Myeong Min Lee

WEREWOLF, a MYB-Related Protein in Arabidopsis, Is a Position-Dependent Regulator of Epidermal Cell Patterning

The formation of the root epidermis of Arabidopsis provides a simple and elegant model for the analysis of cell patterning. A novel gene, WEREWOLF (WER), is described here that is required for position-dependent patterning of the epidermal cell types. The WER gene encodes a MYB-type protein and is preferentially expressed within cells destined to adopt the non-hair fate. Furthermore, WER is shown to regulate the position-dependent expression of the GLABRA2 homeobox gene, to interact with a bHLH protein, and to act in opposition to the CAPRICE MYB. These results suggest a simple model to explain the specification of the two root epidermal cell types, and they provide insight into the molecular mechanisms used to control cell patterning.

The bHLH genes GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) specify epidermal cell fate in the Arabidopsis root

The position-dependent specification of the hair and non-hair cell types in the Arabidopsis root epidermis provides a simple model for the study of cell fate determination in plants. Several putative transcriptional regulators are known to influence this cell fate decision. Indirect evidence from studies with the maize R gene has been used to suggest that a bHLH transcription factor also participates in this process. We show that two Arabidopsis genes encoding bHLH proteins, GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3), act in a partially redundant manner to specify root epidermal cell fates. Plants homozygous for mutations in both genes fail to specify the non-hair cell type, whereas plants overexpressing either gene produce ectopic non-hair cells. We also find that these genes are required for appropriate transcription of the non-hair specification gene GL2 and the hair cell specification gene CPC, showing that GL3 and EGL3 influence both epidermal cell fates. Furthermore, we show that these bHLH proteins require a functional WER MYB protein for their action, and they physically interact with WER and CPC in the yeast two-hybrid assay. These results suggest a model in which GL3 and EGL3 act together with WER in the N cell position to promote the non-hair cell fate, whereas they interact with the incomplete MYB protein CPC in the H position, which blocks the non-hair pathway and leads to the hair cell fate.

A Gene Regulatory Network for Root Epidermis Cell Differentiation in Arabidopsis

The root epidermis of Arabidopsis provides an exceptional model for studying the molecular basis of cell fate and differentiation. To obtain a systems-level view of root epidermal cell differentiation, we used a genome-wide transcriptome approach to define and organize a large set of genes into a transcriptional regulatory network. Using cell fate mutants that produce only one of the two epidermal cell types, together with fluorescence-activated cell-sorting to preferentially analyze the root epidermis transcriptome, we identified 1,582 genes differentially expressed in the root-hair or non-hair cell types, including a set of 208 “core” root epidermal genes. The organization of the core genes into a network was accomplished by using 17 distinct root epidermis mutants and 2 hormone treatments to perturb the system and assess the effects on each genes transcript accumulation. In addition, temporal gene expression information from a developmental time series dataset and predicted gene associations derived from a Bayesian modeling approach were used to aid the positioning of genes within the network. Further, a detailed functional analysis of likely bHLH regulatory genes within the network, including MYC1, bHLH54, bHLH66, and bHLH82, showed that three distinct subfamilies of bHLH proteins participate in root epidermis development in a stage-specific manner. The integration of genetic, genomic, and computational analyses provides a new view of the composition, architecture, and logic of the root epidermal transcriptional network, and it demonstrates the utility of a comprehensive systems approach for dissecting a complex regulatory network.

Functional diversification of MYB23 and GL1 genes in trichome morphogenesis and initiation

The functional diversification of duplicated genes is one of the driving forces in evolution. To understand the molecular mechanisms of gene diversification, we studied the functional relationship of the two Arabidopsis paralogous MYB-related genes GL1 and MYB23. We show that MYB23 controls trichome branching and trichome initiation at leaf edges. The latter is controlled redundantly together with GL1. We show that the two proteins are functionally equivalent during trichome initiation but not during trichome branching. RT-PCR and reporter construct analysis revealed spatial, temporal and genetic differences in transcriptional regulation of the GL1 and MYB23 genes. Presented data indicate that the diversification of GL1 and MYB23 gene functions occurred at the level of cis-regulatory sequences with respect to trichome initiation, and that, in parallel, the diversification with respect to regulation of trichome branching also involved changes in respective proteins.

Large-scale analysis of the GRAS gene family in Arabidopsis thaliana

GRAS proteins belong to a plant-specific transcription factor family. Currently, 33 GRAS members including a putative expressed pseudogene have been identified in the Arabidopsis genome. With a reverse genetic approach, we have constructed a “phenome-ready unimutant collection” of the GRAS genes in Arabidopsis thaliana. Of this collection, we focused on loss-of-function mutations in 23 novel GRAS members. Under standard conditions, homozygous mutants have no obvious morphological phenotypes compared with those of wild-type plants. Expression analysis of GRAS genes using quantitative real-time RT-PCR (qRT-PCR), microarray data mining, and promoter::GUS reporter fusions revealed their tissue-specific expression patterns. Our analysis of protein–protein interaction and subcellular localization of individual GRAS members indicated their roles as transcription regulators. In our yeast two-hybrid (Y2H) assay, we confirmed the protein–protein interaction between SHORT-ROOT (SHR) and SCARECROW (SCR). Furthermore, we identified a new SHR-interacting protein, SCARECROW-LIKE23 (SCL23), which is the most closely related to SCR. Our large-scale analysis provides a comprehensive evaluation on the Arabidopsis GRAS members, and also our phenome-ready unimutant collection will be a useful resource to better understand individual GRAS proteins that play diverse roles in plant growth and development.

Funneling of gibberellin signaling by the GRAS transcription regulator SCARECROW-LIKE 3 in the Arabidopsis root

During plant development, because no cell movement takes place, control of the timing and extent of cell division and coordination of the direction and extent of cell expansion are particularly important for growth and development. The plant hormone gibberellins (GAs) play key roles in the control of these developmental processes. However, little is known about the molecular components that integrate the generic GA signaling into a specific cell/tissue to coordinate cell division and cell expansion. Here we report that SCARECROW-LIKE 3 (SCL3), a GRAS protein, acts as a positive regulator to integrate and maintain a functional GA pathway by attenuating the DELLA repressors in the root endodermis. The tissue-specific maintenance of GA signaling in the root endodermis plays distinct roles along the longitudinal root axis. While in the elongation/differentiation zone (EDZ), the endodermis-confined GA pathway by SCL3 controls primarily coordination of root cell elongation; in the meristem zone (MZ) SCL3 in conjunction with the SHORT-ROOT/SCARECROW (SHR/SCR) pathway controls GA-modulated ground tissue maturation. Our findings highlight the regulatory network of the GRAS transcription regulators (SCL3, DELLAs, and SHR/SCR) in the root endodermis, shedding light on how GA homeostasis is achieved and how the maintenance of GA signaling controls developmental processes in roots.

POL and PLL1 phosphatases are CLAVATA1 signaling intermediates required for Arabidopsis shoot and floral stem cells

The post-embryonic development of above-ground tissues in plants is dependent upon the maintenance and differentiation of stem cells at the shoot meristem. The Arabidopsis WUSCHEL (WUS) transcription factor establishes an organizing center within the shoot meristem that is essential for specification of stem-cell identity in overlying cells. The CLAVATA (CLV) signaling pathway, including the CLV1 receptor-kinase, promotes the differentiation of stem cells by limiting the WUS expression domain, yet the mechanism of CLV signaling is largely unknown. Previously, we have shown that mutations in two protein phosphatases, POLTERGEIST (POL) and PLL1, partially suppress clv mutant phenotypes. Here, we demonstrate that POL and PLL1 are integral components of the CLV1 signaling pathway. POL and PLL1 are essential for stem-cell specification, and can also block stem-cell differentiation when overexpressed. We provide extensive evidence that POL and PLL1 act downstream of CLV signaling to maintain WUS expression and that they regulate WUS at a transcriptional level. Our findings suggest that POL and PLL1 are central players in regulating the balance between stem-cell maintenance and differentiation, and are the closest known factors to WUS regulation in the shoot meristem.

The MYB23 Gene Provides a Positive Feedback Loop for Cell Fate Specification in the Arabidopsis Root Epidermis

The specification of cell fates during development requires precise regulatory mechanisms to ensure robust cell type patterns. Theoretical models of pattern formation suggest that a combination of negative and positive feedback mechanisms are necessary for efficient specification of distinct fates in a field of differentiating cells. Here, we examine the role of the R2R3-MYB transcription factor gene, AtMYB23 (MYB23), in the establishment of the root epidermal cell type pattern in Arabidopsis thaliana. MYB23 is closely related to, and is positively regulated by, the WEREWOLF (WER) MYB gene during root epidermis development. Furthermore, MYB23 is able to substitute for the function of WER and to induce its own expression when controlled by WER regulatory sequences. We also show that the MYB23 protein binds to its own promoter, suggesting a MYB23 positive feedback loop. The localization of MYB23 transcripts and MYB23-green fluorescent protein (GFP) fusion protein, as well as the effect of a chimeric MYB23-SRDX repressor construct, links MYB23 function to the developing non-hair cell type. Using mutational analyses, we find that MYB23 is necessary for precise establishment of the root epidermal pattern, particularly under conditions that compromise the cell specification process. These results suggest that MYB23 participates in a positive feedback loop to reinforce cell fate decisions and ensure robust establishment of the cell type pattern in the Arabidopsis root epidermis.

The WEREWOLF MYB protein directly regulates CAPRICE transcription during cell fate specification in the Arabidopsis root epidermis

The Arabidopsis root epidermis is composed of two types of cells, hair cells and non-hair cells, and their fate is determined in a position-dependent manner. WEREWOLF (WER), a R2R3 MYB protein, has been shown genetically to function as a master regulator to control both of the epidermal cell fates. To directly test the proposed role of WER in this system, we examined its subcellular localization and defined its transcriptional activation properties. We show that a WER-GFP fusion protein is functional and accumulates in the nucleus of the N-position cells in the Arabidopsis root epidermis, as expected for a transcriptional regulator. We also find that a modified WER protein with a strong activation domain (WER-VP16) promotes the formation of both epidermal cell types, supporting the view that WER specifies both cell fates. In addition, we used the glucocorticoid receptor (GR) inducible system to show that CPC transcription is regulated directly by WER. Using EMSA, we found two WER-binding sites (WBSs; WBSI and WBSII) in the CPC promoter. WER-WBSI binding was confirmed in vivo using the yeast one-hybrid assay. Binding between the WER protein and both WBSs (WBSI and WBSII), and the importance of the two WBSs in CPC promoter activity were confirmed in Arabidopsis. These results provide experimental support for the proposed role of WER as an activator of gene transcription during the specification of both epidermal cell fates.

Heterologous Expression and Molecular and Cellular Characterization of CaPUB1 Encoding a Hot Pepper U-Box E3 Ubiquitin Ligase Homolog

The U-box motif is a conserved domain found in the diverse isoforms of E3 ubiquitin ligase in eukaryotes. From water-stressed hot pepper (Capsicum annuum L. cv Pukang) plants, we isolated C. annuum putative U-box protein 1 (CaPUB1), which encodes a protein containing a single U-box motif in its N-terminal region. In vitro ubiquitination and site-directed mutagenesis assays revealed that CaPUB1 possessed E3 ubiquitin ligase activity and that the U-box motif was indeed essential for its enzyme activity. RNA gel-blot analysis showed that CaPUB1 mRNA was induced rapidly by a broad spectrum of abiotic stresses, including drought, high salinity, cold temperature, and mechanical wounding, but not in response to ethylene, abscisic acid, or a bacterial pathogen, suggesting its role in the early events in the abiotic-related defense response. Because transgenic work was extremely difficult in hot pepper, in this study we overexpressed CaPUB1 in Arabidopsis (Arabidopsis thaliana) to provide cellular information on the function of this gene in the development and plant responses to abiotic stresses. Transgenic Arabidopsis plants that constitutively expressed the CaPUB1 gene under the control of the cauliflower mosaic virus 35S promoter had markedly longer hypocotyls and roots and grew more rapidly than the wild type, leading to an early bolting phenotype. Microscopic analysis showed that 35S∷CaPUB1 roots had increased numbers of small-sized cells, resulting in disordered, highly populated cell layers in the cortex, endodermis, and stele. In addition, CaPUB1-overexpressing plants displayed increased sensitivity to water stress and mild salinity. These results indicate that CaPUB1 is functional in Arabidopsis cells, thereby effectively altering cell and tissue growth and also the response to abiotic stresses. Comparative proteomic analysis showed that the level of RPN6 protein, a non-ATPase subunit of the 26S proteasome complex, was significantly reduced in 35S∷CaPUB1 seedlings as compared to the wild type. Pull-down and ubiquitination assays demonstrated that RPN6 interacted physically with CaPUB1 and was ubiquitinated in a CaPUB1-dependent manner in vitro. Although the physiological function of CaPUB1 is not yet clear, there are several possibilities for its involvement in a subset of physiological responses to counteract dehydration and high-salinity stresses in transgenic Arabidopsis seedlings.