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Salinization of soil is a prime abiotic stress that limits agriculture productivity worldwide. To Study the mechanisms that halophytes take up to survive under high salt condition is important in engineering salinity stress tolerance in sensitive species. Suaeda nudiflora is a halophyte plant that grows in the saline environment and extreme high tidal belt. The species have high capability to produce high protein biomass in salty soils due to C4 photosynthesis. The physiological and biochemical changes in S. nudiflora under salinity stress were studied by measuring chlorophyll content, electrolytic leakage, level of lipid peroxidation and total soluble sugars. Increased lipid peroxidation and electrolytic leakage was observed in salt stressed S. nudiflora compared to control plants. A suppression subtractive hybridization strategy was employed to identify differentially expressed genes under salt treatment in S. nudiflora. A total of 333 positive clones were identified and screened. Of these, 250 expressed sequence tags were identified. cDNA subtraction library resulted in 33 contigs and 138 singletons. The functional annotation and metabolic pathways identification were performed using the Blast2GO program. In addition, we analyzed the expression patterns of 18 genes associated with salt stress-responsive pathways by semi-quantitative PCR under salt and elevated carbon dioxide (CO2) conditions. Several of the analyzed genes showed an increase in expression levels under different time points of salt treatment and at different concentrations of salt. When the same genes were studied for its expression under elevated CO2 concentrations, most of the known salt responsive genes showed higher expression under the combined treatment of elevated CO2 concentrations (500 ppm) and NaCl treatment (200 mM) compare to ambient condition. This implies that salt responsive genes are enhanced at elevated CO2 concentrations.

Drought transcriptome analysis of finger millet (Eleusine coracana) by cDNA subtraction identified drought responsive genes that have a potential role in drought tolerance. Through virus-induced gene silencing (VIGS) in a related crop species, maize (Zea mays), several genes, including a G-BOX BINDING FACTOR 3 (GBF3) were identified as candidate drought stress response genes and the role of GBF3 in drought tolerance was studied in Arabidopsis thaliana. Overexpression of both EcGBF3 and AtGBF3 in A. thaliana resulted in improved tolerance to osmotic stress, salinity and drought stress in addition to conferring insensitivity to ABA. Conversely, loss of function of this gene increased the sensitivity of A. thaliana plants to drought stress. EcGBF3 transgenic A. thaliana results also suggest that drought tolerance of sensitive plants can be improved by transferring genes from far related crops like finger millet. Our results demonstrate the role of GBF3 in imparting drought tolerance in A. thaliana and indicate the conserved role of this gene in drought and other abiotic stress tolerance in several plant species.

Citrus sudden death (CSD) appears to be a new disease that is a serious problem in Brazil. Symptoms of CSD include yellow stain in the phloem of the rootstock. The cause is not known, but it appears to be infectious and may only affect trees budded on Rangpur lime. In a survey in Brazil, in addition to CSD, we observed numerous trees on Rangpur lime that were obviously declining but had remained in production for several years. Trees with this disease, referred to as Rangpur lime decline (RLD) were different from those with citrus blight (CB). They had near-normal size fruit compared with the small fruit associated with CB and were negative in the serological test for the CB-associated protein (p12). Moreover, they did not have the yellow stain symptom and obviously were declining much more slowly than was reported for CSD. To determine what viruses or virus strains might be associated with CSD, double-stranded (ds)RNAs from fibrous roots of a tree with CSD and stem bark from greenhouse trees infected with Citrus tristeza virus (CTV) isolates T30 and T36 were used to make random primed cDNAs. A Clontech PCR-Select cDNA Subtraction Kit was used to subtract the CSD cDNA with cDNA from an equal mixture of dsRNA from T30 and T36. Of 28 clones that were sequenced, five were found to be significantly different from published CTV sequences. One clone (SDA-1) was found to be only 48% similar to CTV T30 based on amino acid sequence. Using samples collected in October 2001, hybridization assays with a DIG probe of SDA-1 were positive for RNA from roots of declining trees from an area where CSD is reported to occur and from a second area where trees were declining with what had been thought to be CB and are now considered to be RLD. The SDA-1 probe reacted weakly or not at all with RNA from stem bark of trees with CSD, collected in October 2001, or RNA from roots of trees that were declining with CB. Using samples collected in March 2003 from trees with severe decline (nearly dead), the SDA-1 probe reacted with all preparations from both stems and roots. Reactions to the SDA-1 probe also were observed in many stem or root samples from trees with RLD, with early symptoms of CSD, and nonsymptomatic trees. The SDA-1 probe did not react with samples from roots or stems of healthy or CB trees from Florida.

Transmembrane protein 132A (TMEM132A) was first isolated from rat brain using PCR-selected cDNA subtraction, and it was found to be predominantly expressed in the brain. However, the transcriptional regulation of the TMEM132A gene has not been fully characterized. In this study, we characterized the promoter activity of the 880-bp region upstream of the mouse TMEM132A, identifying several putative sites recognized by transcription factors, which are highly conserved between the mouse and human TMEM132A genes. Using four different mouse cell lines (Neuro2a, NSC-34, NIH3T3, and Raw264.7), we first evaluated the intrinsic levels of TMEM132A mRNA and protein expression. Interestingly, TMEM132A mRNA was expressed in all four cell lines, whereas the protein was negligible in Raw264.7 cells even by transfection of TMEM132A gene. Then, we analyzed the TMEM132A promoter activity using serial deleted constructs, finding it was nearly same pattern in all four cell lines. A mutational analysis of the TMEM132A promoter identified a critical region for its activation just upstream of the transcriptional start site. Finally, we investigated the levels of TMEM132A mRNA and protein after exposure to five different neurotoxic stimuli, including thapsigargin, tunicamycin, serum starvation, homocysteine, and hydrogen peroxide. Treatment with thapsigargin, a calcium modulating agent, markedly attenuated the levels of TMEM132A mRNA and protein in NSC-34 cells. These results give new insight into the mechanisms involved in regulating TMEM132A expression, and suggest that several transcriptional and post-transcriptional pathways regulate TMEM132A expression under developmental and pathophysiological conditions.

Some cultivars of cucumbers, melons, pumpkins, and zucchini, which are members of the Cucurbitaceae family, are uniquely subject to contamination by hydrophobic pollutants such as the organohalogen insecticides DDT. However, the molecular mechanisms for the accumulation of these pollutants in cucurbits have not been determined. Here, cDNA subtraction analysis of Cucurbita pepo cultivars that are low and high accumulators of hydrophobic contaminants revealed that a gene for zinc finger proteins (ZFPs) are preferentially expressed in high accumulators. The cloned CpZFP genes were classified into 2 types: (1) the PBG type, which were expressed in C. pepo cultivars Patty Green, Black Beauty, and Gold Rush, and (2) the BG type, which were expressed in Black Beauty and Gold Rush. Expression of these CpZFP genes in transgenic tobacco plants carrying an aryl hydrocarbon receptor-based inducible gene expression system significantly induced β-glucuronidase activity when the plants were treated with a polychlorinated biphenyl (PCB) compound, indicating that highly hydrophobic PCBs accumulated in the plants. In transgenic tobacco plants carrying CpZFPs, accumulation of dioxins and dioxin-like compounds increased in their aerial parts when they were cultivated in the dioxin-contaminated soil. In summary, we propose that addition of CpZFP genes is a promising tool for conferring noncucurbits with the ability to accumulate hydrophobic contaminants.

Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis, S. globosa, S. mexicana, S. pallida, S. schenckii, and S. lurei. S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the dimorphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa, suppression subtractive hybridization (SSH) was used to prepare a complementary DNA (cDNA) subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between differently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China.

The human BCL6 gene encodes a transcriptional repressor that is needed for germinal center B cell development and T follicular helper cell differentiation, and it is best known for its association with certain human lymphomas, especially the diffuse large B-cell type. We devised a cell system in which the BCL6 repressive effects are inhibited with the goal of detecting the consequently upregulated BCL6 target genes. In order to do this, we converted the BCL6 zinc fingers (BCL6ZF), which can bind DNA but lack repressive effects, into a transcriptional activator and used this construct to compete with endogenous BCL6 in BJAB cells (Burkitt lymphoma cell line) which express high levels of BCL6. We used subtractive hybridization methodology to amplify differentially expressed sequences and identified the Pontin gene (also called Pontin 52, RUVBL1, TIP49, NMP238) as a potential target of BCL6 repression. We confirmed Pontin as a BCL6 target. Northern blots prepared from BJAB cells that had been transfected with the BCL6 ZF construct or the vector in which it was cloned were hybridized with the cDNA fragment of Pontin obtained from cDNA subtraction and amplification. Scanning densitometry, used to normalize relative band intensity to β -actin, indicated that differential expression of Pontin as compared with the vector control was 2.6-fold (p < 0.02). We found an exact match to 7 (out of 8) bases described as a preferential BCL6 binding site in the promoter region of the Pontin gene. ChIP assays indicated that BCL6 was bound to this site, but the appropriate controls did not enrich DNA from this region. Transfection assays showed that the BCL6 protein represses transcription from the BCL6 consensus binding site in the Pontin promoter (Fig. 1, 6.8-fold as compared with the control, p < 0.0001). Transfections of siRNAs targeting the BCL6 protein in BJAB cells led to a significant increase in endogenous Pontin (Fig. 2, 2-fold, p < 0.02). Finally, immunohistochemistry performed on 21 randomly selected B and T cell lymphomas showed an inverse relationship between BCL6 and Pontin in a majority of the tumors. Pontin is a transcriptional cofactor with ATPase activity that is part of the AAA+ (ATPases Associated with diverse cellular Activities) superfamily which includes a large group of ring-shaped complexes that are involved in multiple cellular processes. The Pontin protein is expressed virtually ubiquitously, is evolutionarily conserved, and is expressed in both the cell nucleus and cytoplasm. The BCL6 protein, also expressed ubiquitously and evolutionarily conserved, is upregulated 3 to 34 times in the nucleus of lymph node germinal center B cells as compared with resting B cells. It has been called a "master regulator" of the germinal center. It is possible, judging from the many essential functions of Pontin, that it, too, is a kind of "master regulator." Further understanding of the relationship between BCL6 and Pontin may help to provide new insights concerning the initiation and progression of B and T cell lymphomas. Figure 1. Transfection assays: The BCL6 Protein represses transcription from the Pontin Promoter Figure 1. Transfection assays: The BCL6 Protein represses transcription from the Pontin Promoter Figure 2. Knockdown of BCL6 protein levels by siRNA increases Pontin protein expression Figure 2. Knockdown of BCL6 protein levels by siRNA increases Pontin protein expression Disclosures No relevant conflicts of interest to declare.