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In JoVE (1)
Other Publications (9)
Articles by John J. Finer in JoVE
JoVE General
Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues
1Department of Horticulture and Crop Science, The Ohio State University, 2Department of Plant Pathology, North Carolina State University
We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.
Other articles by John J. Finer on PubMed
Ectopic Expression of a Soybean Phytase in Developing Seeds of Glycine Max to Improve Phosphorus Availability
A transgenic approach was used to alter soybean seed phytate content by expressing a soybean phytase gene (GmPhy) during seed development to degrade accumulating phytic acid (IP6). An expression vector containing the soybean phytase cDNA controlled by the seed-specific beta-conglycinin promoter (alpha'-subunit) was used to transform embryogenic soybean cultures. Plants from four independent transgenic lines were analyzed for transgene integration and seed IP6 levels. The reduction in IP6 levels in transgenic seeds compared to control 'Jack' soybeans ranged from 12.6 to 24.8 as determined by HPLC. A low copy transformant was propagated to the T4 generation and examined in more detail for phytase expression and enzyme activity during seed development. Expression of phytase mRNA and phytase activity increased during seed development, consistent with the use of an embryo-specific promoter. Ectopic phytase expression during seed development offers potential as an effective strategy for reducing phytate content in soybean seed.
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.
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.
Quantification and Extension of Transient GFP Expression by the Co-introduction of a Suppressor of Silencing
Using particle bombardment, a DNA expression vector containing the green fluorescent protein (GFP) reporter gene was introduced into plant cells. Expression of the GFP gene was transient; resulting in peak GFP Expression about 24 h post introduction and a rapid decline thereafter. This well known decline in gene expression has previously been attributed to pre-integrative DNA events that involved the loss of introduced DNA or cell death. Here, we show that post-transcriptional gene silencing (PTGS) is also involved. Introduction of a GFP expression vector alone resulted in a rapid decline in transient expression after 30 h. However, if GFP was expressed as a translational fusion to the RNA silencing suppressor protein HCPro from tobacco etch potyvirus, transgene expression was extended to well over 100 h. Mutant analyses of HCPro showed that a functional HCPro protein was required for this extension of transient expression. Various deletion and translational fusion analyses confirmed that the C-terminal region of the protein was important for suppressor activity and the entire protein was required for optimal suppression of host silencing. The transient nature of gene expression during particle bombardment appears to result from induction of PTGS, which can be mitigated by the presence of a suppressor of silencing. The use of RNA silencing suppressor proteins may make particle bombardment-mediated transient expression assays more useful for evaluating factors that effect gene expression.
Quantitative Evaluation of Six Different Viral Suppressors of Silencing Using Image Analysis of Transient GFP Expression
The effects of six different plant viral suppressors of gene silencing were compared using an automated image collection and analysis system developed for continual monitoring of GFP expression. Suppressors were introduced into lima bean cotyledonary tissues either as 3'-GFP translational fusions or as co-introductions with the GFP gene on a separate plasmid. The resultant transient expression profiles for each suppressor depended on whether the suppressor was introduced as a fusion or co-introduced on separate plasmids. As co-introductions, the silencing suppressors HCPro (from Tobacco etch virus), p19 (from Tomato bushy stunt virus), gammab (from Barley stripe mosaic virus) and p21 (from Beet yellows virus) led to an almost twofold increase in initial GFP expression levels, followed by a rapid decline. In contrast, fusions of HCPro, p19, and gammab to the 3'-end of GFP resulted in slightly lower but more prolonged GFP expression. Compared with the co-introductions, all GFP::Suppressor translational fusions gave reduced GFP fluorescence levels, suggesting interference of the fusion partner with GFP fluorescence. Regardless of the configuration, introductions of the silencing suppressors AL2 (from Tomato golden mosaic virus) and 126-kDa protein (from Tobacco mosaic virus) resulted in very low GFP fluorescence. This is the first report that directly compares the effects of a large number of viral suppressors of silencing on transient transgene expression using both translational fusions and co-introductions.
A Soybean (Glycine Max) Polyubiquitin Promoter Gives Strong Constitutive Expression in Transgenic Soybean
The success of plant genetic transformation relies greatly on the strength and specificity of the promoters used to drive genes of interest. In this study, we analyzed gfp gene expression mediated by a polyubiquitin promoter (Gmubi) from soybean (Glycine max) in stably transformed soybean tissues. Strong GFP expression was observed in stably transformed proliferative embryogenic tissues. In whole transgenic plants, GFP expression was observed in root tips, main and lateral roots, cotyledons and plumules in young plants as well as in leaf veins, petioles, flower petals, pollen, pods and developing seeds in mature plants. GFP expression was localized mainly in epidermal cells, leaf mesophyll, procambium and vascular tissues. Introduction of an intron-less version of the Gmubi promoter (Gmupri) displayed almost the same GFP expression pattern albeit at lower intensities. The Gmubi promoter showed high levels of constitutive expression and represents an alternative to viral promoters for driving gene expression in soybean.
The Arabidopsis Tandem Zinc Finger Protein AtTZF1 Traffics Between the Nucleus and Cytoplasmic Foci and Binds Both DNA and RNA
Processing bodies (PBs) are specialized cytoplasmic foci where mRNA turnover and translational repression can take place. Stress granules are related cytoplasmic foci. The CCCH tandem zinc finger proteins (TZFs) play pivotal roles in gene expression, cell fate specification, and various developmental processes. Human TZF binds AU-rich elements at the 3' untranslated region and recruits decapping, deadenylation, and exonucleolytic enzymes to PBs for RNA turnover. Recent genetic studies indicate that plant TZFs are involved in gene regulation and hormone-mediated environmental responses. It is unknown if plant TZFs can bind RNA and be localized to PBs or stress granules. The Arabidopsis (Arabidopsis thaliana) AtTZF1/AtCTH/AtC3H23 was identified as a sugar-sensitive gene in a previous microarray study. It is characterized by a TZF motif that is distinct from the human TZF. Higher plants such as Arabidopsis and rice (Oryza sativa) each have a gene family containing this unique TZF motif. Here, we show that AtTZF1 can traffic between the nucleus and cytoplasmic foci. AtTZF1 colocalizes with markers of PBs, and the morphology of these cytoplasmic foci resembles that of mammalian PBs and stress granules. AtTZF1-associated cytoplasmic foci are dynamic and tissue specific. They can be induced by dark and wound stresses and are preferentially present in actively growing tissues and stomatal precursor cells. Since AtTZF1 can bind both DNA and RNA in vitro, it raises the possibility that AtTZF1 might be involved in DNA and/or RNA regulation.
High Level Transgenic Expression of Soybean (Glycine Max) GmERF and Gmubi Gene Promoters Isolated by a Novel Promoter Analysis Pipeline
Although numerous factors can influence gene expression, promoters are perhaps the most important component of the regulatory control process. Promoter regions are often defined as a region upstream of the transcriptional start. They contain regulatory elements that interact with regulatory proteins to modulate gene expression. Most genes possess their own unique promoter and large numbers of promoters are therefore available for study. Unfortunately, relatively few promoters have been isolated and characterized; particularly from soybean (Glycine max).
WRKY Transcription Factors: Key Components in Abscisic Acid Signalling
WRKY transcription factors (TFs) are key regulators of many plant processes, including the responses to biotic and abiotic stresses, senescence, seed dormancy and seed germination. For over 15 years, limited evidence has been available suggesting that WRKY TFs may play roles in regulating plant responses to the phytohormone abscisic acid (ABA), notably some WRKY TFs are ABA-inducible repressors of seed germination. However, the roles of WRKY TFs in other aspects of ABA signalling, and the mechanisms involved, have remained unclear. Recent significant progress in ABA research has now placed specific WRKY TFs firmly in ABA-responsive signalling pathways, where they act at multiple levels. In Arabidopsis, WRKY TFs appear to act downstream of at least two ABA receptors: the cytoplasmic PYR/PYL/RCAR-protein phosphatase 2C-ABA complex and the chloroplast envelope-located ABAR-ABA complex. In vivo and in vitro promoter-binding studies show that the target genes for WRKY TFs that are involved in ABA signalling include well-known ABA-responsive genes such as ABF2, ABF4, ABI4, ABI5, MYB2, DREB1a, DREB2a and RAB18. Additional well-characterized stress-inducible genes such as RD29A and COR47 are also found in signalling pathways downstream of WRKY TFs. These new insights also reveal that some WRKY TFs are positive regulators of ABA-mediated stomatal closure and hence drought responses. Conversely, many WRKY TFs are negative regulators of seed germination, and controlling seed germination appears a common function of a subset of WRKY TFs in flowering plants. Taken together, these new data demonstrate that WRKY TFs are key nodes in ABA-responsive signalling networks.
