Novel transcription factors PvBMY1 and PvBMY3 increase biomass yield in greenhouse-grown switchgrass (Panicum virgatum L.).

Increasing crop yield requires the coordination of multiple metabolic pathways spanning photosynthetic carbon fixation, central carbon metabolism, and finally targeted carbon deposition to end product. In this study, we used a transcriptome-based gene regulatory association network to search for transcription factor genes that could play a role in increasing carbon flow through pathways associated with these processes to increase biomass yield in switchgrass. Two novel switchgrass transcription factors, PvBMY1 (BioMass Yield 1, belonging to the APETALA2/Ethylene Response Factor family of transcription factors) and PvBMY3 (BioMass Yield 3, a member of the Nuclear-Factor Y family of transcription factors), with predicted roles in the regulation of photosynthesis and related metabolism were identified. These genes were overexpressed in switchgrass to determine their impact on biomass yield. A significant increase in both aboveground and root biomass was observed in transgenic greenhouse grown plants compared to wild-type control plants with the best line producing 160% more aboveground biomass than controls. Transgenic lines with elevated electron transport rate of photosystems I and II as well as increased levels of starch and soluble sugars were identified.

Transgene autoexcision in switchgrass pollen mediated by the Bxb1 recombinase.

BACKGROUND: Switchgrass (Panicum virgatum L.) has a great potential as a platform for the production of biobased plastics, chemicals and energy mainly because of its high biomass yield on marginal land and low agricultural inputs. During the last decade, there has been increased interest in the genetic improvement of this crop through transgenic approaches. Since switchgrass, like most perennial grasses, is exclusively cross pollinating and poorly domesticated, preventing the dispersal of transgenic pollen into the environment is a critical requisite for the commercial deployment of this important biomass crop. In this study, the feasibility of controlling pollen-mediated gene flow in transgenic switchgrass using the large serine site-specific recombinase Bxb1 has been investigated. RESULTS: A novel approach utilizing co-transformation of two separate vectors was used to test the functionality of the Bxb1/att recombination system in switchgrass. In addition, two promoters with high pollen-specific activity were identified and thoroughly characterized prior to their introduction into a test vector explicitly designed for both autoexcision and quantitative analyses of recombination events. Our strategy for developmentally programmed precise excision of the recombinase and marker genes in switchgrass pollen resulted in the generation of transgene-excised progeny. The autoexcision efficiencies were in the range of 22-42% depending on the transformation event and assay used. CONCLUSION: The results presented here mark an important milestone towards the establishment of a reliable biocontainment system for switchgrass which will facilitate the development of this crop as a biorefinery feedstock through advanced biotechnological approaches.

Sphingosine-1-phosphate receptor 1 in classical Hodgkin lymphoma: assessment of expression and role in cell migration.

Classical Hodgkin lymphoma (CHL), a neoplasm of abnormal B lymphocytes (Hodgkin-Reed-Sternberg (HRS) cells), has been described to have a typical pattern of clinical presentation and dissemination often involving functionally contiguous lymph nodes. Despite the progress made in understanding CHL pathophysiology, the factors that regulate the spread of lymphoma cells in CHL are poorly understood. Sphingosine-1-phosphate (S1P), a bioactive sphingolipid present at high concentrations in the plasma and lymphatic fluid, is known to have a critical role in regulating lymphocyte trafficking mainly through sphingosine-1-phosphate receptor 1 (S1PR1). In this study, we explore the role of the S1P-S1PR1 axis in Hodgkin lymphoma cell migration and the expression of S1PR1 in CHL cell lines and clinical cases. We found that S1PR1 is present in the KM-H2 and SUP-HD1 Hodgkin lymphoma cell lines at the mRNA and protein level. In addition, functionally, S1P potently stimulated migration of both cell lines. S1P-induced migration was inhibited by the S1PR1 antagonist, VPC44116, and the S1PR1 functional antagonist, FTY720-P, but was potentiated by the S1PR2-specific antagonist, JTE013. We also determined that S1PR1 induced migration in the KM-H2 and SUP-HD1 cells via the heterotrimeric G-protein Gi and the phosphatidylinositol-3-kinase pathway. Immunohistochemical assessment of the tissue from CHL samples revealed that a subset of cases (7/57; 12%) show strong, membranous staining for S1PR1 in HRS cells. Altogether, our data indicate that S1PR1 is a functional receptor on HRS cells, which governs tumor cell migration and is expressed in a subset of CHL cases. Given the availability of S1PR1 antagonists, some of which are used clinically for modulation of the immune system, these results suggest that S1PR1 could be a future therapeutic target in the treatment of those cases of S1PR1-positive, refractory/recurrent CHL.

Quantitative effect of suboptimal codon usage on translational efficiency of mRNA encoding HIV-1 gag in intact T cells.

BACKGROUND: The sequences of wild-isolate strains of Human Immunodeficiency Virus-1 (HIV-1) are characterized by low GC content and suboptimal codon usage. Codon optimization of DNA vectors can enhance protein expression both by enhancing translational efficiency, and by altering RNA stability and export. Although gag codon optimization is widely used in DNA vectors and experimental vaccines, the actual effect of altered codon usage on gag translational efficiency has not been quantified. METHODOLOGY AND PRINCIPAL FINDINGS: To quantify translational efficiency of gag mRNA in live T cells, we transfected Jurkat cells with increasing doses of capped, polyadenylated synthetic mRNA corresponding to wildtype or codon-optimized gag sequences, measured Gag production by quantitative ELISA and flow cytometry, and estimated the translational efficiency of each transcript as pg of Gag antigen produced per microg of input mRNA. We found that codon optimization yielded a small increase in gag translational efficiency (approximately 1.6 fold). In contrast when cells were transfected with DNA vectors requiring nuclear transcription and processing of gag mRNA, codon optimization resulted in a very large enhancement of Gag production. CONCLUSIONS: We conclude that suboptimal codon usage by HIV-1 results in only a slight loss of gag translational efficiency per se, with the vast majority of enhancement in protein expression from DNA vectors due to altered processing and export of nuclear RNA.