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Heisler GroupPublications

Live-imaging of the Arabidopsis inflorescence meristem.
Heisler, M.G. & Ohno, C.
Methods Mol Biol. 2014;1110:431-40. doi: 10.1007/978-1-4614-9408-9_25.
The aboveground tissues of higher plants are derived from a small population of stem cells located at the shoot apex within a structure called the shoot apical meristem (SAM). The SAM not only includes the stem cells but also incorporates a region from which lateral organs arise. The SAM is therefore of prime interest for understanding plant growth and development. In this chapter we outline methods for using confocal microscopy to image the Arabidopsis inflorescence SAM. This method enables detailed examination of cell division and growth patterns (Reddy et al., Development 131:4225-4237, 2004) as well as gene expression and protein localization patterns over time (Heisler et al. Curr Biol 15:1899-1911, 2005). When combined with perturbation approaches, the method offers an extremely powerful system for investigating SAM function in great detail.
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Accounting for technical noise in single-cell RNA-seq experiments.
Brennecke, P., Anders, S., Kim, J.K., Kolodziejczyk, A.A., Zhang, X., Proserpio, V., Baying, B., Benes, V., Teichmann, S.A., Marioni, J.C. & Heisler, M.G.
Nat Methods. 2013 Nov;10(11):1093-5. doi: 10.1038/nmeth.2645. Epub 2013 Sep 22.
Single-cell RNA-seq can yield valuable insights about the variability within a population of seemingly homogeneous cells. We developed a quantitative statistical method to distinguish true biological variability from the high levels of technical noise in single-cell experiments. Our approach quantifies the statistical significance of observed cell-to-cell variability in expression strength on a gene-by-gene basis. We validate our approach using two independent data sets from Arabidopsis thaliana and Mus musculus.
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Genome-wide identification of KANADI1 target genes.
Merelo, P., Xie, Y., Brand, L., Ott, F., Weigel, D., Bowman, J.L., Heisler, M.G. & Wenkel, S.
PLoS One. 2013 Oct 14;8(10):e77341. doi: 10.1371/journal.pone.0077341.eCollection 2013.
Plant organ development and polarity establishment is mediated by the action of several transcription factors. Among these, the KANADI (KAN) subclade of the GARP protein family plays important roles in polarity-associated processes during embryo, shoot and root patterning. In this study, we have identified a set of potential direct target genes of KAN1 through a combination of chromatin immunoprecipitation/DNA sequencing (ChIP-Seq) and genome-wide transcriptional profiling using tiling arrays. Target genes are over-represented for genes involved in the regulation of organ development as well as in the response to auxin. KAN1 affects directly the expression of several genes previously shown to be important in the establishment of polarity during lateral organ and vascular tissue development. We also show that KAN1 controls through its target genes auxin effects on organ development at different levels: transport and its regulation, and signaling. In addition, KAN1 regulates genes involved in the response to abscisic acid, jasmonic acid, brassinosteroids, ethylene, cytokinins and gibberellins. The role of KAN1 in organ polarity is antagonized by HD-ZIPIII transcription factors, including REVOLUTA (REV). A comparison of their target genes reveals that the REV/KAN1 module acts in organ patterning through opposite regulation of shared targets. Evidence of mutual repression between closely related family members is also shown.
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Live-imaging of plant development: latest approaches.
Sappl, P.G. & Heisler, M.G.
Curr Opin Plant Biol. 2013 Feb;16(1):33-40. doi: 10.1016/j.pbi.2012.10.006. Epub2012 Nov 26.
Development is a dynamic process occurring at the microscopic scale. The ability to see how it unfolds in detail is invaluable not only for helping us appreciate its full complexity but also to experimentally dissect its mechanisms. The sophistication of experimental approaches and imaging technologies has increased over the past decade at an astounding pace. In this review we highlight and discuss several studies that illustrate the latest advances in the application of live-imaging to dissect plant development.
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Plant stem cell maintenance involves direct transcriptional repression of differentiation program.
Yadav, R.K., Perales, M., Gruel, J., Ohno, C., Heisler, M., Girke, T., Jonsson, H. & Reddy, G.V.
Mol Syst Biol. 2013;9:654. doi: 10.1038/msb.2013.8.
In animal systems, master regulatory transcription factors (TFs) mediate stem cell maintenance through a direct transcriptional repression of differentiation promoting TFs. Whether similar mechanisms operate in plants is not known. In plants, shoot apical meristems serve as reservoirs of stem cells that provide cells for all above ground organs. WUSCHEL, a homeodomain TF produced in cells of the niche, migrates into adjacent cells where it specifies stem cells. Through high-resolution genomic analysis, we show that WUSCHEL represses a large number of genes that are expressed in differentiating cells including a group of differentiation promoting TFs involved in leaf development. We show that WUS directly binds to the regulatory regions of differentiation promoting TFs; KANADI1, KANADI2, ASYMMETRICLEAVES2 and YABBY3 to repress their expression. Predictions from a computational model, supported by live imaging, reveal that WUS-mediated repression prevents premature differentiation of stem cell progenitors, being part of a minimal regulatory network for meristem maintenance. Our work shows that direct transcriptional repression of differentiation promoting TFs is an evolutionarily conserved logic for stem cell regulation.
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Integrated genetic and computation methods for in planta cytometry.
Federici, F., Dupuy, L., Laplaze, L., Heisler, M. & Haseloff, J.
Nat Methods. 2012 Apr 1;9(5):483-5. doi: 10.1038/nmeth.1940.
We present the coupled use of specifically localized fluorescent gene markers and image processing for automated quantitative analysis of cell growth and genetic activity across living plant tissues. We used fluorescent protein markers to identify cells, create seeds and boundaries for the automatic segmentation of cell geometries and ratiometrically measure gene expression cell by cell in Arabidopsis thaliana.
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Cytokinin signaling as a positional cue for patterning the apical-basal axis of the growing Arabidopsis shoot meristem.
Chickarmane, V.S., Gordon, S.P., Tarr, P.T., Heisler, M.G. & Meyerowitz, E.M.
Proc Natl Acad Sci U S A. 2012 Mar 6;109(10):4002-7. Epub 2012 Feb 15.
The transcription factor WUSCHEL (WUS) acts from a well-defined domain within the Arabidopsis thaliana shoot apical meristem (SAM) to maintain a stem cell niche. A negative-feedback loop involving the CLAVATA (CLV) signaling pathway regulates the number of WUS-expressing cells and provides the current paradigm for the homeostatic maintenance of stem cell numbers. Despite the continual turnover of cells in the SAM during development, the WUS domain remains patterned at a fixed distance below the shoot apex. Recent work has uncovered a positive-feedback loop between WUS function and the plant hormone cytokinin. Furthermore, loss of function of the cytokinin biosynthetic gene, LONELY GUY (LOG), results in a wus-like phenotype in rice. Herein, we find the Arabidopsis LOG4 gene is expressed in the SAM epidermis. We use this to develop a computational model representing a growing SAM to suggest the plausibility that apically derived cytokinin and CLV signaling, together, act as positional cues for patterning the WUS domain within the stem cell niche. Furthermore, model simulations backed by experimental data suggest a previously unknown negative feedback between WUS function and cytokinin biosynthesis in the Arabidopsis SAM epidermis. These results suggest a plausible dynamic feedback principle by which the SAM stem cell niche is patterned.
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Alignment between PIN1 polarity and microtubule orientation in the shoot apical meristem reveals a tight coupling between morphogenesis and auxin transport.
Heisler, M.G.*, Hamant, O.*, Krupinski, P.*, Uyttewaal, M., Ohno, C., Jonsson, H., Traas, J. & Meyerowitz, E.M.
PLoS Biol. 2010 Oct 19;8(10):e1000516.
*equal contribution
Morphogenesis during multicellular development is regulated by intercellular signaling molecules as well as by the mechanical properties of individual cells. In particular, normal patterns of organogenesis in plants require coordination between growth direction and growth magnitude. How this is achieved remains unclear. Here we show that in Arabidopsis thaliana, auxin patterning and cellular growth are linked through a correlated pattern of auxin efflux carrier localization and cortical microtubule orientation. Our experiments reveal that both PIN1 localization and microtubule array orientation are likely to respond to a shared upstream regulator that appears to be biomechanical in nature. Lastly, through mathematical modeling we show that such a biophysical coupling could mediate the feedback loop between auxin and its transport that underlies plant phyllotaxis.
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Developmental patterning by mechanical signals in Arabidopsis.
Hamant*, O., Heisler*, M.G., Jonsson*, H., Krupinski, P., Uyttewaal, M., Bokov, P., Corson, F., Sahlin, P., Boudaoud, A., Meyerowitz, E.M., Couder, Y. & Traas, J.
Science. 2008 Dec 12;322(5908):1650-5.
*equal contribution
A central question in developmental biology is whether and how mechanical forces serve as cues for cellular behavior and thereby regulate morphogenesis. We found that morphogenesis at the Arabidopsis shoot apex depends on the microtubule cytoskeleton, which in turn is regulated by mechanical stress. A combination of experiments and modeling shows that a feedback loop encompassing tissue morphology, stress patterns, and microtubule-mediated cellular properties is sufficient to account for the coordinated patterns of microtubule arrays observed in epidermal cells, as well as for patterns of apical morphogenesis.
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Pattern formation during de novo assembly of the Arabidopsis shoot meristem.
Gordon, S.P., Heisler, M.G., Reddy, G.V., Ohno, C., Das, P. & Meyerowitz, E.M.
Development. 2007 Oct;134(19):3539-48.
Most multicellular organisms have a capacity to regenerate tissue after wounding. Few, however, have the ability to regenerate an entire new body from adult tissue. Induction of new shoot meristems from cultured root explants is a widely used, but poorly understood, process in which apical plant tissues are regenerated from adult somatic tissue through the de novo formation of shoot meristems. We characterize early patterning during de novo development of the Arabidopsis shoot meristem using fluorescent reporters of known gene and protein activities required for shoot meristem development and maintenance. We find that a small number of progenitor cells initiate development of new shoot meristems through stereotypical stages of reporter expression and activity of CUP-SHAPED COTYLEDON 2 (CUC2), WUSCHEL (WUS), PIN-FORMED 1 (PIN1), SHOOT-MERISTEMLESS (STM), FILAMENTOUS FLOWER (FIL, also known as AFO), REVOLUTA (REV), ARABIDOPSIS THALIANA MERISTEM L1 LAYER (ATML1) and CLAVATA 3 (CLV3). Furthermore, we demonstrate a functional requirement for WUS activity during de novo shoot meristem initiation. We propose that de novo shoot meristem induction is an easily accessible system for the study of patterning and self-organization in the well-studied model organism Arabidopsis.
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Antagonistic regulation of PIN phosphorylation by PP2A and PINOID directs auxin flux.
Michniewicz, M., Zago, M.K., Abas, L., Weijers, D., Schweighofer, A., Meskiene, I., Heisler, M.G., Ohno, C., Zhang, J., Huang, F., Schwab, R., Weigel, D., Meyerowitz, E.M., Luschnig, C., Offringa, R. & Friml, J.
Cell. 2007 Sep 21;130(6):1044-56.
In plants, cell polarity and tissue patterning are connected by intercellular flow of the phytohormone auxin, whose directional signaling depends on polar subcellular localization of PIN auxin transport proteins. The mechanism of polar targeting of PINs or other cargos in plants is largely unidentified, with the PINOID kinase being the only known molecular component. Here, we identify PP2A phosphatase as an important regulator of PIN apical-basal targeting and auxin distribution. Genetic analysis, localization, and phosphorylation studies demonstrate that PP2A and PINOID both partially colocalize with PINs and act antagonistically on the phosphorylation state of their central hydrophilic loop, hence mediating PIN apical-basal polar targeting. Thus, in plants, polar sorting by the reversible phosphorylation of cargos allows for their conditional delivery to specific intracellular destinations. In the case of PIN proteins, this mechanism enables switches in the direction of intercellular auxin fluxes, which mediate differential growth, tissue patterning, and organogenesis.
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Modelling meristem development in plants.
Heisler, M.G. & Jonsson, H.
Curr Opin Plant Biol. 2007 Feb;10(1):92-7. Epub 2006 Nov 30.
Meristems continually supply new cells for post-embryonic plant development and coordinate the initiation of new organs, such as leaves and flowers. Meristem function is regulated by a large and interconnected dynamic system that includes transcription networks, intercellular protein signalling, polarized transport of hormones and a constantly changing cellular topology. Mathematical modelling, in which the dynamics of a system are simulated using explicitly defined interactions, can serve as a powerful tool for examining the expected behaviour of such a system given our present knowledge and assumptions. Modelling can also help to investigate new hypotheses in silico both to validate ideas and to obtain inspiration for new experiments. Several recent studies have used new molecular data together with modelling and computational techniques to investigate meristem function.
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Apical-basal polarity: why plant cells don't stand on their heads.
Friml, J., Benfey, P., Benkova, E., Bennett, M., Berleth, T., Geldner, N., Grebe, M., Heisler, M., Hejatko, J., Jurgens, G., Laux, T., Lindsey, K., Lukowitz, W., Luschnig, C., Offringa, R., Scheres, B., Swarup, R., Torres-Ruiz, R., Weijers, D. & Zazimalova, E.
Trends Plant Sci. 2006 Jan;11(1):12-4. Epub 2005 Dec 13. Europe PMC

An auxin-driven polarized transport model for phyllotaxis.
Jonsson*, H., Heisler*, M.G., Shapiro, B.E., Meyerowitz, E.M. & Mjolsness, E.
Proc Natl Acad Sci U S A. 2006 Jan 31;103(5):1633-8. Epub 2006 Jan 13.
Recent studies show that plant organ positioning may be mediated by localized concentrations of the plant hormone auxin. Auxin patterning in the shoot apical meristem is in turn brought about by the subcellular polar distribution of the putative auxin efflux mediator, PIN1. However, the question of what signals determine PIN1 polarization and how this gives rise to regular patterns of auxin concentration remains unknown. Here we address these questions by using mathematical modeling combined with confocal imaging. We propose a model that is based on the assumption that auxin influences the polarization of its own efflux within the meristem epidermis. We show that such a model is sufficient to create regular spatial patterns of auxin concentration on systems with static and dynamic cellular connectivities, the latter governed by a mechanical model. We also optimize parameter values for the PIN1 dynamics by using a detailed auxin transport model, for which parameter values are taken from experimental estimates, together with a template consisting of cell and wall compartments as well as PIN1 concentrations quantitatively extracted from confocal data. The model shows how polarized transport can drive the formation of regular patterns.
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In situ hybridization for mRNA detection in Arabidopsis tissue sections.
Brewer, P.B., Heisler, M.G., Hejatko, J., Friml, J. & Benkova, E.
Nat Protoc. 2006;1(3):1462-7.
Plant biology is currently confronted with an overflow of expression profile data provided by high-throughput microarray transcription analyses. However, the tissue and cellular resolution of these techniques is limited. Thus, it is still necessary to examine the expression pattern of selected candidate genes at a cellular level. Here we present an in situ mRNA hybridization method that is routinely used in the analysis of gene expression patterns. The protocol is optimized for mRNA localizations in sectioned tissue of Arabidopsis seedlings including embryos, roots, hypocotyls, young primary leaves and flowers. The detailed protocol, recommended controls and troubleshooting are presented along with examples of application. The total time for the process is 10 days.
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Patterns of auxin transport and gene expression during primordium development revealed by live imaging of the Arabidopsis inflorescence meristem.
Heisler, M.G., Ohno, C., Das, P., Sieber, P., Reddy, G.V., Long, J.A. & Meyerowitz, E.M.
Curr Biol. 2005 Nov 8;15(21):1899-911.
BACKGROUND: Plants produce leaf and flower primordia from a specialized tissue called the shoot apical meristem (SAM). Genetic studies have identified a large number of genes that affect various aspects of primordium development including positioning, growth, and differentiation. So far, however, a detailed understanding of the spatio-temporal sequence of events leading to primordium development has not been established. RESULTS: We use confocal imaging of green fluorescent protein (GFP) reporter genes in living plants to monitor the expression patterns of multiple proteins and genes involved in flower primordial developmental processes. By monitoring the expression and polarity of PINFORMED1 (PIN1), the auxin efflux facilitator, and the expression of the auxin-responsive reporter DR5, we reveal stereotypical PIN1 polarity changes which, together with auxin induction experiments, suggest that cycles of auxin build-up and depletion accompany, and may direct, different stages of primordium development. Imaging of multiple GFP-protein fusions shows that these dynamics also correlate with the specification of primordial boundary domains, organ polarity axes, and the sites of floral meristem initiation. CONCLUSIONS: These results provide new insight into auxin transport dynamics during primordial positioning and suggest a role for auxin transport in influencing primordial cell type.
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Modeling the organization of the WUSCHEL expression domain in the shoot apical meristem.
Jonsson, H., Heisler, M., Reddy, G.V., Agrawal, V., Gor, V., Shapiro, B.E., Mjolsness, E. & Meyerowitz, E.M.
Bioinformatics. 2005 Jun;21 Suppl 1:i232-40.
MOTIVATION: The above-ground tissues of higher plants are generated from a small region of cells situated at the plant apex called the shoot apical meristem. An important genetic control circuit modulating the size of the Arabidopsis thaliana meristem is a feed-back network between the CLAVATA3 and WUSCHEL genes. Although the expression patterns for these genes do not overlap, WUSCHEL activity is both necessary and sufficient (when expressed ectopically) for the induction of CLAVATA3 expression. However, upregulation of CLAVATA3 in conjunction with the receptor kinase CLAVATA1 results in the downregulation of WUSCHEL. Despite much work, experimental data for this network are incomplete and additional hypotheses are needed to explain the spatial locations and dynamics of these expression domains. Predictive mathematical models describing the system should provide a useful tool for investigating and discriminating among possible hypotheses, by determining which hypotheses best explain observed gene expression dynamics. RESULTS: We are developing a method using in vivo live confocal microscopy to capture quantitative gene expression data and create templates for computational models. We present two models accounting for the organization of the WUSCHEL expression domain. Our preferred model uses a reaction-diffusion mechanism in which an activator induces WUSCHEL expression. This model is able to organize the WUSCHEL expression domain. In addition, the model predicts the dynamical reorganization seen in experiments where cells, including the WUSCHEL domain, are ablated, and it also predicts the spatial expansion of the WUSCHEL domain resulting from removal of the CLAVATA3 signal. AVAILABILITY: An extended description of the model framework and image processing algorithms can be found at http://www.computableplant.org, together with additional results and simulation movies. SUPPLEMENTARY INFORMATION: http://www.computableplant.org/ and alternatively for a direct link to the page, http://computableplant.ics.uci.edu/bti1036 can be accessed.
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Real-time lineage analysis reveals oriented cell divisions associated with morphogenesis at the shoot apex of Arabidopsis thaliana.
Reddy, G.V., Heisler, M.G., Ehrhardt, D.W. & Meyerowitz, E.M.
Development. 2004 Sep;131(17):4225-37. Epub 2004 Jul 27.
Precise knowledge of spatial and temporal patterns of cell division, including number and orientation of divisions, and knowledge of cell expansion, is central to understanding morphogenesis. Our current knowledge of cell division patterns during plant and animal morphogenesis is largely deduced from analysis of clonal shapes and sizes. But such an analysis can reveal only the number, not the orientation or exact rate, of cell divisions. In this study, we have analyzed growth in real time by monitoring individual cell divisions in the shoot apical meristems (SAMs) of Arabidopsis thaliana. The live imaging technique has led to the development of a spatial and temporal map of cell division patterns. We have integrated cell behavior over time to visualize growth. Our analysis reveals temporal variation in mitotic activity and the cell division is coordinated across clonally distinct layers of cells. Temporal variation in mitotic activity is not correlated to the estimated plastochron length and diurnal rhythms. Cell division rates vary across the SAM surface. Cells in the peripheral zone (PZ) divide at a faster rate than in the central zone (CZ). Cell division rates in the CZ are relatively heterogeneous when compared with PZ cells. We have analyzed the cell behavior associated with flower primordium development starting from a stage at which the future flower comprises four cells in the L1 epidermal layer. Primordium development is a sequential process linked to distinct cellular behavior. Oriented cell divisions, in primordial progenitors and in cells located proximal to them, are associated with initial primordial outgrowth. The oriented cell divisions are followed by a rapid burst of cell expansion and cell division, which transforms a flower primordium into a three-dimensional flower bud. Distinct lack of cell expansion is seen in a narrow band of cells, which forms the boundary region between developing flower bud and the SAM. We discuss these results in the context of SAM morphogenesis.
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The Arabidopsis JAGGED gene encodes a zinc finger protein that promotes leaf tissue development.
Ohno, C.K., Reddy, G.V., Heisler, M.G. & Meyerowitz, E.M.
Development. 2004 Mar;131(5):1111-22.
Important goals in understanding leaf development are to identify genes involved in pattern specification, and also genes that translate this information into cell types and tissue structure. Loss-of-function mutations at the JAGGED (JAG) locus result in Arabidopsis plants with abnormally shaped lateral organs including serrated leaves, narrow floral organs, and petals that contain fewer but more elongate cells. jag mutations also suppress bract formation in leafy, apetala1 and apetala2 mutant backgrounds. The JAG gene was identified by map-based cloning to be a member of the zinc finger family of plant transcription factors and encodes a protein similar in structure to SUPERMAN with a single C(2)H(2)-type zinc finger, a proline-rich motif and a short leucine-rich repressor motif. JAG mRNA is localized to lateral organ primordia throughout the plant but is not found in the shoot apical meristem. Misexpression of JAG results in leaf fusion and the development of ectopic leaf-like outgrowth from both vegetative and floral tissues. Thus, JAG is necessary for proper lateral organ shape and is sufficient to induce the proliferation of lateral organ tissue.
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SPATULA, a gene that controls development of carpel margin tissues in Arabidopsis, encodes a bHLH protein.
Heisler, M.G., Atkinson, A., Bylstra, Y.H., Walsh, R. & Smyth, D.R.
Development. 2001 Apr;128(7):1089-98.
Studies involving mutants of the gene SPATULA indicate that it promotes the growth of carpel margins and of pollen tract tissues derived from them. We show that it encodes a new member of the basic-helix-loop-helix family of transcription factors. SPATULA is expressed in marginal and pollen tract tissues throughout their development confirming its role in regulating their growth. It is also expressed in many other tissues where it may act redundantly to control growth, including the peripheral zone of the shoot apical meristem, and specific tissues within leaves, petals, stamens and roots. Expression in the stomium, funiculus and valve dehiscence zone indicates an additional role in abscission. SPATULA expression does not require the function of the other carpel development genes CRABS CLAW and AGAMOUS, although its expression is repressed in first whorl organs by the A function gene APETALA2. Further, we have shown that disruptions to gynoecial pattern formation seen in ettin mutants can largely be attributed to ectopic SPATULA action. ETTIN's role seems to be to negatively regulate SPATULA expression in abaxial regions of the developing gynoecium. SPATULA is the first basic-helix-loop-helix gene in plants known to play a role in floral organogenesis.
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Molecular and evolutionary aspects of self-incompatibility in flowering plants.
Mau, S.L., Anderson, M.A., Heisler, M., Haring, V., McClure, B.A. & Clarke, A.E.
Symp Soc Exp Biol. 1991;45:245-69.
Self-incompatibility (SI) is widely distributed in flowering plants. In this review, early work on the biology, genetics and distribution of SI is summarized. Approaches to understanding the molecular genetics of SI have been made in two systems-Solanaceous species, for example Nicotiana alata, which have gametophytic systems of SI, and Brassica spp, which have sporophytic systems of SI. The information in both systems is derived from cDNAs that encode pistil glycoproteins (S-glycoproteins) that segregate with S-genotype. Comparison of the sequence data indicates that the gametophytic and sporophytic systems of SI probably arose independently during the evolution of angiosperms. The S-glycoproteins of a solanaceous plant Nicotiana alata, are ribonucleases (RNases). Whether the RNase activity is directly involved in the characteristic arrest of pollen tube growth during self-(incompatible) pollination, is not known. An alternative possibility is that the RNase was 'recruited' during evolution for a function in SI, without involvement of its catalytic function. The nature of the S-gene in pollen is not yet known for either the gametophytic or sporophytic SI systems. This is a key piece of information that will be required to progress our understanding of how the growth of a pollen tube bearing a particular S-allele is arrested within the style bearing an identical S-allele, but is not arrested within the style bearing other S-alleles.
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