR-ABL mRNA transcript amount and is useful in prediction of the hematologic relapse. RQ-PCR can successfully distinguish the group of patients with high risk of relapse from two groups without need for additional therapy (persistent disease/lack of disease).

WT-1 Over-Expression Could Be Related to " High Metaplastic Index" in Myelofibrosis with Myeloid Metaplasia (MMM) Patients. A Preliminary Report.

WT1 is a tumor-suppressor gene coding for a zinc-finger transcription factor located on chromosome 11p13, which was originally identified for its involvement in the pathogenesis of the Wilms’ tumor. In normal bone marrow and peripheral blood(PB)WT1 expression is extremely low and often undetectable even by RT-PCR. By contrast WT1 expression has been previously reported to be increased in MDS, AML, ALL and PNH. In CMPDs, little data are available only in CML. With this background we analyze by RQ-PCR the WT1 transcript amount in 13 patients with myelofibrosis with myeloid metaplasia (MMM) in comparison with a group of 32 age- and sex-matched healthy controls. All measurements were carried out only on PB and performed as previous published. All MMM patients were under cytoreductive treatment and at different time from diagnosis ( range 1 – 7 yrs). In MMM WT1 transcript amount is consistently higher (range 23–2518 WT1 copies/104 ABL copies, mean 382±692) than controls ( range 0 – 22, mean 5,6 ± 5,87) ( P < 0.0001 Mann-Whitney U test). To assess the significance of the WT1 expression as a marker for disease features, the following variables were analyzed and compared : age, hemoglobin level, white blood cell count, circulating blasts, LDH serum levels, number of peripheral CD 34 cells, Dupriez score and, according to Barosi published data, myeloproliferation index, myelodepletion index, severity score. We did not found any significant correlation between WT1 and all these variables, probably because the patients were studied during the course of their disease. Moreover, cytoreductive treatment might have modified the disease characteristics. Stricking, when analyzed WT1 expression according to the therapeutic need, we found a subset of patients with a WT1 overexpression ( range 132–2518, mean 758±911; p <0.06) and spleen size between 4 – 7 cm despite receiving > 1,5 gr/daily of hydroxyurea or more than 1 drug. In such of patients WT1 over-expression allowed an almost complete discrimination between MMM in stable phase and MMM with “high metaplastic index”. In conclusion, our results, although based on a relatively small number of patients, provide an interesting profile of WT1 expression in MMM. The possible clinico-therapeutic implications of these observations are worthy of investigation in well-designed studies.