[Show abstract][Hide abstract] ABSTRACT: Pre-mitotic establishment of polarity is a key event in the preparation of mother cells for asymmetric cell divisions that produce daughters of distinct fates, and ensures correct cellular patterning of tissues and eventual organ function. Previous work has shown that two receptor-like kinases, PANGLOSS2 (PAN2) and PAN1, and the small GTPase RHO GTPASE OF PLANTS (ROP) promote mother cell polarity and subsequent division asymmetry in developing maize stomata. PAN proteins become polarized prior to asymmetric cell division, however, the mechanism of this polarization is unknown. Here we show that the SCAR/WAVE regulatory complex, which activates the actin-nucleating ARP2/3 complex, is the first known marker of polarity in this asymmetric division model and is required for PAN polarization. These findings implicate actin, and specifically branched actin networks, in PAN polarization and asymmetric cell division.
[Show abstract][Hide abstract] ABSTRACT: Fluorescent proteins (FP) have significantly impacted the way that we study plants in the past two decades. In the post-genomics era, these FP tools are in higher demand by plant scientists for studying the dynamics of protein localization, function, and interactions, and to translate sequence information to biological knowledge that can benefit humans. Although FP tools have been widely used in the model plant Arabidopsis, few FP resources have been developed for maize, one of the most important food crops worldwide, and an ideal species for genetic and developmental biology research. In an effort to provide the maize and cereals research communities with a comprehensive set of FP resources for different purposes of study, we generated more than 100 stable transformed maize FP marker lines, which mark most compartments in maize cells with different FPs. Additionally, we are generating driver and reporter lines, based on the principle of the pOp-LhG4 transactivation system, allowing specific expression or mis-expression of any gene of interest to precisely study protein functions. These marker lines can be used not only for static protein localization studies, but will be useful for studying protein dynamics and interactions using kinetic microscopy methods, such as fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and fluorescence resonance energy transfer (FRET). All of the constructs and maize marker lines are publicly available through our website, http://maize.jcvi.org/cellgenomics/index.php.
Full-text · Article · Oct 2013 · The International journal of developmental biology
[Show abstract][Hide abstract] ABSTRACT: Maize (Zea mays) transformation routinely produces stable transgenic lines essential for functional genomics; however, transient expression of target proteins in maize cells is not yet routine. Such techniques are critical for rapid testing of transgene constructs and for experimental studies. Here, we report bombardment methods that depend on leaf developmental stage and result in successful expression with broad applications. Fluorescent marker genes were constructed and bombarded into five developmental regions in a growing maize leaf. Expression efficiency was highest in the basal-most 3 cm above the ligule of an approximately 50-cm growing adult leaf. Straightforward dissection procedures provide access to the receptive leaf regions, increasing efficiency from less than one transformant per cm(2) to over 21 transformants per cm(2). Successful expression was routine for proteins from full genomic sequences driven by native regulatory regions and from complementary DNA sequences driven by the constitutive maize polyubiquitin promoter and a heterologous terminator. Four tested fusion proteins, maize PROTEIN DISULFIDE ISOMERASE-Yellow Fluorescent Protein, GLOSSY8a-monomeric Red Fluorescent Protein and maize XYLOSYLTRANSFERASE, and maize Rho-of-Plants7-monomeric Teal Fluorescent Protein, localized as predicted in the endoplasmic reticulum, Golgi, and plasma membrane, respectively. Localization patterns were similar between transient and stable modes of expression, and cotransformation was equally successful. Coexpression was also demonstrated by transiently transforming cells in a stable line expressing a second marker protein, thus increasing the utility of a single stable transformant. Given the ease of dissection procedures, this method replaces heterologous expression assays with a more direct, native, and informative system, and the techniques will be useful for localization, colocalization, and functional studies.
[Show abstract][Hide abstract] ABSTRACT: Plant Rho family GTPases (ROPs) have been investigated primarily for their functions in polarized cell growth. We previously showed that the maize (Zea mays) Leu-rich repeat receptor-like protein PANGLOSS1 (PAN1) promotes the polarization of asymmetric subsidiary mother cell (SMC) divisions during stomatal development. Here, we show that maize Type I ROPs 2 and 9 function together with PAN1 in this process. Partial loss of ROP2/9 function causes a weak SMC division polarity phenotype and strongly enhances this phenotype in pan1 mutants. Like PAN1, ROPs accumulate in an asymmetric manner in SMCs. Overexpression of yellow fluorescent protein-ROP2 is associated with its delocalization in SMCs and with aberrantly oriented SMC divisions. Polarized localization of ROPs depends on PAN1, but PAN1 localization is insensitive to depletion and depolarization of ROP. Membrane-associated Type I ROPs display increased nonionic detergent solubility in pan1 mutants, suggesting a role for PAN1 in membrane partitioning of ROPs. Finally, endogenous PAN1 and ROP proteins are physically associated with each other in maize tissue extracts, as demonstrated by reciprocal coimmunoprecipitation experiments. This study demonstrates that ROPs play a key role in polarization of plant cell division and cell growth and reveals a role for a receptor-like protein in spatial localization of ROPs.
[Show abstract][Hide abstract] ABSTRACT: We present the application of remote focusing to multiphoton laser scanning microscopy and
utilize this technology to demonstrate simultaneous, programmable multi-layer imaging. Remote
focusing is used to independently control the axial location of multiple focal planes that can
be simultaneously imaged with single element detection. This facilitates volumetric multiphoton
imaging in scattering specimens and can be practically scaled to a large number of focal
planes. Further, it is demonstrated that the remote focusing control can be synchronized with
the lateral scan directions, enabling imaging in orthogonal scan planes.
Full-text · Article · Jan 2010 · Biomedical Optics Express
[Show abstract][Hide abstract] ABSTRACT: Genomic resources have significantly impacted plant biology research in recent years. Cell biology has been further enabled by an ongoing revolution in visualization technologies. Using fluorescent proteins (FPs), we now have unprecedented views of cellular architecture, and we can study real-time dynamics of cell structure, function, and protein localization. To date, these technologies have been most widely used in Arabidopsis (Arabidopsis thaliana); however, the grasses provide a unique opportunity to study the underlying mechanisms and inter-related controls of cell growth, morphogenesis, and physiology in leading crop models. Here, we present a resource that leverages the emerging maize (Zea mays) genome sequence to develop tools to study protein structure and function in a cellular context. Traditionally, such studies relied on fixed tissue or FP fusions driven by constitutive promoters, which can lead to significant artifacts. The maize genome sequence now provides access to regulatory regions that can be used to drive native expression. We have developed streamlined methods to generate maize FP-tagged lines using these regulatory elements, allowing analysis of tissue-specific expression and localized function. Identification of diverse proteins that function in specific subcellular compartments will provide the tools for understanding basic developmental, biochemical, and physiological processes in maize, with direct application potential for crop improvement.
[Show abstract][Hide abstract] ABSTRACT: The use of fluorescent proteins to localize gene products in living cells has revolutionized cell biology. Although maize has excellent genetics resources, the use of fluorescent proteins in maize cell biology has not been well developed. To date, protein localization in this species has mostly been performed using immunolocalization with specific antibodies, when available, or by overexpression of fluorescent protein fusions. Localization of tagged proteins using native regulatory elements has the advantage that it is less likely to generate artifactual results, and also reports tissue-specific expression patterns for the gene of interest. Fluorescent protein tags can also be used for other applications, such as protein-protein interaction studies and purification of protein complexes. This chapter describes methods to generate and characterize fluorescent protein-tagged maize lines driven by their native regulatory elements.
Full-text · Article · Feb 2009 · Methods in Molecular Biology