Firing of transcription and compartmentalization of splicing factors in tomato radicle nuclei during germination¹

Abstract

Background information. Germination is a well-characterized process in which embryo cells of seeds experience a programmed transition from quiescence to proliferation. For this reason they constitute a very good system to analyse nuclear evolution from a dehydrated practically inactive state until the steady state of proliferation. We analysed the temporal and spatial organization of transcription and splicing factors in nuclei of tomato radicle cells during germination. To address this issue we performed in situ immunodetection of several markers of these processes: the Z-DNA stretches forming behind the active RNA polymerases, the splicing proteins U2B and Sm, and the trimethyl guanosin cap of small nuclear RNA. The concomitant structural changes of the different nuclear compartments were studied in meristematic nuclei by electron microscopy and high-resolution cytochemistry for DNA and ribonucleoproteins.Results. In quiescent cells practically no Z-DNA stretches were detected and splicing components localized mainly to one or two Cajal bodies associated to the nucleolus. In early germination, a massive de-condensation of chromatin and nucleolar Z-DNA conformation stretches were first detected, followed by the relocation of scarce splicing components to the small interchromatin spaces. Nucleoplasmic Z-DNA stretches were not detected until 4 h of imbibition and were accompanied by an important increase of splicing components in this nuclear domain. Soon after the post-germination stage, transcription and splicing topology and nuclear organization in meristematic nuclei resemble those in steady state growing tomato roots.Conclusions. Our results demonstrate that, in tomato, dormant nuclei splicing factors are stored in nucleolar Cajal bodies. In early germination, RNA polymerase I transcription is first activated, whereas mRNA transcription is fired later and is accompanied by a massive de-condensation of chromatin and accumulation of splicing factors in the interchromatin domains. Nucleoplasmic Cajal bodies appear later in germination.

Stigma/style cell cycle inhibitor 1 (SCI1), a tissue‐specific cell cycle regulator that controls upper pistil development

“…Although the SCI1 localization pattern is unique, a similar pattern has been observed for a cyclin A in Arabidopsis (Boruc et al, 2010). Additionally, changes in the architecture and functional organization of the nuclear bodies accompany the switch of the developmental program and the activation of proliferative activity in plant cells (Testillano et al, 2005;Echeverría et al, 2007). Therefore, SCI1-GFP distribution in the nucleolus and its relocation to nuclear bodies may be linked to different cell cycle phases, which is consistent with SCI1 involvement in cell proliferation ⁄ differentiation control.…”

“…Additionally, changes in the architecture and functional organization of the nuclear bodies accompany the switch of the developmental program and the activation of proliferative activity in plant cells (Testillano et al. , 2005; Echeverría et al. , 2007).…”

Periodic expression of Sm proteins parallels formation of nuclear Cajal bodies and cytoplasmic snRNP-rich bodies

“…A western immunoblot analysis was performed to examine the specificity of the mouse Y12 antibody, in Larix decidua which recognizes Sm B′/B and D1 proteins in plants (Hirakata et al 1993; Echeverría et al 2007). As shown in Fig.…”

mRNA accumulation in the Cajal bodies of the diplotene larch microsporocyte

“…Sm proteins were also detected by incubating with a primary anti-Sm Y12 mouse antibody, which recognizes Sm B′/B and D1 proteins in plants (Hirakata et al 1993; Echeverría et al 2007), according to the method in Zienkiewicz et al (2008b), in 0.1% acetylated BSA in PBS (1:10) in a humidified chamber at 8°C overnight and an anti-mouse Alexa 488 (Invitrogen) secondary antibody. For all experiments (Sm–mRNA, m3G snRNA–mRNA), immunocytochemistry always preceded in situ hybridization because immunofluorescent signals were extremely weak if ISH was applied first.…”