Anne W. Sylvester

Invited Review Article: Imaging techniques for harmonic and multiphoton absorption fluorescence microscopy

We review the current state of multiphoton microscopy. In particular, the requirements and limitations associated with high-speed multiphoton imaging are considered. A description of the different scanning technologies such as line scan, multifoci approaches, multidepth microscopy, and novel detection techniques is given. The main nonlinear optical contrast mechanisms employed in microscopy are reviewed, namely, multiphoton excitation fluorescence, second harmonic generation, and third harmonic generation. Techniques for optimizing these nonlinear mechanisms through a careful measurement of the spatial and temporal characteristics of the focal volume are discussed, and a brief summary of photobleaching effects is provided. Finally, we consider three new applications of multiphoton microscopy: nonlinear imaging in microfluidics as applied to chemical analysis and the use of two-photon absorption and self-phase modulation as contrast mechanisms applied to imaging problems in the medical sciences.

The liguleless-1 gene acts tissue specifically in maize leaf development

The liguleless-1 (lg1) gene affects maize leaf development. In a normal maize leaf, a ligule and auricles separate the blade and sheath. The recessive lg1 mutation prevents formation of ligules and auricles during leaf development. To determine the timing and site of lg1 gene action, we compared development of wild-type and lg1 mutant leaves, and analyzed genetic mosaics composed of wild-type and lg1 mutant cells. In wild-type leaves the first sign of differentiation of the ligular region is a series of specialized anticlinal divisions in the adaxial epidermis. This establishes a distinct band of cells, from which the ligule arises via periclinal divisions. The anticlinal divisions preceding ligule formation are altered in the mutant; therefore, the gene acts early in development, before the periclinal divisions, and possibly during basipetal vascularization. Genetic mosaic analysis indicates that the lg1 gene has at least two functions with different tissue specificities: The Lg1+ wild-type allele acts autonomously in the adaxial epidermis for normal ligule development, and in internal tissues for auricle formation. Wild-type internal tissue in direct contact with lg1 epidermis appears able to induce the mutant epidermis to form a rudimentary ligule. The results indicate that the lg1 gene acts tissue specifically in an early step of ligule and auricle initiation.

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.

Leaf shape and anatomy as indicators of phase change in the grasses: Comparison of maize, rice, and bluegrass

Leaf morphology and anatomy during vegetative phase change was compared in bluegrass, rice, and maize. Maize juvenile leaves are coated with epicuticular wax, lack specialized cells, such as trichomes and bulliform cells, and epidermal cell walls stain a uniform purple color. Adult maize leaves are pubescent, lack epicuticular waxes, and have crenulated epidermal cell walls that stain purple and blue. All bluegrass and rice blades are pubescent, coated with epicuticular waxes, and show purple and blue wall staining. In all three grasses, blade width steadily increases at each node until a threshold size is achieved several nodes before reproductive competence is acquired. Blade-to-sheath length showed a similar trend of continuous change followed by discontinuous change prior to reproduction. Analysis of leaf development demonstrated that maize primordia initiate more rapidly relative to blade and sheath growth than do either bluegrass or rice. We conclude that leaf shape, as defined by blade width and blade-to-sheath ratio, is a reliable indicator of phase, whereas anatomy is not a universal indicator of phase change in the grasses. We speculate that different growth patterns among these grasses may be attributed to changes in the timing of embryonic and postembryonic development.

Division decisions and the spatial regulation of cytokinesis.

Cytokinesis in plant cells in accomplished when a membranous cell plate is guided to a pre-established division site. The orientation of the new wall establishes the starting position of a cell in a growing tissue, but the impact of this position on future development varies. Recently, proteins have been identified that participate in forming, stabilizing and guiding the cell plate to the correct division site. Mutations that affect cytokinesis with varying impacts on plant development are providing information about the mechanics of cytokinesis and also about how the division site is selected.

Identification of PAN2 by Quantitative Proteomics as a Leucine-Rich Repeat–Receptor-Like Kinase Acting Upstream of PAN1 to Polarize Cell Division in Maize

PAN2 functions with PAN1, a Leu-rich repeat–receptor-like kinase (LRR-RLK) to polarize the divisions that form stomatal subsidiary cells in maize. Quantitative proteomics was used to identify PAN2 as a second LRR-RLK. PAN2 functions upstream of PAN1, potentially perceiving extracellular cues that initiate or amplify premitotic subsidiary mother cell polarity. Mechanisms governing the polarization of plant cell division are poorly understood. Previously, we identified pangloss1 (PAN1) as a leucine-rich repeat–receptor-like kinase (LRR-RLK) that promotes the polarization of subsidiary mother cell (SMC) divisions toward the adjacent guard mother cell (GMC) during stomatal development in maize (Zea mays). Here, we identify pangloss2 (PAN2) as a second LRR-RLK promoting SMC polarization. Quantitative proteomic analysis identified a PAN2 candidate by its depletion from membranes of pan2 single and pan1;pan2 double mutants. Genetic mapping and sequencing of mutant alleles confirmed the identity of this protein as PAN2. Like PAN1, PAN2 has a catalytically inactive kinase domain and accumulates in SMCs at sites of GMC contact before nuclear polarization. The timing of polarized PAN1 and PAN2 localization is very similar, but PAN2 acts upstream because it is required for polarized accumulation of PAN1 but is independent of PAN1 for its own localization. We find no evidence that PAN2 recruits PAN1 to the GMC contact site via a direct or indirect physical interaction, but PAN2 interacts with itself. Together, these results place PAN2 at the top of a cascade of events promoting the polarization of SMC divisions, potentially functioning to perceive or amplify GMC-derived polarizing cues.

Cellulose Synthase-Like D1 is integral to normal cell division, expansion, and leaf development in maize

The Cellulose Synthase-Like D (CslD) genes have important, although still poorly defined, roles in cell wall formation. Here, we show an unexpected involvement of CslD1 from maize (Zea mays) in cell division. Both division and expansion were altered in the narrow-organ and warty phenotypes of the csld1 mutants. Leaf width was reduced by 35%, due mainly to a 47% drop in the number of cell files across the blade. Width of other organs was also proportionally reduced. In leaf epidermis, the deficiency in lateral divisions was only partially compensated by a modest, uniform increase in cell width. Localized clusters of misdivided epidermal cells also led to the formation of warty lesions, with cell clusters bulging from the epidermal layer, and some cells expanding to volumes 75-fold greater than normal. The decreased cell divisions and localized epidermal expansions were not associated with detectable changes in the cell wall composition of csld1 leaf blades or epidermal peels, yet a greater abundance of thin, dense walls was indicated by high-resolution x-ray tomography of stems. Cell-level defects leading to wart formation were traced to sites of active cell division and expansion at the bases of leaf blades, where cytokinesis and cross-wall formation were disrupted. Flow cytometry confirmed a greater frequency of polyploid cells in basal zones of leaf blades, consistent with the disruption of cytokinesis and/or the cell cycle in csld1 mutants. Collectively, these data indicate a previously unrecognized role for CSLD activity in plant cell division, especially during early phases of cross-wall formation.

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.

Light Microscopy I: Dissection and Microtechnique

THE microscope is one of the primary tools of the cell biologist. In this first section we set forth a number of approaches that can be used to study maize using the stereo, compound, light, and epifluorescence microscopes. Successful cellular analysis is dependent on three factors: proper specimen preparation, careful use of the instrument, and appropriate observation techniques. Furthermore, any cellular analysis must be conducted within the context of the whole organism; hence, we begin in Part I by describing methods of dissection and observation using the whole plant. Then we present several methods of specimen preparation. Standard methods of observation using transmitted light and epifluorescence microscopy and for recording and analyzing the image are presented in part II.