[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.
The International journal of developmental biology 01/2013; 57(6-7-8):535-543. · 2.16 Impact Factor
[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.
The Plant Cell 06/2011; 23(6):2273-84. · 9.25 Impact Factor
[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.
[Show abstract][Hide abstract] ABSTRACT: About 25,000 rice T-DNA insertional mutant lines were generated using the vector pCAS04 which has both promoter-trapping and activation-tagging function. Southern blot analysis revealed that about 40% of these mutants were single copy integration and the average T-DNA insertion number was 2.28. By extensive phenotyping in the field, quite a number of agronomically important mutants were obtained. Histochemical GUS assay with 4,310 primary mutants revealed that the GUS-staining frequency was higher than that of the previous reports in various tissues and especially high in flowers. The T-DNA flanking sequences of some mutants were isolated and the T-DNA insertion sites were mapped to the rice genome. The flanking sequence analysis demonstrated the different integration pattern of the right border and left border into rice genome. Compared with Arabidopsis and poplar, it is much varied in the T-DNA border junctions in rice.
Journal of Genetics and Genomics 06/2009; 36(5):267-76. · 2.08 Impact Factor
[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.
[Show abstract][Hide abstract] ABSTRACT: Elongation of rice internodes is one of the most important agronomic traits, which determines the plant height and underlies the grain yield. It has been shown that the elongation of internodes is under genetic control, and various factors are implicated in the process. Here, we report a detailed characterization of an elongated uppermost internode1 (eui1) mutant, which has been used in hybrid rice breeding. In the eui1-2 mutant, the cell lengths in the uppermost internodes are significantly longer than that of wild type and thus give rise to the elongated uppermost internode. It was found that the level of active gibberellin was elevated in the mutant, whereas its growth in response to gibberellin is similar to that of the wild type, suggesting that the higher level accumulation of gibberellin in the eui1 mutant causes the abnormal elongation of the uppermost internode. Consistently, the expression levels of several genes which encode gibberellin biosynthesis enzymes were altered. We cloned the EUI1 gene, which encodes a putative cytochrome P450 monooxygenase, by map-based cloning and found that EUI1 was weakly expressed in most tissues, but preferentially in young panicles. To confirm its function, transgenic experiments with different constructs of EUI1 were conducted. Overexpression of EUI1 gave rise to the gibberellin-deficient-like phenotypes, which could be partially reversed by supplementation with gibberellin. Furthermore, apart from the alteration of expression levels of the gibberellin biosynthesis genes, accumulation of SLR1 protein was found in the overexpressing transgenic plants, indicating that the expression level of EUI1 is implicated in both gibberellin-mediated SLR1 destruction and a feedback regulation in gibberellin biosynthesis. Therefore, we proposed that EUI1 plays a negative role in gibberellin-mediated regulation of cell elongation in the uppermost internode of rice.
Plant and Cell Physiology 03/2006; 47(2):181-91. · 4.13 Impact Factor