Research Interests

 Detecting and responding to environmental perturbations are important for all living organisms.  One of the most important distinguishing features of plants is that they are sessile and thus have to endure environmental challenges.  Our lab is interested in the molecular mechanisms underlying plant responses to harsh environments such as soil salinity, drought and cold temperatures.  In addition, we are interested in the mechanisms of transcriptional gene silencing and in the role of epigenetic gene regulation in stress adaptation.  We use a combination of genetic, biochemical, genomic and proteomic approaches to analyze various levels of gene regulation (chromatin level/epigenetic, transcriptional, posttranscriptional, and protein activity) and to understand stress signaling and stress tolerance.  Our long-term goals are to elucidate the signaling pathways used by plants in responding to environmental stresses and to identify key genes for modifying the responses of crops to environmental stresses which ultimately will lead to major contributions to agriculture and the environment. 

Salt stress and the SOS pathway

Soil salinity is a severe and increasing constraint on the productivity of agricultural crops.  High concentrations of salts in the soil have a strongly inhibitory effect on the growth and harvestable yield of all crop species.  Salinization of arable land arising from poor water management has led to the decline of past civilizations, and it threatens the long-term sustainability of many present large-scale irrigation systems.  A critical aspect of salt tolerance is for plant cells to maintain a low concentration of the toxic sodium ion (Na⁺) in the cytosol.  The regulatory pathways controlling intracellular Na⁺ homeostasis are not well understood in higher eukaryotic organisms.  We are interested in the signaling cascades controlling Na⁺ homeostasis in the model multicellular organism Arabidopsis thaliana.  Recently, through the identification of Arabidopsis mutants that are salt overly sensitive (sos) and the cloning and characterization of the SOS genes, we have discovered a novel signaling pathway that mediates ion homeostasis and salt tolerance in Arabidopsis (Figure 1).  In this pathway, a myristoylated calcium-binding protein, SOS3, senses cytosolic calcium changes elicited by salt stress.  SOS3 physically interacts with and activates the protein kinase, SOS2.  The SOS3/SOS2 kinase complex phosphorylates and activates the transport activity of the plasma membrane Na⁺/H⁺ exchanger encoded by the SOS1 gene.  In addition to its transport function, preliminary results suggest that SOS1 may also have a regulatory role and may even be a novel sensor for Na⁺.  Our current research is focused on the putative sensory role of SOS1, and the characterization of additional regulatory components as well as new targets of the SOS signaling pathway.

Using the SOS pathway as a paradigm, we have extended our work to the entire family of 9 SOS3-like calcium-binding proteins (designated as SCaBPs) and 24 SOS2-like protein kinases (PKS) in Arabidopsis.  Members of the two protein families interact specifically to form distinct protein kinase complexes, and our work has implicated several of them in decoding calcium signals elicited by various environmental and hormonal stimuli.  The function of the remaining SCaBP and PKS proteins are being investigated using biochemical and reverse genetics approaches.  

Figure 1. Regulation of Na⁺ homeostasis by the SOS pathway.  High Na⁺ stress initiates a calcium signal that stimulates the SOS3-SOS2 protein kinase complex, which then activates the Na⁺/H⁺ exchange activity of SOS1 and regulates the expression of some salt-responsive genes.  In addition, SOS3-SOS2 may activate or suppress the activities of other transporters involved in Na⁺ homeostasis.

Drought and abscisic acid signaling

Drought is the most significant limiting factor for plant agriculture worldwide.  Upon drought stress, plants accumulate the phytohormone abscisic acid ( ABA ), which in turn controls many adaptive responses.  Our current research is focused on how plant cells perceive drought stress and the signal transduction cascade leading to the induction of ABA biosynthesis genes (Figure 2).  In addition, we are interested in the mechanisms of ABA perception and ABA signal transduction.

To facilitate genetic analysis, we have constructed transgenic Arabidopsis plants with drought stress- and/or ABA-inducible bioluminescence by introducing into plants chimeric genes consisting of drought/ABA-responsive promoters fused with the firefly luciferase reporter gene.  A large collection of mutants that respond abnormally to water stress or ABA were recovered, and recent characterization of some of these mutants has provided many new insights into osmosensing and osmotolerance.  For example, we have demonstrated a paramount role of ABA in osmotic stress-responsive gene expression, and provided evidence that this hormone is required not only for the ABA-dependent pathway, but also for the supposedly “ABA-independent” pathway of osmotic stress signaling.  We cloned LOS5/ABA3, a major genetic locus controlling ABA biosynthesis, and showed that LOS5/ABA3 and several other ABA biosynthetic genes are positively regulated by the end product, ABA (Figure 2).  Our work on sad1 (sensitive to ABA and drought 1) and several related mutants contributed to the discovery of a surprising role of RNA metabolism in regulating ABA sensitivity and biosynthesis.  In addition, our work on the fiery1 mutant provided the first mutational evidence supporting that inositol-1, 4, 5-triphosphate is a second messenger for ABA as well as for osmotic and cold stress signaling.

Figure 2. Self-regulation and osmotic stress regulation of ABA biosynthesis.

Cold stress signaling and tolerance

Many plants can increase their freezing tolerance by a pre-exposure to low, non-freezing temperatures, a process known as cold acclimation.  During cold acclimation, the expression of hundreds of genes is either up- or down-regulated.  Many of the cold up-regulated genes are also up-regulated by drought, high salt or ABA .  These genes encode proteins that presumably protect cellular structures from dehydration caused by extracellular ice formation or by salt/drought stress.  The induction of these genes by cold is achieved through a transcriptional cascade (Figure 3).

Facilitated by the firefly luciferase reporter gene driven by cold-responsive promoters (e.g. RD29A, ZAT10 or CBFs), we have isolated many Arabidopsis mutants that are defective in cold signal transduction and cold tolerance.  The characterization and cloning of some of the mutations have led to the discovery of several novel regulators of cold-responsive gene transcription, and of chilling and freezing tolerance.  For example, we have cloned an important negative regulator of cold responsive gene expression, HOS1, and found that it is a RING finger protein with an ubiquitin E3 ligase activity, thus implicating a critical role of protein degradation in cold signaling.  HOS1 also provides the first example of a cellular protein that exhibits cold-regulated nucleocytoplasmic partitioning.  More recently, we have identified the ICE1 protein, a key upstream transcription factor that binds to the CBF3 promoter and controls the expression of CBF genes in the cold (Figure 3).  Other work in our laboratory has shown a complex regulation of cold signaling and tolerance by an RNA helicase, a bifunctional enolase, and by the functional state of mitochondria.

Figure 3. Cold-activated transcriptional cascade in Arabidopsis.  SNOW is a partner protein of ICE1 (unpublished).

Gene silencing and stress adaptation

Epigenetic control of gene expression plays vital roles in development as well as in cellular responses to viruses, transposons and transgenes in eukaryotes.  The silencing of transgenes and endogenous genes can occur at either the transcriptional (transcriptional gene silencing, TGS) or posttranscriptional (posttranscriptional gene silencing, PTGS) levels.  While there has been tremendous progress in the understanding of PTGS in recent years, the mechanism of TGS is not well understood.  Little is known about the initial trigger for DNA methylation that is important for stable TGS.  In particular, the cellular mechanisms for the active suppression of TGS are not known.   We have developed a unique TGS system in the model organism Arabidopsis thaliana.  In this system, an active transgene and a homologous endogenous gene become silenced when cellular ROS (repressor of silencing) factors are mutated (Figure 4).  We have shown that ROS1 encodes a DNA glycosylase/lyase that prevents the hypermethylation and TGS of the homologous genes by active DNA demethylation via a base excision repair mechanism.  We hypothesize that double stranded RNA (or its small RNA products) from the transgene repeat triggers the silencing of the homologous genes and the ROS factors counter the production or action of the silencing RNA to prevent RNA-dependent DNA methylation or participate in the active demethylation of the DNA (Figure 4).  To test this hypothesis, we plan to characterize the putative DNA demethylation activity of ROS1, to clone other ROS loci, to identify ROS1-interacting proteins, and to isolate and clone ros1 suppressor mutations. In related projects, we are investigating the potential role of miRNAs and other small RNAs in the regulation of stress-responsive genes and in stress adaptation.

Figure 4. Suppression of transcriptional gene silencing by ROS (repressor of silencing) proteins.  The RD29A-LUC transgene repeat generates small RNAs that are proposed to be the diffusible signal for triggering the hypermethylation of the RD29A promoter at both the transgene and endogenous loci on two different chromosomes.  ROS1 is proposed to counter the silencing activity of the small RNAs by active demethylation of the promoter DNA.  ROS2 and ROS3 have not been cloned, and may encode proteins that act together with ROS1 in a base excision DNA repair pathway for demethylation, or function in suppressing the production or action of the silencing dsRNA or small RNAs.