experimental-design-for-laser-microdissection-rna-seq-lessons-from-an

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

University of Wyoming

Advancing cell biology and functional genomics in maize using fluorescent protein-tagged 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.

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

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.

Invited review article: Imaging techniques for harmonic and multiphoton absorption fluorescence microscopy.

First discovered in jellyfish, fluorescent proteins (FPs) have been successfully optimized for use as effective biomarkers within living plant cells. When exposed to light, FPs fused to a protein or regulatory element will fluoresce, and non-invasively mark expression and protein localization, which allows for the in vivo monitoring of diverse cellular processes. In this review, we discuss how FP technology has evolved from small-scale analysis of individual genes to more high-throughput techniques for global expression and functional profiling in plants.

Illuminating plant biology: using fluorescent proteins for high-throughput analysis of protein localization and function in plants.

&#x975E;&#x7EBF;&#x6027;&#x6210;&#x50CF;&#x5728;&#x6D3B;&#x7EC4;&#x7EC7;&#x9762;&#x4E34;&#x7684;&#x6311;&#x6218;&#x662F;&#x8981;&#x6700;&#x5927;&#x9650;&#x5EA6;&#x5730;&#x4ECE;&#x6BCF;&#x4E2A;&#x8367;&#x5149;&#x8367;&#x5149;&#x603B;&#x4EA7;&#x91CF;&#x3002;&#x6211;&#x4EEC;&#x8C03;&#x67E5;&#x7684;&#x4E09;&#x4E2A;&#x8367;&#x5149;-&#x7F57;&#x4E39;&#x660E; B&#x3001; &#x7EA2;&#x8272;&#x8367;&#x5149;&#x86CB;&#x767D;&#x548C; CdSe &#x91CF;&#x5B50;&#x70B9;&#x7684;&#x6392;&#x653E;&#x7387;-&#x540C;&#x65F6;&#x64CD;&#x7EB5;&#x6FC0;&#x5149;&#x6FC0;&#x53D1;&#x8109;&#x51B2;&#x5728;&#x7126;&#x70B9;&#x7684;&#x9636;&#x6BB5;&#x3002;&#x5728;&#x6240;&#x6709;&#x60C5;&#x51B5;&#x4E0B;&#x53D8;&#x6362;&#x6709;&#x9650;&#x8109;&#x51B2;&#x6700;&#x5927;&#x5316;&#x603B;&#x7684;&#x4EA7;&#x91CF;&#xFF0C;&#x4EE5;&#x786E;&#x4FDD;&#x6700;&#x9AD8;&#x7684;&#x4FE1;&#x53F7;&#x7684;&#x4FE1;&#x566A;&#x6BD4;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x6211;&#x4EEC;&#x53D1;&#x73B0;&#x8BC1;&#x636E;&#x7684;&#x8367;&#x5149; antibleaching &#x91CF;&#x5B50;&#x70B9;&#x6837;&#x54C1;&#x4E2D;&#x3002;

&#x4F18;&#x5316;&#x53CC;&#x5149;&#x5B50;&#x6FC0;&#x5149;&#x626B;&#x63CF;&#x663E;&#x5FAE;&#x955C;&#x4E0E;&#x8272;&#x6563;&#x8865;&#x507F;&#x7684;&#x6536;&#x76CA;&#xFF0C;&#x8367;&#x5149;&#x3002;

A challenge for nonlinear imaging in living tissue is to maximize the total fluorescent yield from each fluorophore. We investigated the emission rates of three fluorophores-rhodamine B, a red fluorescent protein, and CdSe quantum dots-while manipulating the phase of the laser excitation pulse at the focus. In all cases a transform-limited pulse maximized the total yield to insure the highest signal-to-noise ratio. Further, we find evidence of fluorescence antibleaching in quantum dot samples.

Optimizing the fluorescent yield in two-photon laser scanning microscopy with dispersion compensation.

Un d&#xE9;fi pour l&#x26;#39;imagerie non-lin&#xE9;aire dans les tissus vivants est de maximiser le rendement total fluorescent de chaque fluorophore. On &#xE9;tudi&#xE9; les taux d&#x26;#39;&#xE9;mission de trois fluorophores-rhodamine B, une prot&#xE9;ine fluorescente rouge et CdSe points quantiques-tout en manipulant le phase de l&#x26;#39;impulsion d&#x26;#39;excitation au laser de la mise au point. Dans tous les cas une impulsion transform&#xE9;e limit&#xE9;e maximis&#xE9;e le rendement total le plus &#xE9;lev&#xE9; pour assurer le signal-sur-bruit. En outre, nous trouvons des preuves de la fluorescence dans les &#xE9;chantillons antibleaching points quantiques.

Optimisation du rendement de fluorescence &#xE0; deux photons de microscopie &#xE0; balayage laser avec compensation de dispersion

Eine Herausforderung f&uuml;r nichtlineare Imaging in lebendes Gewebe ist der fluoreszierenden Gesamtertrag aus jeder Fluorophor zu maximieren. Wir untersuchten die Emissionsraten von drei Fluorophore-Rhodamin B, einem rot fluoreszierende Protein und CdSe Quantenpunkte-beim Bearbeiten von die Phase des Laserimpulses Erregung im Mittelpunkt. In allen F&auml;llen maximiert ein Transform-begrenzte Puls der Gesamtertrag um das h&ouml;chste Signal-Rausch-Verh&auml;ltnis zu gew&auml;hrleisten. Des weiteren finden wir Beweise f&uuml;r Fluoreszenz-Antibleaching in Quantum Dot Proben.

Optimieren die fluoreszierende Rendite in zwei-Photonen-Laser-scanning-Mikroskopie mit Dispersion Ausgleich.

Una sfida per l&#39;imaging non lineare in tessuto vivente &egrave; quello di massimizzare il rendimento totale fluorescente da ogni fluorophore. Abbiamo esaminato i tassi di emissione delle tre fluorofori-Rodamina B, una proteina fluorescente rossa e punti quantici CdSe-durante la manipolazione la fase di impulso di eccitazione laser al fuoco. In tutti i casi un impulso di trasformazione-limited massimizzato il rendimento totale per assicurare il pi&ugrave; alto rapporto segnale-rumore. Ulteriormente, troviamo prove di fluorescenza antibleaching nei campioni di quantum dot.

Ottimizzando il rendimento fluorescente in due-fotone laser scanning microscopy con compensazione della dispersione.

&#x751F;&#x4F53;&#x7D44;&#x7E54;&#x306E;&#x975E;&#x7DDA;&#x5F62;&#x30A4;&#x30E1;&#x30FC;&#x30B8;&#x30F3;&#x30B0;&#x306E;&#x305F;&#x3081;&#x306E;&#x8AB2;&#x984C;&#x306F;&#x3001;&#x305D;&#x308C;&#x305E;&#x308C;&#x306E;&#x86CD;&#x5149;&#x4F53;&#x304B;&#x3089;&#x306E;&#x7DCF;&#x86CD;&#x5149;&#x53CE;&#x7387;&#x3092;&#x6700;&#x5927;&#x5316;&#x3059;&#x308B;&#x3053;&#x3068;&#x3067;&#x3042;&#x308B;&#x3002;&#x6211;&#x3005;&#x306F;3&#x3064;&#x306E;&#x767A;&#x5149;&#x7387;&#x306F;&#x30D5;&#x30EB;&#x30AA;&#x30ED;&#x30D5;&#x30A9;&#x30A2; - &#x30ED;&#x30FC;&#x30C0;&#x30DF;&#x30F3;B&#x3001;&#x8D64;&#x8272;&#x86CD;&#x5149;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x3001;&#x304A;&#x3088;&#x3073;CdSe&#x91CF;&#x5B50;&#x7126;&#x70B9;&#x3067;&#x306E;&#x30EC;&#x30FC;&#x30B6;&#x30FC;&#x52B1;&#x8D77;&#x30D1;&#x30EB;&#x30B9;&#x306E;&#x4F4D;&#x76F8;&#x3092;&#x64CD;&#x4F5C;&#x3059;&#x308B;&#x30C9;&#x30C3;&#x30C8;&#x9593;&#x3092;&#x8ABF;&#x3079;&#x305F;&#x3002;&#x3059;&#x3079;&#x3066;&#x306E;&#x30B1;&#x30FC;&#x30B9;&#x3067;&#x5909;&#x63DB;&#x9650;&#x754C;&#x30D1;&#x30EB;&#x30B9;&#x306F;&#x3001;&#x6700;&#x9AD8;&#x306E;&#x4FE1;&#x53F7;&#x5BFE;&#x96D1;&#x97F3;&#x6BD4;&#x3092;&#x78BA;&#x4FDD;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x5168;&#x53CE;&#x7387;&#x3092;&#x6700;&#x5927;&#x5316;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x6211;&#x3005;&#x306F;&#x91CF;&#x5B50;&#x30C9;&#x30C3;&#x30C8;&#x8A66;&#x6599;&#x4E2D;&#x306E;&#x86CD;&#x5149;antibleaching&#x306E;&#x8A3C;&#x62E0;&#x3092;&#x898B;&#x3064;&#x3051;&#x308B;&#x3002;

&#x5206;&#x6563;&#x88DC;&#x511F;&#x3068;&#x4E8C;&#x5149;&#x5B50;&#x30EC;&#x30FC;&#x30B6;&#x30FC;&#x8D70;&#x67FB;&#x9855;&#x5FAE;&#x93E1;&#x3067;&#x86CD;&#x5149;&#x53CE;&#x7387;&#x306E;&#x6700;&#x9069;&#x5316;

&#xAC01; fluorophore&#xC5D0;&#xC11C; &#xCD1D; &#xD615;&#xAD11; &#xC218;&#xC728;&#xC744; &#xADF9;&#xB300;&#xD654; &#xD558;&#xAE30; &#xC704;&#xD574; &#xC870;&#xC9C1; &#xC0DD;&#xD65C;&#xC5D0;&#xC11C;&#xC5D0;&#xC11C; &#xBE44;&#xC120;&#xD615; &#xC601;&#xC0C1;&#xC5D0; &#xB300; &#xD55C; &#xB3C4;&#xC804;&#xC774;&#xC785;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC; &#xC138; fluorophores rhodamine B, &#xBE68;&#xAC04;&#xC0C9; &#xD615;&#xAD11; &#xB2E8;&#xBC31;&#xC9C8; &#xBC0F; CdSe &#xC591;&#xC790; &#xC810; &#xB4E4;&#xC758; &#xBC29;&#xCD9C; &#xC18D;&#xB3C4; &#xC870;&#xC0AC;-&#xCD08;&#xC810;&#xC5D0;&#xC11C; &#xB808;&#xC774;&#xC800; &#xC790;&#xADF9; &#xD384;&#xC2A4;&#xC758; &#xB2E8;&#xACC4;&#xB97C; &#xC870;&#xC791; &#xD558;&#xB294; &#xB3D9;&#xC548;. &#xBAA8;&#xB4E0; &#xACBD;&#xC6B0;&#xC5D0; &#xBCC0;&#xD658; &#xC81C;&#xD55C; &#xD384;&#xC2A4; &#xAC00;&#xC7A5; &#xB192;&#xC740; &#xC2E0;&#xD638; &#xB300; &#xC7A1;&#xC74C; &#xBE44;&#xC728;&#xC744; &#xD655;&#xC2E4;&#xD788; &#xD558;&#xAE30; &#xC704;&#xD558;&#xC5EC; &#xCD1D; &#xC218;&#xC775;&#xB960; &#xADF9;&#xB300;&#xD654;. &#xB610;&#xD55C;, &#xC6B0;&#xB9AC;&#xAC00; &#xC591;&#xC790; &#xC810; &#xC0D8;&#xD50C;&#xC758; &#xD615;&#xAD11; antibleaching&#xC758; &#xC99D;&#xAC70;&#xB97C; &#xBC1C;&#xACAC;.

&#xB450; &#xAD11;&#xC790; &#xB808;&#xC774;&#xC800; &#xC2A4;&#xCE90;&#xB2DD; &#xD604;&#xBBF8;&#xACBD; &#xBD84;&#xC0B0; &#xBCF4;&#xC0C1;&#xC5D0; &#xD615;&#xAD11; &#xC218;&#xC728; &#xCD5C;&#xC801;&#xD654;.

Un desaf&#xED;o para formaci&#xF3;n de im&#xE1;genes no lineal en el tejido vivo es maximizar el rendimiento total fluorescente de cada fluor&#xF3;foro. Se investigaron las tasas de emisi&#xF3;n de tres fluor&#xF3;foros rodamina-B, una prote&#xED;na fluorescente de color rojo, y la cuant&#xED;a CdSe puntos-, mientras que la manipulaci&#xF3;n de la fase del pulso de excitaci&#xF3;n l&#xE1;ser en el foco. En todos los casos un pulso de transformaci&#xF3;n limitada maximiza el rendimiento total para asegurar la m&#xE1;s alta relaci&#xF3;n se&#xF1;al-ruido. Adem&#xE1;s, encontramos evidencia de antibleaching fluorescencia en las muestras de puntos cu&#xE1;nticos.

Optimizaci&#xF3;n del Rendimiento de marcaje de dos fotones microscop&#xED;a de barrido l&#xE1;ser con compensaci&#xF3;n de dispersi&#xF3;n

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.

Remote focusing for programmable multi-layer differential multiphoton microscopy.

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.

ROP GTPases act with the receptor-like protein PAN1 to polarize asymmetric cell division 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.

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

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.

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

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.

Identification of PAN2 by quantitative proteomics as a leucine-rich repeat-receptor-like kinase acting upstream of PAN1 to polarize cell division in maize.

Development of multicellular organisms proceeds via the correct interpretation of positional information to establish boundaries that separate developmental fields with distinct identities. The maize (Zea mays) leaf is an ideal system to study plant morphogenesis as it is subdivided into a proximal sheath and a distal blade, each with distinct developmental patterning. Specialized ligule and auricle structures form at the blade-sheath boundary. The auricles act as a hinge, allowing the leaf blade to project at an angle from the stem, while the ligule comprises an epidermally derived fringe. Recessive liguleless1 mutants lack ligules and auricles and have upright leaves. We used laser microdissection and RNA sequencing to identify genes that are differentially expressed in discrete cell/tissue-specific domains along the proximal-distal axis of wild-type leaf primordia undergoing ligule initiation and compared transcript accumulation in wild-type and liguleless1-R mutant leaf primordia. We identified transcripts that are specifically upregulated at the blade-sheath boundary. A surprising number of these "ligule genes" have also been shown to function during leaf initiation or lateral branching and intersect multiple hormonal signaling pathways. We propose that genetic modules utilized in leaf and/or branch initiation are redeployed to regulate ligule outgrowth from leaf primordia.

Transcriptomic analyses indicate that maize ligule development recapitulates gene expression patterns that occur during lateral organ initiation.

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.

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

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.

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

Multifocal multiphoton microscopy (MMM) in the biological and medical sciences has become an important tool for obtaining high resolution images at video rates. While current implementations of MMM achieve very high frame rates, they are limited in their applicability to essentially those biological samples that exhibit little or no scattering. In this paper, we report on a method for MMM in which imaging detection is not necessary (single element point detection is implemented), and is therefore fully compatible for use in imaging through scattering media. Further, we demonstrate that this method leads to a new type of MMM wherein it is possible to simultaneously obtain multiple images and view differences in excitation parameters in a single shot.

Differential Multiphoton Laser Scanning Microscopy.

A sensitive and dynamically responsive auxin signaling reporter based on the DII domain of the INDOLE-3-ACETIC ACID28 (IAA28, DII) protein from Arabidopsis (Arabidopsis thaliana) was modified for use in maize (Zea mays). The DII domain was fused to a yellow fluorescent protein and a nuclear localization sequence to simplify quantitative nuclear fluorescence signal. DII degradation dynamics provide an estimate of input signal into the auxin signaling pathway that is influenced by both auxin accumulation and F-box coreceptor concentration. In maize, the DII-based marker responded rapidly and in a dose-dependent manner to exogenous auxin via proteasome-mediated degradation. Low levels of DII-specific fluorescence corresponding to high endogenous auxin signaling occurred near vasculature tissue and the outer layer and glume primordia of spikelet pair meristems and floral meristems, respectively. In addition, high DII levels were observed in cells during telophase and early G1, suggesting that low auxin signaling at these stages may be important for cell cycle progression.

Anne W. Sylvester

Department of Developmental Genetics

University of Wyoming

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development