Genetically modifying Arabidopsis thaliana with a gene from Drought-tolerant Xerophyte Larrea tridentata (Creosote Bush)

L. tridentata, or desert creosote bush, is a xerophytic C3 plant native to the American Southwest, and is known to have evolutionarily developed sophisticated cellular mechanisms to deal with periods of intense abiotic stress. Particularly, complex signaling pathways in L. tridentata allow it to survive in periods of severe water deficiency. Through the findings of Zou et al. [5,6], LtWRKY21 synergistically works with abscisic acid (ABA) to transactivate both ABA-inducible HVA1 and HVA22 promoters. In addition, as ABA and gibberellic acid (GA) pathways are known to act antagonistically. Expectantly, the findings of Zou et al. suggest that LtWRKY21 activates ABA signaling pathways and represses GA signaling pathways [5,6]. More importantly, the LtWRKY21 transcription factor’s synergy with ABA is directly linked to some remarkable molecular adaptations of L. tridentata, some of which include stomatal closure to prevent transpiration, and slowing down gene expression to withstand dehydration [6]. To examine some of these mechanisms, the model plant Arabidopsis thaliana will be transformed with the LtWRKY21 coding region via Agrobacterium-mediated transformation. Successful transformants will be selected and the subsequent generation of transgenic plants will be assayed. Both phenotypic (screening) and genotypic (qRT-PCR and Southern Blot) examination will allow the function and expression patterns of LtWRKY21 to be elucidated under simulated drought. In order for LtWRKY21 to be successfully transformed into Arabidopsis, a tumor-inducing (Ti) plasmid must be engineered to carry LtWRKY21

A \u3cem\u3eWRKY\u3c/em\u3e Gene from Creosote Bush Encodes an Activator of the Abscisic Acid Signaling Pathway

The creosote bush (Larrea tridentata) is a xerophytic evergreen C3 shrub thriving in vast arid areas of North America. As the first step toward understanding the molecular mechanisms controlling the drought tolerance of this desert plant, we have isolated a dozen genes encoding transcription factors, including LtWRKY21 that encodes a protein of 314 amino acid residues. Transient expression studies with the GFP-LtWRKY21 fusion construct indicate that the LtWRKY21 protein is localized in the nucleus and is able to activate the promoter of an abscisic acid (ABA)-inducible gene, HVA22, in a dosage-dependent manner. The transactivating activity of LtWRKY21 relies on the C-terminal sequence containing the WRKY domain and a N-terminal motif that is essential for the repression activity of some regulators in ethylene signaling. LtWRKY21 interacts synergistically with ABA and transcriptional activators VP1 and ABI5 to control the expression of the HVA22 promoter. Co-expression of VP1, ABI5, and LtWRKY21 leads to a much higher expression of the HVA22 promoter than does the ABA treatment alone. In contrast, the Lt-WRKY21-mediated transactivation is inhibited by two known negative regulators of ABA signaling: 1-butanol, an inhibitor of phospholipase D, and abi1-1, a dominant negative mutant protein phosphatase. Interestingly, abi1-1 does not block the synergistic effect of LtWRKY21, VP1, and ABI5 co-expression, indicating that LtWRKY21, VP1, and ABI5 may form a complex that functions downstream of ABI1 to control ABA-regulated expression of genes

Decoding the protein interaction network - an approach integrating biology and math

The WRKY super family is known to play a major role during the plant stress response and development. My project focuses on the protein-protein interaction of an Oryzasativa (rice) transcription factor, OsWRKY71 which functions as the repressor of gibberellins signaling pathway. Previous literature revealed that OsWRKY71 can interact with itself or OsWRKY51 to form dimmers by using bimolecular fluorescence complementation (BiFC). To confirm this result, we use yeast two-hybrid system. As our data showed, OsWRKY71 seems to suppress the reporter gene expression of the conventional yeast two-hybrid system, so we use a modified yeast two-hybrid, Mating-based Split Ubiquitin System (MbSUS). The result confirms OsWRKY71 can interact with another OsWRKY71, so this system can be used for future studies of protein-protein interaction of OsWRKY71. Images from Confocal microscopy show OsWRKY71 proteins are anchored on to the membrane through the membrane adaptor, and the Support Vector Machine software confirms the protein-protein interaction of OsWRKY71. The next step of this project is to construct the full length cDNA library of rice to screen suspicious proteins in a larger scale

An Algorithm for Identifying Novel Targets of Transcription Factor Families: Application to Hypoxia-inducible Factor 1 Targets

Efficient and effective analysis of the growing genomic databases requires the development of adequate computational tools. We introduce a fast method based on the suffix tree data structure for predicting novel targets of hypoxia-inducible factor 1 (HIF-1) from huge genome databases. The suffix tree data structure has two powerful applications here: one is to extract unknown patterns from multiple strings/sequences in linear time; the other is to search multiple strings/sequences using multiple patterns in linear time. Using 15 known HIF-1 target gene sequences as a training set, we extracted 105 common patterns that all occur in the 15 training genes using suffix trees. Using these 105 common patterns along with known subsequences surrounding HIF-1 binding sites from the literature, the algorithm searches a genome database that contains 2,078,786 DNA sequences. It reported 258 potentially novel HIF-1 targets including 25 known HIF-1 targets. Based on microarray studies from the literature, 17 putative genes were confirmed to be upregulated by HIF-1 or hypoxia inside these 258 genes. We further studied one of the potential targets, COX-2, in the biological lab; and showed that it was a biologically relevant HIF-1 target. These results demonstrate that our methodology is an effective computational approach for identifying novel HIF-1 targets

The evolution of WRKY transcription factors

BACKGROUND: The availability of increasing numbers of sequenced genomes has necessitated a re-evaluation of the evolution of the WRKY transcription factor family. Modern day plants descended from a charophyte green alga that colonized the land between 430 and 470 million years ago. The first charophyte genome sequence from Klebsormidium flaccidum filled a gap in the available genome sequences in the plant kingdom between unicellular green algae that typically have 1-3 WRKY genes and mosses that contain 30-40. WRKY genes have been previously found in non-plant species but their occurrence has been difficult to explain. RESULTS: Only two WRKY genes are present in the Klebsormidium flaccidum genome and the presence of a Group IIb gene was unexpected because it had previously been thought that Group IIb WRKY genes first appeared in mosses. We found WRKY transcription factor genes outside of the plant lineage in some diplomonads, social amoebae, fungi incertae sedis, and amoebozoa. This patchy distribution suggests that lateral gene transfer is responsible. These lateral gene transfer events appear to pre-date the formation of the WRKY groups in flowering plants. Flowering plants contain proteins with domains typical for both resistance (R) proteins and WRKY transcription factors. R protein-WRKY genes have evolved numerous times in flowering plants, each type being restricted to specific flowering plant lineages. These chimeric proteins contain not only novel combinations of protein domains but also novel combinations and numbers of WRKY domains. Once formed, R protein WRKY genes may combine different components of signalling pathways that may either create new diversity in signalling or accelerate signalling by short circuiting signalling pathways. CONCLUSIONS: We propose that the evolution of WRKY transcription factors includes early lateral gene transfers to non-plant organisms and the occurrence of algal WRKY genes that have no counterparts in flowering plants. We propose two alternative hypotheses of WRKY gene evolution: The “Group I Hypothesis” sees all WRKY genes evolving from Group I C-terminal WRKY domains. The alternative “IIa + b Separate Hypothesis” sees Groups IIa and IIb evolving directly from a single domain algal gene separate from the Group I-derived lineage. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12870-015-0456-y) contains supplementary material, which is available to authorized users

Comparative Nutritional Assessment and Metabolomics of a WRKY Rice Mutant with Enhanced Germination Rates

Rice is the primary staple food for half the world’s population. Climate change challenges and food insecurity supports the need for rice with agronomically advantageous traits. We report on a transposon insertional rice mutant with enhanced germination rates. This trait is advantageous for rice growth in limited water regions and to reduce yield constraints caused by weed and bird competition. Evaluations of vital nutritional components, compositional analysis, and comparative metabolomics on threshed grain samples are performed, as these assays are those used to assess the safety of foods from genetically modified crops. Compared with the wild type (cv. Nipponbare), oswrky71 mutant grains have a similar size, shape, amount of crude fiber, crude fat, and ash content but higher crude protein. Mineral analyses reveal higher contents of phosphorus and zinc but lower calcium, potassium, sodium, and manganese in the mutant. Analysis of B vitamins reveals significantly higher riboflavin concentrations but lower choline chloride, calcium pantothenate, and thiamine. In addition, untargeted metabolomics analyses identify approximately 50 metabolites whose levels differed between the mutant and its wild type. Physical traits and compositional parameters analyzed are mostly similar and within the range or very close to being considered safe for consumption by the International Life Sciences Institute Crop Composition Database. Further agronomic evaluation and cooked rice sensory properties assessment are needed before positioning this mutant for human consumption

Transcriptomics analyses of soybean leaf and root samples during water-deficit

AbstractDrought being a major challenge for crop productivity and yield affects multigenic and quantitative traits. It is also well documented that water stress shows a cross talk with other abiotic stresses such as high temperature and high light intensities (Tripathi et al., 2013) [1]. In this report, we documented the details of the methods and quality controls used and considered in our time course-based transcriptome profile of soybean plants under water deficit conditions using microarray technology. The findings of this study are recently published by the Rushton lab in BMC Genomics for a comparative study of tobacco and Soybean (Rabara et al., 2015) [2]. The raw microarray data set is deposited in GEO database with accession number GSE49537

Three WRKY transcription factors additively repress abscisic acid and gibberellin signaling in aleurone cells

Members of the WRKY transcription factor superfamily are essential for the regulation of many plant pathways. Functional redundancy due to duplications of WRKY transcription factors, however, complicates genetic analysis by allowing single-mutant plants to maintain wild-type phenotypes. Our analyses indicate that three group I WRKY genes, OsWRKY24, -53, and -70, act in a partially redundant manner. All three showed characteristics of typical WRKY transcription factors: each localized to nuclei and yeast one-hybrid assays indicated that they all bind to W-boxes, including those present in their own promoters. Quantitative real time-PCR (qRT-PCR) analyses indicated that the expression levels of the three WRKY genes varied in the different tissues tested. Particle bombardment-mediated transient expression analyses indicated that all three genes repress the GA and ABA signaling in a dosage-dependent manner. Combination of all three WRKY genes showed additive antagonism of ABA and GA signaling. These results suggest that these WRKY proteins function as negative transcriptional regulators of GA and ABA signaling. However, different combinations of these WRKY genes can lead to varied strengths in suppression of their targets

Structure and Evolution of WRKY Transcription Factors

In the light of increasing numbers of sequenced plant genomes we have re-evaluated the evolution of the WRKY transcription factor family. In particular, the publication of the first charophyte genome sequence from Klebsormidium flaccidum filled a gap in the available genome sequences in the plant kingdom between unicellular green algae such as Chlamydomonas reinhardtii that typically have 1–3 WRKY genes and mosses such as Physcomitrella patens that have 30–40 genes. The K. flaccidum genome contains just two WRKY genes but the presence of a Group IIb WRKY gene was unexpected and rewrites the previously accepted evolution of WRKY transcription factors. Similarly, the presence of WRKY transcription factor genes outside of the plant lineage in some diplomonads, social amoebae, fungi incertae sedis, and amoebozoa also sheds new light on the early evolution of WRKY genes. This patchy distribution of WRKY genes outside the plant lineage suggests that lateral gene transfer has been responsible for WRKY gene transfer to nonplant species. During the evolution of flowering plants, one other type of WRKY genes evolved that contain domains typical for both intracellular type-R proteins (NBS–LRR proteins) and WRKY transcription factors. These NBS–LRR–WRKY-like proteins have been divided into eight R protein–WRKY groups (RW1–RW8). They are not found in all plant genomes but have evolved numerous times in flowering plants. These chimeric proteins also contain not only novel combinations of protein domains but also novel combinations and numbers of WRKY domains. The formation of these R protein–WRKY genes is recent with classes being restricted to specific flowering plant lineages. Once formed, R protein–WRKY genes may be selected for as they combine different components of signaling pathways that may either create new diversity in signaling or accelerate signaling by short-circuiting signaling pathways

Understanding water-stress responses in Soybean using Hydroponics system - A Systems Biology Perspective

The deleterious changes in environmental conditions such as water stress bring physiological and biochemical changes in plants, which results in crop loss. Thus, combating water stress is important for crop improvement to manage the needs of growing population. Utilization of hydroponics system in growing plants is questionable to some researchers, as it does not represent an actual field condition. However, trying to address a complex problem like water stress we have to utilize a simpler growing condition like the hydroponics system wherein every input given to the plants can be controlled. With the advent of high-throughput technologies, it is still challenging to address all levels of the genetic machinery whether a gene, protein, metabolite, and promoter. Thus, using a system of reduced complexity like hydroponics can certainly direct us towards the right candidates, if not completely help us to resolve the issue