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Horticulture & Landscape Architecture > Jian-Kang Zhu Lab

​About Dr. Jian-Kang Zhu

Distinguished Professor of Plant Biology​, Departments of Horticulture and Landscape Architecture and Biochemistry. Dr. Zhu earned his bachelor's degree in soils and agricultural chemistry from Beijing Agricultural University; his master's degree in botany from the University of California, Riverside, and his doctorate in plant physiology from Purdue. He comes to Purdue from the University of California, Riverside, where he was the Jane Johnson Chair Professor in the Institute for Integrative Genome Biology and Department of Botany and Plant Sciences. He is internationally renowned for his creative and path-breaking research that has sought to elucidate the signaling pathways in plants that govern their responses to environmental stresses. His research has contributed fundamentally to current understanding of the molecular-genetic mechanisms underlying salinity tolerance, drought tolerance, and low temperature stress in plants. Dr. Zhu’s laboratory has been central in the effort to identify key genes that could be manipulated to modify crop responses to abiotic stresses, with the ultimate goals of both enhanced agriculture productivity and decreased degradation of the environment.


Research Interests​

Detecting and responding to environmental perturbations are important for all living organisms.  One of the most important distinguishing features of p​lants is that they are sessile and thus have to endure environmental challenges. We are interested in understanding the genetic and epigenetic basis of plant resistance to environmental stresses and in identifying key genes for modifying the responses of crops to environmental stresses which ultimately will lead to major contributions to agriculture and the environment.

Plant agriculture must change fundamentally by mid-century, w​hen 9 billion people are expected to inhabit the planet, consuming 70–100% more food than is currently available (Godfray et al., 2010).  Water is the primary limiting factor in global agriculture, yet water availability and quality are diminishing for crops as cities grow and as irrigation and land-clearing salinize the soil and the underlying water tables (Sophocleous, 2004).  The problems of water deficit, salt, and other abiotic stresses are exacerbated by global warming and climate change (Fedoroff et al., 2010). The looming gap between water supply and demand creates a need for major advances in crop adaptation to drought and salt stresses through increased water-use efficiency and tolerance to saline soil. Our current and future research is aimed at improving our understand​ing of the drought, cold and heat, and salt-stress signaling pathways and resistance mechanisms. Increasing evidence suggests that plant adaptation to these abiotic stresses, in addition to being under genetic control, is also under epigenetic regulation. Accordingly, we are interested in epigenetic mechanisms of gene regulation and their roles in abiotic stress resistance. Furthermore, we are interested in developing and applying TALE nuclease technologies and other genome engineering technologies for crop improvement.  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 resistance.  Our long-term goals are to elucidate the sensing and signaling pathways used by plants in responding to environmental stresses and to identify and utilize key genes for improving the stress resistance of crops.

 

Drought-stress signaling and resistance mechanisms

The phytohormone abscisic acid (ABA) is a central regulator of plant drought resistance.  Under osmotic stress imposed by drought, cellular ABA content increases dramatically.  Significant pr​​ogress has been made in the elucidation of ABA biosynthesis and signaling but little is known about how osmotic stress is sensed to induce ABA production and other adaptive responses.  We are addressing this question with the following three approaches. 1) Identifying regulatory factors for osmotic-stress induced expression of NCED3 which encodes the rate-limiting enzyme in ABA biosynthesis. 2) Investigating the mechanisms of SnRK2 protein kinase activation by osmotic stress. Three of the 10 members of the SnRK2 family of protein kinases are activated by ABA, while virtually all 10 members can be activated by osmotic stress independently of ABA. We have recently constructed an Arabidopsis decuple mutant in which all 10 SnRK2 kinases are mutated. Analysis of this decuple mutant revealed that the SnRK2 kinases are essential for osmotic stress tolerance and for osmotic stress induction of NCED3 expression and ABA production. We are using biochemical and genetic approaches to identify the regulatory factors controlling the activation of SnRK2s under osmotic stress. 3) Applying chemical genetics to osmotic-stress signaling. We are screening synthetic chemical libraries for small molecules that inhibit the expression of the RD29A-LUC reporter gene under osmotic stress. Chemicals that perturb osmotic-stress signaling will be characterized and their target proteins in plants identified using genetic and molecular approaches.


Salt-stress sensing and tolerance mechanisms

 

A major advance in understanding plant salt tolerance has been our discovery of the SOS pathway in which the myristoylated calcium-binding protein SOS3 senses salt-elicited Ca2+ signal and translates it to downstream responses by interacting with and activating the protein kinase SOS2 (Sanchez-Barrena et al., 2005; Ishitani et al., 2000; Liu and Zhu, ​1998; Halfter et al., 2000; Liu et al, 20​00).  SOS2 and SOS3 regulate the expression and/or activities of various ion transporters including the plasma membrane Na+/H+ antiporter SOS1 (Shi et al., 2000; Qiu et al., 2002). High Na+ stress is known to elicit a cytosolic calcium signal (Knight et al., 1997) but how Na+ is sensed and how the sensing leads to a cytosolic calcium transient is not known in any organism.  Based on our recent results, we hypothesize that the Na+/H+ antiporter SOS1 may sense Na+ and generate a microdomain Ca2+ signal. This and other related hypotheses are being tested.



Cold and heat stress responses

 

Using stress-responsive promoter-driven firefly luciferase reporter genes (e.g. RD29A promoter::LUC and CBF3 promoter::LUC), we have identified several regulators of plant cold stress responses. For example, we discovered ICE1 and HOS1, two upstream regulators of the cold-responsive CBF regulon, and found that HOS1 is an ubiquitin E3 ligase that targets the ICE1 transcription factor for proteosomal degradation (reviewed in Chinnusamy et al., 2007; Zhu et al., 2007a). We are identifying additional regulators of cold as well as heat stress tolerance and are also studying cold stress sensing and signaling, events that are upstream of ICE1.

 


DNA demethylation and RNA-directed DNA methylation

Epigenetic changes caused by DNA methylation, histone modifications, and histone variants are critical for the regulation of chromatin structure, genome stability, and gene expression in response to developmental and environmental cues. In several systems, small non-coding RNAs can direct transcriptional gene silencing (TGS) by guiding sequence-specific DNA methylation (RNA-directed DNA methylation, RdDM) and/or heterochromatic histone modifications. The status of DNA methylation is determined by both methylation and demethylation reactions. DNA methylation by methyltransferases and the interplay between DNA methylation, siRNAs, and histone modification patterns have been extensively studied.  In contrast, the mechanism of active DNA demethylation and its relationship with non-coding RNAs and histone modifications are poorly understood. We have developed a unique TGS system in Arabidopsis, in which an active transgene (RD29A promoter::LUC reporter gene) and a homologous endogenous gene (endogenous RD29A gene) become silenced when cellular ROS (repressor of silencing) factors are mutated.  ROS1 encodes a 5-methycytosine-specific DNA glycosylase/lyase that prevents the hypermethylation of the homologous genes and other genes throughout the genome by active DNA demethylation through a base-excision repair pathway (Gong et al., 2002; Agius et al., 2006; Zhu et al., 2007b).  ROS3 encodes a single-stranded RNA-binding protein that also functions in preventing DNA hypermethylation (Zheng et al., 2008).  Recently, we reported another regulator of active DNA demethylation, IDM1, which reads multiple epigenetic marks (DNA methylation, unmethylated H3K4 and H3R2) and creates new epigenetic marks (histone H3K18 and H3K23 acetylation) to attract the DNA demethylation machinery to selected genomic loci to prevent their hypermethylation and transcriptional silencing. Our current efforts are focused on the following projects:

1) Search for other enzymes and regulatory factors of active DNA demethylation. The 5-methylcytosine DNA glycosylase and lyase activities of ROS1 in active DNA demethylation yield a gapped DNA with a 5’ fragment that has a 3’ phosphate (Zhu, 2009). 


In collaboration with Dr. Roldan-Arjona’s group, we have recently found a DNA 3’-phosphatase that removes the 3’-phosphate to generate a 3’-OH group for subsequent actions by DNA polymerase and ligase. Our results suggest that following ROS1 action, demethylation can also proceed via alternative mechanisms. We are studying these alternative mechanisms as well as other downstream enzymes in the base excision repair pathway for active DNA demethylation. In addition, we are characterizing several other regulatory factors of active DNA demethylation as well as other new anti-silencing factors recently identified from DNA methylation and reporter gene based forward genetic screens.

2) Dissect the RdDM pathway using ros1 suppressors. The TGS in ros1 mutant plants depends on the RdDM pathway, and second-site suppressors of the ros1-1 mutant have led to the identification of several new components of the RdDM pathway such as the histone H2B deubiquitination enzyme UBP26 (Sridhar et al., 2007), the AGO4- and scaffold RNA-binding protein KTF1 (He et al., 2009a), the single-stranded methyl-DNA-binding protein RDM1 (Gao et al., 2010), AGO6 (Zheng et al., 2007), a shared subunit of Pol IV and Pol V (He et al., 2009b), and a transcription factor for Pol II and Pol V (He et al., 2009c). We have additional ros1 suppressor mutants that we are in the process of analyzing, which is expected to yield significant new insights into the RdDM pathway.


Epigenetics and stress responses


Environmental stress may trigger epigenetic changes that contribute to stress resistance (Chinnusamy and Zhu, 2009). A few cases of transgenerational memory of stress tolerance and transgenerational inheritance of putative epigenetic changes have been reported (reviewed in Chinnusamy and Zhu, 2009). However, the extent of transgenerational memory of stress responses is unclear, and the exact epigenetic marks that show mitotic or meiotic inheritance have not been defined. We have been using our expertise in both stress biology and epigenetics to investigate the role of epigenetic regulation in stress resistance and mechanisms of transgenerational epigenetic inheritance. To study the roles of epigenetic factors in stress acclimation, we have been examining the responses of mutants of various epigenetic pathways (DNA methylation, histone modifications, chromatin remodeling, non-coding RNAs) to drought, salt, and other abiotic stresses. Various epigenetic mutants show reduced silencing of transposons and other repetitive elements and increased genomic recombination, indicating a decreased genomic stability. This decreased genomic stability could be important for stress adaptation. We have begun to test this by subjecting suspension-cultured cells from various epigenetic mutants to stepwise increases in stress intensity over many culture cycles. It is well known that wild-type cells in culture can adapt to very high levels of salt (~500 mM NaCl) and other stresses by this long-term stepwise stress challenge (Binzel et al., 1985). Perhaps some epigenetic factors are required for such adaptation. To determine the precise epigenetic marks that may be responsive to environmental challenge, we are performing genome-wide profiling of DNA methylation, histone modifications, and small non-coding RNAs in Arabidopsis plants exposed to various stress-treatment intensities and durations. Monitoring of epigenetic changes in subsequent generations may reveal any transgenerational effects of stress treatments. These studies could improve not only our understanding of stress resistance mechanisms but may also help resolve long-standing and controversial questions about the role of the environment in directing phenotypic changes in addition to its passive role in selecting the fittest. 


Using genetically tractable extremophile plants

 

There are many plants in nature that are adapted to grow in extremely dry (xerophytes and resurrection plants) or saline (halophytes) environment, but the genetic and epigenetic basis of their extremophile lifestyle is poorly understood. We have developed the halophytic Arabidopsis relative, Thellungiella halophila, as a genetically tractable model system to understand how plants tolerate extremely high levels of salinity (Zhu, 2001; Inan et al., 2004; Oh et al., 2010). We are interested in investigating the genomes and epigenomes of this and other extremophiles with the intention of learning how they flourish in extreme environments.


Epigenetic basis of hybrid vigor

 
 

Hybrid vigor, also known as heterosis, refers to the superior performance of hybrid plants or animals relative to their homozygous parental lines. Despite its paramount agronomic importance and its close relationship with stress resistance, the molecular basis of heterosis is enigmatic. Although heterosis has been most studied in corn and other crops, hybrids between some Arabidopsis ecotypes, such as Col and C24, also exhibit robust heterosis, especially in early seedling growth and abiotic stress resistance (Rohde et al., 2004). We are interested in testing the effect of genetic mutations in epigenetic pathways on the heterosis between Col and C24. Various mutants disrupted in DNA methylation, histone modifications, or non-coding RNAs are available in the Col ecotype. We also have alleles of these mutants in the C24 ecotype because our genetic screens for epigenetic pathway mutants have been conducted in the C24 ecotype (e.g., Gong et al., 2002; He et al., 2009b). We are thus in a unique position to cross homozygous epigenetic pathway mutants in the Col ecotype to their corresponding homozygous alleles in the C24 ecotype in order to test the effect of the homozygous mutations on heterosis in the F1 progeny.


TALENs and genome engineering

 
 

Transcription activator-like effectors (TALE) from plant pathogenic bacteria have been shown to bind DNA with a defined code and thus can be designed to bind any DNA sequence of interests. We and other researchers have fused designer TALEs to a DNA nuclease and other functional proteins or domains for targeted genome disruption, and gene activation and repression. We are interested in further developing and applying the TALE nuclease (TALEN) technology and other technologies (e.g. CRISPR) for targeted gene mutations, gene addition and gene correction in plants.  

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