Mona B. Damaj

Sugarcane DIRIGENT and O-METHYLTRANSFERASE promoters confer stem-regulated gene expression in diverse monocots.

Transcription profiling analysis identified Saccharum hybrid DIRIGENT (SHDIR16) and Ο-METHYLTRANSFERASE (SHOMT), putative defense and fiber biosynthesis-related genes that are highly expressed in the stem of sugarcane, a major sucrose accumulator and biomass producer. Promoters (Pro) of these genes were isolated and fused to the β-glucuronidase (GUS) reporter gene. Transient and stable transgene expression analyses showed that both ProDIR16:GUS and ProOMT:GUS retain the expression characteristics of their respective endogenous genes in sugarcane and function in orthologous monocot species, including rice, maize and sorghum. Furthermore, both promoters conferred stem-regulated expression, which was further enhanced in the stem and induced in the leaf and root by salicylic acid, jasmonic acid and methyl jasmonate, key regulators of biotic and abiotic stresses. ProDIR16 and ProOMT will enable functional gene analysis in monocots, and will facilitate engineering monocots for improved carbon metabolism, enhanced stress tolerance and bioenergy production.

Enhanced Transgene Expression in Sugarcane by Co-Expression of Virus-Encoded RNA Silencing Suppressors

Post-transcriptional gene silencing is commonly observed in polyploid species and often poses a major limitation to plant improvement via biotechnology. Five plant viral suppressors of RNA silencing were evaluated for their ability to counteract gene silencing and enhance the expression of the Enhanced Yellow Fluorescent Protein (EYFP) or the β-glucuronidase (GUS) reporter gene in sugarcane, a major sugar and biomass producing polyploid. Functionality of these suppressors was first verified in Nicotiana benthamiana and onion epidermal cells, and later tested by transient expression in sugarcane young leaf segments and protoplasts. In young leaf segments co-expressing a suppressor, EYFP reached its maximum expression at 48–96 h post-DNA introduction and maintained its peak expression for a longer time compared with that in the absence of a suppressor. Among the five suppressors, Tomato bushy stunt virus-encoded P19 and Barley stripe mosaic virus-encoded γb were the most efficient. Co-expression with P19 and γb enhanced EYFP expression 4.6-fold and 3.6-fold in young leaf segments, and GUS activity 2.3-fold and 2.4-fold in protoplasts compared with those in the absence of a suppressor, respectively. In transgenic sugarcane, co-expression of GUS and P19 suppressor showed the highest accumulation of GUS levels with an average of 2.7-fold more than when GUS was expressed alone, with no detrimental phenotypic effects. The two established transient expression assays, based on young leaf segments and protoplasts, and confirmed by stable transgene expression, offer a rapid versatile system to verify the efficiency of RNA silencing suppressors that proved to be valuable in enhancing and stabilizing transgene expression in sugarcane.

Reproducible RNA Preparation from Sugarcane and Citrus for Functional Genomic Applications

High-throughput functional genomic procedures depend on the quality of the RNA used. Copurifying molecules can negatively impact the functionality of some plant RNA preparations employed in these procedures. We present a simplified, rapid, and scalable SDS/phenol-based method that provides the high-quantity and -quality RNA required by the newly emerging biotechnology applications. The method is applied to isolating RNA from tissues of two biotechnologically important crop plants, sugarcane and citrus, which provide a challenge due to the presence of fiber, polysaccharides, or secondary metabolites. The RNA isolated by this method is suitable for several downstream applications including northern blot hybridization, microarray analysis, and quantitative RT-PCR. This method has been used in a diverse range of projects ranging from screening plant lines overexpressing mammalian genes to analyzing plant responses to viral infection and defense signaling molecules.

Genetic Engineering of Saccharum

Over the last two decades, substantial progress has been made in the genetic engineering of sugarcane (Saccharum spp.) through improvements in tissue culture procedures, allowing a higher efficiency of generating transgenic plants using Agrobacterium-mediated and biolistic gene transfers. Elucidation of gene function and development of varieties with improved yield, sugar level, fiber content, and other desirable traits and products for superior performance have been possible through transgenic technologies. Researchers are now focusing on optimizing existing methodologies and developing new technologies for the production of elite varieties, enhancement of transgene expression, and manipulation of metabolic pathways for improved molecular breeding and commercial exploitation. At present, no transgenic sugarcane has been released to the commercial market, but with the aid of large investments from the private sector, the commercialization of this major sugar- and biomass-producing crop should be accelerated.

Evaluation of Processing Options for Transgenic Sugarcane Tissue Expressing Bovine Lysozyme

Bovine lysozyme, an antimicrobial enzyme, was expressed in sugarcane using constitutive promoters. As a result, both stalks and leaves express this enzyme, albeit at different levels. The goal of this work was to assess the various options available for processing transgenic sugarcane tissue expressing bovine lysozyme and ultimately develop a process for efficient recovery of this enzyme from sugarcane tissue. Since sugarcane stalk is traditionally pressed for sucrose release, we wanted to assess whether the enzyme could also be released through conventional stalk processing. Single pressing of shredded / chopped sugarcane stalks was partially (~50%) effective in releasing the enzyme from stalk tissue. This finding was weighed versus extraction of stalk and leaf tissue. In general, lysozyme extraction from leaves and stalks required particle size reduction, high-shear mixing and in some cases pH and ionic strength adjustment. We ultimately selected stalk extraction conditions that gave efficient release of active enzyme with a minimal processing time.

Evidence for nitric oxide involvement in experimental autoimmune encephalomyelitis and adjuvant-induced arthritis in Lewis rat

Nitric oxide (NO) has been implicated as an important pathogenic mediator in several inflammatory and autoimmune diseases. We have developed experimental autoimmune encephalomyelitis (EAE) and adjuvant-induced arthritis in the Lewis rat as experimental models for multiple sclerosis, and rheumatoid arthritis, respectively, in order to investigate the role of NO in the inflammatory process. We have used the monoclonal antibody anti-NO-cysteine (Cys)-G-bovine serum albumin (BSA) developed in our laboratory as an inhibitor of the toxic effect of NO to determine the implication of NO in the creation of neoepitopes and therefore in the symptomatology of these two diseases. In addition, we have detected a significant level of circulating endogenous antibodies directed against nitrosylated and nitrated epitopes in the sera of preimmunized rats. Our data demonstrate the ability of the monoclonal anti-NO-Cys-G-BSA antibody to modulate these diseases through NO neutralization, and further provide evidence for the in vivo synthesis of nitrosylated and nitrated neoepitopes that are stable enough to be highly immunogenic, thereby inducing inflammatory injuries and a humoral immune response. Our findings provide further evidence for the implication of the NO pathway in the pathogenesis of EAE and adjuvant-induced arthritis. summary