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An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in <em>Arabidopsis</em>

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

University of Zürich

The variation of expression pattern exhibited by a transgene as a result of random integration, known as position effect, is, among other mechanisms, a particular challenge to reverse genetics. We present a strategy to counteract position effect in Arabidopsis thaliana by flanking the transgenes with the gypsy insulator from Drosophila melanogaster. In addition, Suppressor of Hairy-wing [Su(Hw)], the binding protein of the gypsy insulator, was coexpressed. Results indicated that the gypsy insulators could efficiently improve the expression levels of reporter genes driven by various kinds of promoters by 8- to 13-fold. Coexpression of the Su(Hw) protein led to a more uniform expression level of transgenes, as the coefficient of variation of expression levels was reduced further. The gypsy-Su(Hw) system enhanced expression levels, but did not alter the specificity of promoter activities, as experimentally evidenced by the promoters of the PIN and the AFB gene families. Interestingly, the gypsy insulator was also able to improve the expression of a selectable marker gene outside the insulated region, which facilitated the screen of transformants. Our system will likely decrease the number of lines that experimenters need to create and examine for a given transgene by contributing to relatively high and precise expression of transgenes in plants. Certain features of the gypsy insulator in Arabidopsis also provide new perspectives on the insulator field.

The gypsy insulator of Drosophila melanogaster, together with its binding protein suppressor of Hairy-wing, facilitate high and precise expression of transgenes in Arabidopsis thaliana.

Among the three primary auxin-induced gene families, Auxin/Indole-3-Acetic Acid (Aux/IAA), Gretchen Hagen3 (GH3) and SMALL AUXIN UP RNA (SAUR), the function of SAUR genes remains unclear. Arabidopsis SAUR genes have been phylogenetically classified into three clades. Recent work has suggested that SAUR19 (clade II) and SAUR63 (clade I) promote cell expansion through the modulation of auxin transport. Herein, we present our work on SAUR41, a clade III SAUR gene with a distinctive expression pattern in root meristems. SAUR41 was normally expressed in the quiescent center and cortex/endodermis initials; upon auxin stimulation, the expression was provoked in the endodermal layer. During lateral root development, SAUR41 was expressed in prospective stem cell niches of lateral root primordia and in expanding endodermal cells surrounding the primordia. SAUR41-EGFP (enhanced green fluorescent protein) fusion proteins localized to the cytoplasm. Overexpression of SAUR41 from the Cauliflower mosaic virus 35S promoter led to pleiotropic auxin-related phenotypes, including long hypocotyls, increased vegetative biomass and lateral root development, expanded petals and twisted inflorescence stems. Ectopic SAUR41 proteins were able to promote auxin transport in hypocotyls. Tissue-specific expression of SAUR41 from the PIN1, WOX5, PLT2 and ACR4 promoters induced the formation of new auxin accumulation/signaling peaks above the quiescent centers, whereas tissue-specific expression of SAUR41 from the PIN2 and PLT2 promoters enhanced root gravitropic growth. Cells in the root stem cell niches of these transgenic seedlings were differentially enlarged. The distinctive expression pattern of the SAUR41 gene and the explicit function of SAUR41 proteins implied that further investigations on the loss-of-function phenotypes of this gene in root development and environmental responses are of great interest.

Tissue-specific expression of SMALL AUXIN UP RNA41 differentially regulates cell expansion and root meristem patterning in Arabidopsis.

The life cycle of flowering plants is marked by several post-embryonic developmental transitions during which novel cell fates are established. Notably, the reproductive lineages are first formed during flower development. The differentiation of spore mother cells, which are destined for meiosis, marks the somatic-to-reproductive fate transition. Meiosis entails the formation of the haploid multicellular gametophytes, from which the gametes are derived, and during which epigenetic reprogramming takes place. Here we show that in the Arabidopsis female megaspore mother cell (MMC), cell fate transition is accompanied by large-scale chromatin reprogramming that is likely to establish an epigenetic and transcriptional status distinct from that of the surrounding somatic niche. Reprogramming is characterized by chromatin decondensation, reduction in heterochromatin, depletion of linker histones, changes in core histone variants and in histone modification landscapes. From the analysis of mutants in which the gametophyte fate is either expressed ectopically or compromised, we infer that chromatin reprogramming in the MMC is likely to contribute to establishing postmeiotic competence to the development of the pluripotent gametophyte. Thus, as in primordial germ cells of animals, the somatic-to-reproductive cell fate transition in plants entails large-scale epigenetic reprogramming.

Wenjing She

Institute of Plant Biology and Zürich-Basel Plant Science Center

University of Zürich

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

Wenjing She

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute of Plant Biology and Zürich-Basel Plant Science Center

University of Zürich

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

Institute of Plant Biology and Zürich-Basel Plant Science Center