Anding Luo

Advancing Cell Biology and Functional Genomics in Maize Using Fluorescent Protein-Tagged Lines

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.

ROP GTPases Act with the Receptor-Like Protein PAN1 to Polarize Asymmetric Cell Division in Maize

This study demonstrates a role for Rho family GTPases (ROPs) in asymmetric cell division in maize. Functional and localization studies together with analysis of physical interactions demonstrate that ROPs function cooperatively with the receptor-like protein PAN1 to promote the premitotic polarization of subsidiary mother cells during stomatal complex development. 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.

RNA Interference Knockdown of BRASSINOSTEROID INSENSITIVE1 in Maize Reveals Novel Functions for Brassinosteroid Signaling in Controlling Plant Architecture

Brassinosteroid signaling is central to controlling auricle development and establishing the blade-sheath boundary in maize leaf. Brassinosteroids (BRs) are plant hormones involved in various growth and developmental processes. The BR signaling system is well established in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) but poorly understood in maize (Zea mays). BRASSINOSTEROID INSENSITIVE1 (BRI1) is a BR receptor, and database searches and additional genomic sequencing identified five maize homologs including duplicate copies of BRI1 itself. RNA interference (RNAi) using the extracellular coding region of a maize zmbri1 complementary DNA knocked down the expression of all five homologs. Decreased response to exogenously applied brassinolide and altered BR marker gene expression demonstrate that zmbri1-RNAi transgenic lines have compromised BR signaling. zmbri1-RNAi plants showed dwarf stature due to shortened internodes, with upper internodes most strongly affected. Leaves of zmbri1-RNAi plants are dark green, upright, and twisted, with decreased auricle formation. Kinematic analysis showed that decreased cell division and cell elongation both contributed to the shortened leaves. A BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1-yellow fluorescent protein (BES1-YFP) transgenic line was developed that showed BR-inducible BES1-YFP accumulation in the nucleus, which was decreased in zmbri1-RNAi. Expression of the BES1-YFP reporter was strong in the auricle region of developing leaves, suggesting that localized BR signaling is involved in promoting auricle development, consistent with the zmbri1-RNAi phenotype. The blade-sheath boundary disruption, shorter ligule, and disrupted auricle morphology of RNAi lines resemble KNOTTED1-LIKE HOMEOBOX (KNOX) mutants, consistent with a mechanistic connection between KNOX genes and BR signaling.

The SCAR/WAVE complex polarizes PAN receptors and promotes division asymmetry in maize.

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.

Sucrose Transporter ZmSut1 Expression and Localization Uncover New Insights into Sucrose Phloem Loading

Maize SUCROSE TRANSPORTER1 functions to load sucrose into phloem companion cells, restrict its accumulation in the apoplasm, and prevent its loss during long-distance transport. Sucrose transporters (SUTs) translocate sucrose (Suc) across cellular membranes, and in eudicots, multiple SUTs are known to function in Suc phloem loading in leaves. In maize (Zea mays), the Sucrose Transporter1 (ZmSut1) gene has been implicated in Suc phloem loading based upon RNA expression in leaves, electrophysiological experiments, and phenotypic analysis of zmsut1 mutant plants. However, no previous studies have examined the cellular expression of ZmSut1 RNA or the subcellular localization of the ZmSUT1 protein to assess the gene’s hypothesized function in Suc phloem loading or to evaluate its potential roles, such as phloem unloading, in nonphotosynthetic tissues. To this end, we performed RNA in situ hybridization experiments, promoter-reporter gene analyses, and ZmSUT1 localization studies to elucidate the cellular expression pattern of the ZmSut1 transcript and protein. These data showed that ZmSut1 was expressed in multiple cell types throughout the plant and indicated that it functions in phloem companion cells to load Suc and also in other cell types to retrieve Suc from the apoplasm to prevent its accumulation and loss to the transpiration stream. Additionally, by comparing a phloem-mobile tracer with ZmSut1 expression, we determined that developing maize leaves dynamically switch from symplasmic to apoplasmic phloem unloading, reconciling previously conflicting reports, and suggest that ZmSut1 does not have an apparent function in either unloading process. A model for the dual roles for ZmSut1 function (phloem loading and apoplasmic recycling), Sut1 evolution, and its possible use to enhance Suc export from leaves in engineering C3 grasses for C4 photosynthesis is discussed.

Fluorescent protein marker lines in maize: generation and applications

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).

Reliable transient transformation of intact maize leaf cells for functional genomics and experimental study

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 cm2 to over 21 transformants per cm2. 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.

Remote focusing for programmable multi-layer differential multiphoton microscopy.

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.

Methods for generation and analysis of fluorescent protein-tagged maize lines.

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.

A Maize Database Resource that Captures Tissue-Specific and Subcellular-Localized Gene Expression, via Fluorescent Tags and Confocal Imaging (Maize Cell Genomics Database)

Maize is a global crop and a powerful system among grain crops for genetic and genomic studies. However, the development of novel biological tools and resources to aid in the functional identification of gene sequences is greatly needed. Towards this goal, we have developed a collection of maize marker lines for studying native gene expression in specific cell types and subcellular compartments using fluorescent proteins (FPs). To catalog FP expression, we have developed a public repository, the Maize Cell Genomics (MCG) Database, (http://maize.jcvi.org/cellgenomics), to organize a large data set of confocal images generated from the maize marker lines. To date, the collection represents major subcellular structures and also developmentally important progenitor cell populations. The resource is available to the research community, for example to study protein localization or interactions under various experimental conditions or mutant backgrounds. A subset of the marker lines can also be used to induce misexpression of target genes through a transactivation system. For future directions, the image repository can be expanded to accept new image submissions from the research community, and to perform customized large-scale computational image analysis. This community resource will provide a suite of new tools for gaining biological insights by following the dynamics of protein expression at the subcellular, cellular and tissue levels.