Research Interests

 Detecting andresponding to environmental perturbations are important for all livingorganisms.  Oneof the most important distinguishing features of plants is that they are sessileand thus have to endure environmental challenges. Our lab is interested in the molecular mechanisms underlying plantresponses to harsh environments such as soil salinity, drought and coldtemperatures.  In addition, we areinterested in the mechanisms of transcriptional gene silencing and in the roleof epigenetic gene regulation in stress adaptation. We use a combination of genetic, biochemical, genomic and proteomicapproaches to analyze various levels of gene regulation (chromatinlevel/epigenetic, transcriptional, posttranscriptional, and protein activity)and to understand stress signaling and stress tolerance. Ourlong-term goals are to elucidate the signaling pathways used by plants inresponding to environmental stresses and to identify key genes for modifying theresponses of crops to environmental stresses which ultimately will lead to majorcontributions to agriculture and the environment. 

Salt stress and the SOS pathway

Soilsalinity is a severe and increasing constraint on the productivity ofagricultural crops.  Highconcentrations of salts in the soil have a strongly inhibitory effect on thegrowth and harvestable yield of all crop species. Salinization of arable land arising from poor water management has led tothe decline of past civilizations, and it threatens the long-term sustainabilityof many present large-scale irrigation systems. A critical aspect of salt tolerance is for plant cells to maintain a lowconcentration 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 Arabidopsisthaliana.  Recently, through theidentification of Arabidopsis mutants that are salt overly sensitive (sos)and the cloning and characterization of the SOSgenes, we have discovered a novel signaling pathway that mediates ionhomeostasis and salt tolerance in Arabidopsis (Figure 1). In this pathway, a myristoylated calcium-binding protein, SOS3, sensescytosolic 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 transportactivity of the plasma membrane Na⁺/H⁺ exchanger encodedby the SOS1 gene. In addition to its transport function, preliminary results suggest thatSOS1 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, andthe characterization of additional regulatory components as well as new targetsof the SOS signaling pathway.

Using the SOS pathway as aparadigm, we have extended our work to the entire family of 9 SOS3-likecalcium-binding proteins (designated as SCaBPs) and 24 SOS2-like protein kinases(PKS) in Arabidopsis.  Members of thetwo protein families interact specifically to form distinct protein kinasecomplexes, and our work has implicated several of them in decoding calciumsignals elicited by various environmental and hormonal stimuli. The function of the remaining SCaBP and PKS proteins are beinginvestigated 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 theSOS3-SOS2 protein kinase complex, which then activates the Na⁺/H⁺exchange activity of SOS1 and regulates the expression of some salt-responsivegenes.  In addition, SOS3-SOS2 mayactivate or suppress the activities of other transporters involved in Na⁺homeostasis.

Drought and abscisic acid signaling

Droughtis 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 droughtstress and the signal transduction cascade leading to the induction of ABA biosynthesis genes (Figure 2).  Inaddition, we are interested in the mechanisms of ABA perception and ABA signal transduction.

Tofacilitate genetic analysis, we have constructed transgenicArabidopsis plants with drought stress- and/or ABA-inducible bioluminescence byintroducing into plants chimeric genes consisting of drought/ABA-responsivepromoters fused with the firefly luciferase reporter gene. A largecollection of mutants that respond abnormally to water stress or ABA were recovered, and recent characterization of some of these mutants hasprovided 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 thishormone is required not only for the ABA-dependent pathway, but also for thesupposedly “ABA-independent” pathway of osmotic stress signaling. We cloned LOS5/ABA3, a majorgenetic locus controlling ABA biosynthesis, and showed that LOS5/ABA3and 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 asurprising role of RNA metabolism in regulating ABA sensitivity and biosynthesis.  Inaddition, our work on the fiery1mutant 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

Manyplants 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 eitherup- or down-regulated.  Many of thecold up-regulated genes are also up-regulated by drought, high salt or ABA.  These genes encode proteins thatpresumably protect cellular structures from dehydration caused by extracellularice formation or by salt/drought stress.  Theinduction of these genes by cold is achieved through a transcriptional cascade(Figure 3).

Facilitatedby the firefly luciferase reporter gene driven by cold-responsive promoters(e.g. RD29A, ZAT10 or CBFs), we have isolated manyArabidopsis mutants that are defective in cold signal transduction and coldtolerance.  The characterization andcloning of some of the mutations have led to the discovery of several novelregulators of cold-responsive gene transcription, and of chilling and freezingtolerance.  For example, we havecloned 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 exhibitscold-regulated nucleocytoplasmic partitioning. More recently, we have identified the ICE1 protein, a key upstreamtranscription factor that binds to the CBF3 promoter and controls theexpression of CBF genes in the cold (Figure 3). Other work in our laboratory has shown a complex regulation of coldsignaling and tolerance by an RNA helicase, a bifunctional enolase, and by thefunctional state of mitochondria.

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

Gene silencing and stress adaptation

Epigeneticcontrol of gene expression plays vital roles in development as well as incellular responses to viruses, transposons and transgenes in eukaryotes. The silencing of transgenes and endogenous genes can occur at either thetranscriptional (transcriptional gene silencing, TGS) or posttranscriptional(posttranscriptional gene silencing, PTGS) levels. While there has been tremendous progress in the understanding of PTGS inrecent years, the mechanism of TGS is not well understood. Little is known about the initial trigger for DNA methylation that isimportant for stable TGS.  Inparticular, the cellular mechanisms for the active suppression of TGS are notknown.   We have developed aunique TGS system in the model organism Arabidopsisthaliana.  In this system, anactive transgene and a homologous endogenous gene become silenced when cellular ROS (repressor ofsilencing) factors are mutated (Figure 4). We have shown that ROS1encodes a DNA glycosylase/lyase that prevents the hypermethylation and TGS ofthe homologous genes by active DNA demethylation via a base excision repairmechanism.  Wehypothesize that double stranded RNA (or its small RNA products) from thetransgene repeat triggers the silencing of the homologous genes and the ROSfactors counter the production or action of the silencing RNA to preventRNA-dependent DNA methylation or participate in the active demethylation of theDNA (Figure 4).  To test thishypothesis, we plan to characterize the putative DNA demethylation activity ofROS1, to clone other ROS loci, toidentify ROS1-interacting proteins, and to isolate and clone ros1suppressor mutations. In related projects, we are investigating the potentialrole of miRNAs and other small RNAs in the regulation of stress-responsive genesand in stress adaptation.

Figure 4. Suppression of transcriptional gene silencing byROS (repressor of silencing) proteins.  TheRD29A-LUC transgene repeat generatessmall RNAs that are proposed to be the diffusible signal for triggering thehypermethylation of the RD29A promoterat both the transgene and endogenous loci on two different chromosomes. ROS1 is proposed to counter the silencing activity of the small RNAs byactive demethylation of the promoter DNA.  ROS2and ROS3 have not been cloned, and may encode proteins that act together withROS1 in a base excision DNA repair pathway for demethylation, or function insuppressing the production or actionof the silencing dsRNA or small RNAs.