Overall Goals and Rationales
The twentieth century has spawned two revolutions in biological research. The first revolution merged chemistry and physics with biology in the new discipline of molecular biology. The second revolution is integrating the mathematical sciences, and computer sciences with biology, and forming a new interdisciplinary research area, i.e. the so-called "Bioinformatics". The genomes of human beings, plants and animals consist of all genes in a cell. The recent accomplishment in sequencing the genomes of over 2000 organisms, include human being and important crops such as rice, is leading to a revolution in scientific research, medicine discovery and improvement of the quality of our food. Our lab is interested in developing (adopting, modifying and inventing) Bioinformatics tools for genome analyses and gene ontology studies. Gene ontology addresses Biological Process (Why is this, such as cell enlargement, being done?), Molecular Function (What kind of molecule is this? Enzymes or transcription factors?), and Cellular Component (Where is this located? Nuclei or Mitochondria?).
With the Bioinformatics, Genomics and Protemics tools, we are studying the signal transduction pathways mediating plant response to environmental stresses and molecular mechanism controlling seed development, dormancy and germination. In angiosperm, double fertilization initiates the embryogenesis process within a developing seed, which arises from an ovule. The seeds are the carriers of a new plant to be dispersed hence they occupy a critical position in the life history of higher plants. Seeds are also desiccation tolerant; understanding this aspect of seed development will help us decipher the mechanism controlling plant response to biotic stress. The molecular approach of studying seeds requires the identification and isolation of genes involved in seed development and germination, and dissection of signal transduction pathways mediating the induction or repression of these genes. Because plant hormones play important roles in controlling many developmental processes, studying the biosynthesis, metablism and mode of action of related hormones is essential to address the mechanisms regulating seed development, dormancy and germination.
In addition to the hormonal regulation of gene expression during seed development and germination, my research interests also include the mechanism controlling tissue-specific gene expression and gene silencing. Growing evidence suggests that gene silencing might be as important as activation in coordinating the regulation of genes essential for proper development of multicellular organisms. Investigation of gene silencing and gene activation during seed development, dormancy and germination will facilitate our understanding of the molecular mechanisms controlling these important physiological processes.
Physiological, genetic and biochemical studies demonstrate that plant development is regulated by the interaction of several hormones. The molecular foundation of the interaction is the cross-talk of cell signaling which integrates the independent stimuli using connections between biochemical pathways. Signal cross-talk includes gibberellins (GA), brassinosteroids (BR), and abscisic acid (ABA) pathways, ethylene and jasmonic acid (JR) pathways, ethylene and glucose pathways, sugar sensing and light response pathways, and phytochrome and cryptochrome pathways. The Boolean network model is proposed to integrate genetic data into the logical network of biochemical pathway connections deduced from DNA microarray data . The genomics and proteomics technology makes the study of signal cross-talk more practical and will accelerate the process of investigating seed development, dormancy and germination.
With the effort of International Rice Genome Sequencing Project (IRGSP), Beijing Genomics Institute (BGI) and private industry such as Monsanto Company and Syngenta, the genomes (first draft, incomplete) of two rice subspecies, Oryza sativa spp japonica and indica, have been sequenced and assembled. Some of the sequences are made available to the general public through GenBank® and BGI websites. The sequencing of rice genome has provided an unprecedented opportunity for the elucidation of ABA and GA signal transduction. We have taken a bioinformatics-based integrative approach to studying ABA and GA responses. To facilitate the predication of ABA and GA responsive genes in rice, we have developed processes to identify gene structure and extract coding sequences, promoter and terminator regions throughout the rice genomes. The identified rice genes are further characterized by the protein domain family of interest. With an established cis-acting element module, which includes those elements mediating ABA and GA responses (ABRC and GARC respectively), we have identified rice genes putatively responsive to these two plant hormones (see the abstract for ASPB2002). Java programs have been developed to display promoter structures (sequences, positions and orientations of cis-acting elements located on the promoters scanned) in a graphic mode, as shown in the example. To facilitate the prediction of protein functions, we also have developed programs to locate protein motifs and subcellular localization signal (click here to see an example).
"Wet-bench" experiments, of both over-expression and plasmid-based RNA-Interference (RNAi), have been conducted to study some of the signaling molecules predicted to mediate ABA and GA response. By analyzing the genomic sequences of the two rice subspecies, we have identified 241 families of transcription factors, accounting for 10% of predicted rice genes. Among these are over 90 members of the WRKYgene family, which is involved in many different physiological processes including biotic (viruses, bacteria and fungi) and abitoic (wounding, salt, freezing, drought, cold , and heat) stress responses. The WRKY gene annotations were recently further improved by Dr. Robin Buell's group at TIGR. The OsWRKY genes are classified into several subgroups based on the phylogenetic tree constructed with the WRKY domain amino acid sequences (Group Ia proteins, in red; group Ib proteins, in purple; group II proteins, in black, group III proteins, in green color; group IVa proteins, in yellow; group IVb proteins, in blue). This tree is supported by intron positions and intron phases [between two codons (phase 0, red), between the 1st and 2nd nucleotides of a codon (phase 1, blue), or between the 2nd and 3rd nucleotides of a codon (phase 2, black)]. At least seven of these OsWRKY genes encode transcriptional activators and/or repressors for ABA and GA signaling in barley aleurone cells, a homogenous and highly-synchronized cell population in dry seeds. Detailed biochemical studies indicate that OsWRKY51 and OsWRKY71 synergistically suppress GA induction by functionally competing with GAMYB, a transcriptional activator mediating GA-induced alpha-amylase production and pollen development. GA treatments promote the degradation of this repressor, most likely via the 26S proteasome because MG132 (z-Leu-Leu-Leu-CHO), a potent and reversible inhibitor of the proteasome, blocks the GA-promoted degradation of OsWRKY71. Experiments are ongoing to study how many of the rest OsWRKY genes are involved in ABA and GA responses and how do they interact with each other, and with other ABA/GA signaling molecules.
To engineer drought-resistant crops, we are studying creosote bush (Larrea tridentata), a xerophytic evergreen C3 shrub thriving in vast arid areas of North America. We have isolated a dozen genes encoding transcription factors, including LtWRKY21 that encodes a WRKY 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 LtWRKY21-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 gene expression.