Marco T. Buenrostro-Nava

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.

Isolation of two highly active soybean (Glycine max (L.) Merr.) promoters and their characterization using a new automated image collection and analysis system

A novel automated image collection and analysis system was used to compare two new soybean (Glycine max (L.) Merr.) promoters with the cauliflower mosaic virus 35S (CaMV35S) promoter, which was used as an expression standard. For expression comparisons, various permutations of a soybean polyubiquitin (Gmubi) promoter, a soybean heat shock protein 90-like (GmHSP90L) promoter and the CaMV35S promoter were placed upstream of a green fluorescent protein (gfp) gene. DNA constructs were introduced via particle bombardment into excised cotyledons of germinating lima bean (Phaseolus lunatus L.) seeds, which were arranged in Petri dishes for automated image capture and image analysis. The automated system allowed monitoring and quantification of gfp gene expression in the same piece of tissue over time. The Gmubi promoter, with its intronic region intact, showed the highest expression that was over five times stronger than the CaMV35S promoter. When an intronic region was removed from the Gmubi promoter, GFP expression was reduced, but was still over two times greater than with the CaMV35S promoter. The full-length soybean GmHSP90L promoter was four times stronger than the CaMV35S promoter. Truncation of the GmHSP90L promoter resulted in stepwise decreases in promoter strength, which appear to correspond to removal of regulatory elements. Automated image capture and analysis allowed the rapid and efficient evaluation of these new promoters.

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.

Comparative analysis of 35S and lectin promoters in transgenic soybean tissue using an automated image acquisition system and image analysis

Expression of the green fluorescent protein (gfp) gene, under regulatory control of either the constitutive 35S promoter or the developmentally-regulated lectin promoter was monitored and quantified using a newly-developed automated tracking system. The automated system consisted of a computer-controlled two-dimensional robotics table and a programmable image acquisition system, which was used to semi-continuously monitor gfp gene expression during development of transgenic soybean [Glycine max (L.) Merrill] somatic embryos. Quantitative analysis of GFP expression showed that, during somatic embryo proliferation and early development, expression of lectin-GFP was not detected. During late embryo development, expression of lectin-GFP gradually increased until the levels were similar to those of 35S-GFP. The use of an automated image collection system and image analysis facilitated the frequent monitoring and quantification of gfp gene expression on a large number of samples over an extended period of time.

DEVELOPMENT OF AN AUTOMATED IMAGE ACQUISITION SYSTEM FOR MONITORING GENE EXPRESSION AND TISSUE GROWTH

Automated systems can be used to facilitate continual collection of biological information from a large number of samples over long periods of time. The proper combination of automated systems with fluorescent reporter genes offers the potential for non-destructive analysis of gene expression over time. One of the fluorescent genes more commonly used in many different areas of biology is the green fluorescent protein (GFP) gene from jellyfish. The expression of this reporter gene can be detected in the tissues of any transgenic organism by using a microscope equipped with special illumination and emission filters. The main aim of this work was to develop a computer-controlled automated system to monitor GFP gene expression over time. The automated system consists of a two-dimensional belt-driven positioning table, a custom-designed sample holder, and a charged-coupled device (CCD) camera mounted on a stereomicroscope equipped for GFP fluorescence detection. To precisely control the system, software with custom applications was generated. The automated system was used to monitor the response of GFP-expressing Agrobacterium to plant tissues in vitro. Gene expression and bacterial growth were successfully monitored and quantified using image analysis. Our results showed that bacterial colonies growing in the presence of plant tissue had faster growth than those growing in the absence of plant tissues.

Development of an Automated Image Collection System for Generating Time-Lapse Animations of Plant Tissue Growth and Green Fluorescent Protein Gene Expression

Robotics systems have been widely used by the industry to perform tasks that may be hazardous, time consuming, or impossible to perform by humans. In the area of plant developmental biology, these systems have been used to gather information on how plants grow and develop under different environmental conditions. Digital imaging permits the non-destructive evaluation of both plant growth and their response to different environments. Images can either be analyzed as they are acquired (real time) or stored for subsequent analysis. Digital imaging has recently been used for the analysis of gene expression using the jellyfish Green Fluorescent Protein (GFP). GFP detection requires no exogenous cofactors or destructive assays (Haseloff and Siemering, 1998), which greatly simplifies reporter gene detection in living organisms.

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.