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Gene expression profiling of the different stages of Arabidopsis thaliana trichome development on the single cell level

Max Planck Institute of Molecular Plant Physiology, 14476 Potsdam, Germany.
Plant Physiology and Biochemistry (Impact Factor: 2.35). 03/2008; 46(2):160-73. DOI: 10.1016/j.plaphy.2007.11.001
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ABSTRACT Leaf hairs (trichomes) of Arabidopsis thaliana are a model system for studying cell development, differentiation and cell cycle regulation. To exploit this model system with ultimate spatial resolution we applied single cell sampling, thus avoiding the averaging effect induced by complex tissue mixtures. In particular, we analysed gene expression profiles of two selected stages of the developing trichome: trichome initial cells and mature trichomes, as well as pavement cells. Ten single cells per sample were collected by glass microcapillaries and used for the generation of radioactive probes for subsequent hybridization to nylon filters representing approximately 8000 genes of A. thaliana. Functional categorization of genes transcribed in trichome initials, mature trichomes and pavement cells demonstrated involvement of these surface cells in the stress response. In silico promoter analysis of genes preferentially expressed in trichome initials revealed enrichment in MYB-binding sites and presence of elements involved in hormonal, metal, sulphur response and cell cycle regulation. Three candidate genes preferentially expressed in trichome initials were selected for further analysis: At3g16980 (putative RNA polymerase II), At5g15230 (GASA4) and At4g27260 (GH3.5, WES1). Promoter:GUS studies confirmed expression of the putative RNA polymerase II and the gibberellin responsive GASA4 in trichome initials and partially in mature trichomes. Functional implication of the three selected candidates in trichome development and hence in cell cycle regulation in A. thaliana is discussed. We suggest that these genes are involved in differentiation and initiation of endocycling during trichome development.

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    • "Cells in the interior of the resulting trichome clusters undergo little endoreduplication or expansion, and resemble early stage developing trichomes. These trichomes express high levels of GL1 and GASA4, which are markers for early trichome development (Larkin et al., 1993; Kryvych et al., 2008; Marks et al., 2009). Over 50 loss-of-function mutations in Arabidopsis alter trichome cell fate or development (see Marks et al., 2009 and Morohashi and Grotewold, 2009) for recent lists of mutants. "
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    ABSTRACT: Transcriptome analysis using the Affymetrix ATH1 platform has been completed on purified trichomes from the gl3-sst mutant. These trichomes display immature features, such as glassy cell walls and blunted branches. The gl3-sst trichome transcriptome was greatly enriched for genes involved in lipid biosynthesis, including those mediating the synthesis of fatty acids and wax. In addition, gl3-sst trichomes displayed reduced expression of the R3 MYBs TRY and CPC, which normally function to limit trichome development. The expression of the MIXTA-like MYB gene NOK was elevated. Members of the MIXTA-like family promote conical cell outgrowth, and in some cases, trichome initiation in diverse plant species. In contrast, NOK limits trichome outgrowth in wild-type Arabidopsis plants. Similar to other MIXTA-like genes, NOK was required for the expansion of gl3-sst trichomes, as the gl3-sst nok double mutant trichomes were greatly reduced in size. Expression of NOK in nok mutants reduced branch formation, whereas in gl3-sst nok, NOK expression promoted trichome cell outgrowth, illustrating duel roles for NOK in both promoting and limiting trichome development. MIXTA-like genes from phylogenetically diverse plant species could substitute for NOK in both nok and gl3-sst nok backgrounds. These findings suggest that certain aspects of NOK and MIXTA-like gene function have been conserved.
    The Plant Journal 10/2010; 64(2):304-17. DOI:10.1111/j.1365-313X.2010.04329.x · 6.82 Impact Factor
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    • "For collecting single cell sap from leaves of A. thaliana sampling was performed as described previously (Brandt et al. 1999; Ebert et al. 2008; Kryvych et al. 2008). Borosilicate glass capillaries (WPI, 1B100-3: Berlin, Germany) were pulled on a List pipette puller (Darmstadt, Germany) with a tip aperture of 1–10µm. "
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    ABSTRACT: Gene expression levels of several transcription factors from Arabidopsis thaliana that were described previously to be involved in leaf development and trichome formation were analysed in trichome, basal and pavement cells of mature leaves. Single cell samples of these three cells types were collected by glass micro-capillaries. Real-time reverse transcription (RT)-PCR was used to analyse expression patterns of the following transcription factors: MYB23, MYB55, AtHB1, FILAMENTOUS FLOWER (FIL)/YABBY1 (YAB1), TRIPTYCHON (TRY) and CAPRICE (CPC). A difference in the expression patterns of TRY and CPC was revealed. Contrary to the CPC expression pattern, no transcripts of TRY could be detected in pavement cells. FIL/YAB1 was exclusively expressed in trichome cells. AtHB1 was highly expressed throughout all three cell types. MYB55 was higher expressed in basal cells than in trichome and pavement cells. MYB23 showed a pattern of low expression in pavement cells, medium in basal cells and high expression in trichomes. Expression patterns obtained by single cell sampling and real-time RT-PCR were compared to promoter GUS fusions of the selected transcription factors. Therefore, we regenerated two transgenic Arabidopsis lines that expressed the GUS reporter gene under control of the promoters of MYB55 and YAB1. In conclusion, despite their function in leaf morphogenesis, all six transcription factors were detected in mature leaves. Furthermore, single cell sampling and promoter GUS staining patterns demonstrated the predominant presence of MYB55 in basal cells as compared to pavement cells and trichomes.
    Protoplasma 05/2010; 241(1-4):29-36. DOI:10.1007/s00709-009-0099-7 · 3.17 Impact Factor
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    • "Of note, the second highest expressed gene was GASA4, which was expressed over seven times more highly in the double mutant than in wild-type trichomes. Previous studies on immature wild-type trichomes also found that this gene was highly expressed (Kryvych et al., 2008). The highest expressed gene has an unknown function, but we show below that this gene is required for trichome differentiation. "
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    ABSTRACT: Transcriptome analyses have been performed on mature trichomes isolated from wild-type Arabidopsis leaves and on leaf trichomes isolated from the gl3-sst sim double mutant, which exhibit many attributes of immature trichomes. The mature trichome profile contained many highly expressed genes involved in cell wall synthesis, protein turnover, and abiotic stress response. The most highly expressed genes in the gl3-sst sim profile encoded ribosomal proteins and other proteins involved in translation. Comparative analyses showed that all but one of the genes encoding transcription factors previously found to be important for trichome formation, and many other trichome-important genes, were preferentially expressed in gl3-sst sim trichomes. The analysis of genes preferentially expressed in gl3-sst sim led to the identification of four additional genes required for normal trichome development. One of these was the HDG2 gene, which is a member of the HD-ZIP IV transcription factor gene family. Mutations in this gene did not alter trichome expansion, but did alter mature trichome cell walls. Mutations in BLT resulted in a loss of trichome branch formation. The relationship between blt and the phenotypically identical mutant, sti, was explored. Mutations in PEL3, which was previously shown to be required for development of the leaf cuticle, resulted in the occasional tangling of expanding trichomes. Mutations in another gene encoding a protein with an unknown function altered trichome branch formation.
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