Regulation of poly(A) RNA retention in the nucleus as a survival strategy of plants during hypoxia

<p>Last finding indicates that post-transcriptional processes are significant in low-oxygen conditions, but their nature is poorly understood. Here, we localized poly(A) RNA and mRNA coding proteins involved and not involved with resistance to hypoxia in <i>Lupinus luteus</i> and <i>Arabidopsis thaliana</i> during submergence and after recovery of aerobic conditions. We showed a strong nuclear accumulation of poly(A) RNA and 6 of 7 studied mRNAs with a concurrent strong reduction in RNA polymerase II transcription during hypoxia. In this study, the nucleus did not accumulate mRNA of the <i>ADH1</i> (alcohol dehydrogenase 1) gene, which is a core hypoxia gene. The RNA accumulation in the nucleus is among the mechanisms of post-transcriptional gene regulation that prevents translation. However re-aeration was accompanied by a strong increase in the amount of the mRNAs in the cytoplasm and a simultaneous decrease in nuclear mRNAs. This finding indicates that the nucleus is a storage site for those of mRNAs which are not involved in the response to hypoxia for use by the plants after the hypoxic stress. In this study, the highest intensity of RNA accumulation occurred in Cajal bodies (CBs); the intensity of accumulation was inversely correlated with transcription. Under hypoxia, <i>ncb-1</i> mutants of <i>Arabidopsis thaliana</i> with a complete absence of CBs died sooner than wild type (WT), accompanied by a strong reduction in the level of poly(A) RNA in the nucleus. These results suggest that the CBs not only participate in the storage of the nuclear RNA, but they also could take part in its stabilization under low-oxygen conditions.</p

Analysis of the fluorescence intensity resulting from <i>in situ</i> hybridisation of poly(A) RNA in the meristematic cells of lupines roots.

<p>There were significant differences in intensity (p<0.05) between the cytoplasm (MC), nucleus (MN) and nuclear bodies (MNB).</p

Double labelling of poly(A) RNA and the PANA antigen in the chromocentric (<i>Lupinus</i>) (A–C) and reticular (<i>Allium</i>) (D–F) nuclei of root cells.

<p>In the nucleoplasm of both species, poly(A) RNA is present in nuclear structures (arrows) and does not colocalise with speckles. Representative examples of Pearson correlation coefficients for weak and non-colocalisation of poly(A) mRNA with the PANA antigen in <i>Lupinus luteus</i> and <i>Allium cepa</i> cells (G). A scale bar representing 2 µm is shown. The percentages of weak and non-colocalisation of poly(A) RNA-rich bodies with the PANA antigen are indicated by the Pearson correlation coefficient (H). Error bars represent standard error. Double labelling of poly(A) RNA and U2 snRNA in <i>Lupinus</i> (I–K) and <i>Allium</i> (L–N) cells. Accumulation of poly(A) RNA in nuclear bodies rich in U2 snRNA (arrows). Bar, 5 µm. N-nucleus, Nu- nucleolus</p

Localisation of poly(A) RNA in control lupine root cells (A) and after submersion to tap water for 3 h (B).

<p>Arrows indicate CBs, and arrow heads indicate cytoplasmic granules. Bar, 5 µm. Nu- nucleolus. Quantitative analysis of the fluorescence intensity associated with the localisation of poly(A) RNA. Significant differences in the signal intensity in the nuclei and CBs between control and hypoxia-treated cells (C) MNu- control nucleus, MCB control Cajal bodies, HypNu and HypCB- nucleus and Cajal bodies after hypoxia treatment, respectively (p = 0.05).</p

Double labelling of a mixture of four mRNAs (A, C) and distinct genes: cyclin B1 mRNA (D, F), peroxidase mRNA (G, I), cytokinin-specific binding protein mRNA (J, L) and pectin methylesterase mRNA (M, O), with Sm proteins (B, E, H, K, N) in <i>Lupinus</i> cells.

<p>The arrows indicate colocalisation of mRNA transcripts with CBs. A stronger signal was observed in the cytoplasm than in the nuclei after <i>in situ</i> hybridisation with the mixture of probes (A, C) and with a probe against cytokinin-specific binding protein mRNA (J, L). % indicates the percentage of nuclei showing the representative immunolocalisation pattern. Bar, 5 µm. N-nucleus, Nu- nucleolus, C- cytoplasm.</p

Double labelling of poly(A) RNA (A) and the elongation form of RNA polymerase II (B) in <i>Lupinu</i> root cells.

<p>Cajal body (arrow) rich in poly(A) RNA near the nucleolus do not colocalise with RNA polymerase II. Double labelling of a newly formed transcript (E, H) and poly(A) RNA (D, G) in lupine cells. If the transcript does not leave the nucleus, no signal occurs in the CB (arrow) (D–F). In cells in which the newly formed RNA is transported to the cytoplasm, a weak signal is observed in the Cajal body (arrow) (G–I). Bar, 5 µm. Fragment of the nucleus from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111780#pone-0111780-g002" target="_blank">Fig 2I</a> (J). BrU-containing RNA localises at the periphery and in small spots in the middle of CB (J). Bar, 1 µm. The percentage of Cajal bodies rich in poly(A) RNA that colocalise with BrU-containing RNA (K, L). The data for experimental treatments and control were conducted, analyzed and plotted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111780#pone-0111780-g002" target="_blank">Figure 2G–H</a>. A scale bar representing 2 µm is shown. Error bars represent standard error. N- nucleus, Nu- nucleolus, C- cytoplasm.</p

Double labelling of poly(A) RNA and the U2B” protein in <i>Lupinus</i> (A–C) and <i>Allium</i> (D–F) root cells.

<p>Poly(A) RNA present within Cajal bodies stained an anti-U2B” antibody (arrows). Immunogold labelling of poly(A) RNA in the Cajal bodies (CBs) of <i>Lupinus</i> root cells. Gold particles were mainly observed in the Cajal body (CB) (G). Simultaneous localisation of U2B”:GFP and poly(A) RNA in transgenic <i>Arabidopsis thaliana</i> root cells. Strong colocalisation in Cajal bodies (H–J). Localisation of poly(A) RNA with Atcoilin:RFP in <i>Arabidopsis thaliana</i> root cells. Accumulation of poly(A) in Cajal bodies (K–M). Bar, 5 µm. N-nucleus, Nu- nucleolus, C- cytoplasm.</p

Examples of the two types of chromatin morphology.

<p><b>A,B</b>—Feulgen staining. Bars—10 μm; Microsporocyte ultrastructure of contraction stage (<b>C</b>) and diffusion stage (<b>D</b>), anti-DNA localisation at the EM level. Strong and specific labelling was observed within condensed chromatin (<b>C</b>). Dispersed cluster labelling (arrows, <b>D</b>). Gold particles: <b>C</b>—20 nm, <b>D</b>—10 nm. Bars—1 μm (Chr—chromatin; Cyt—cytoplasm; NE—nuclear envelope; Nu—nucleolus). The nuclear and cellular volume during the subsequent diplotene stages <b>(E)</b>.</p

Chromatin morphology and transcriptional activity during microsporocyte development (A—L).

<p>Chromatin morphology detected via DAPI staining during the diffuse (<b>A</b>, <b>G</b>) and contraction (<b>D</b>, <b>J</b>) stages; Newly formed transcripts, detected via BrU incorporation, was performed with a long (90 min) incubation time in diffuse (<b>B</b>, <b>H</b>) and contraction (<b>E</b>, <b>K</b>) stages. <b>C, F</b>, <b>I</b>, <b>L</b>—merge. Bars—10 μm. <b>Total newly formed transcripts contents during microsporocyte development (M)</b>; 1–5—periods of high transcription activity correlated with diffuse stages. <b>Distribution of newly formed transcripts and active polymerase RNA II (N—T)</b>. <b>N—</b>distribution of newly formed transcript during diffuse stages; <b>O—</b>distribution of RNA pol II during diffuse stages using the H5 antibody, which recognises Ser-2-phosphorylated RNA pol II; <b>P—</b>merge. <b>R</b>—distribution of newly formed transcripts during chromatin condensation stages; <b>S</b>—distribution of RNA pol II during chromatin condensation stages (H5 antibody); <b>T</b>—merge. Bars—10 μm.</p

Transcriptional Activity in Diplotene Larch Microsporocytes, with Emphasis on the Diffuse Stage

<div><p>Manuscript provides insights into the biology of long-lived plants, different from Arabidopsis, tomato or grass species that are widely studied. In the European larch the diplotene stage lasts approximately 5 months and it is possible to divide it into several substages and to observe each of them in details. The diplotene stage is a period of intensive microsporocyte growth associated with the synthesis and accumulation of different RNA and proteins. Larch microsporocytes display changes in chromatin morphology during this stage, alternating between 4 short stages of chromatin condensation (contraction) and 5 longer diffusion (relaxation) stages. The occurrence of a diplotene diffusion stage has been observed in many plant species. Interestingly, they have also been observed during spermiogenesis and oogenesis in animals. The aim of this study was to examine whether chromatin relaxation during the diplotene is accompanied by the synthesis and maturation of mRNA. The results reveal a correlation between the diffusion and chromatin decondensation, transcriptional activity. We also found decreasing amount of poly(A) mRNA synthesis in the consecutive diffusion stages. During the early diffusion stages, mRNA is intensively synthesized. In the nuclei large amounts of RNA polymerase II, and high levels of snRNPs were observed. In the late diffusion stages, the synthesized mRNA is not directly subjected to translation but it is stored in the nucleus, and later transported to the cytoplasm and translated. In the last diffusion stage, the level of poly(A) RNA is low, but that of splicing factors is still high. It appears that the mRNA synthesized in early stages is used during the diplotene stage and is not transmitted to dyad and tetrads. In contrast, splicing factors accumulate and are most likely transmitted to the dyad and tetrads, where they are used after the resumption of intense transcription. Similar meiotic process were observed during oogenesis in animals. This indicates the existence of an evolutionarily conserved mechanism of chromatin-based regulation of gene expression during meiotic prophase I.</p></div