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

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

snRNA levels during microsporocyte development.

<p><b>A</b>—mature snRNA (m3G snRNA) levels against background transcriptional activity; <b>B</b>—U2 snRNA levels relative to transcriptional activity; 1–5—diffuse stages. Arrows—contraction stages. <b>snRNA distribution during early and late diffusion diplotene stages. C</b>—distribution of U2 snRNA; <b>D</b>—distribution of m3G snRNA; <b>E</b>—merge. <b>F</b>—m3G snRNA primarily accumulates cytoplasmic clusters (arrows); <b>G</b>—U2 snRNA primarily accumulates in the nucleoplasm, forming numerous large Cajal bodies (CB); <b>H</b>—merge. Bar—10 μm.</p

Poly(A) RNA distribution during all five diffuse stages of diplotene (A—O).

<p>Distribution of newly formed transcripts—BrU incorporation was performed with a long (90 min) incubation time (<b>A</b>, <b>D</b>, <b>G</b>, <b>J</b>, <b>M</b>). Distribution of poly(A) RNA during 5 diffuse stages (FISH with oligo d(T) probe) (<b>B</b>, <b>E</b>, <b>H</b>, <b>K</b>, <b>N</b>). Merge (<b>C</b>, <b>F</b>, <b>I</b>, <b>L</b>, <b>O</b>). Bars—10 μm. <b>Poly(A) RNA levels against background transcriptional activity in larch microsporocytes. P</b>—Poly(A) RNA level. 1st-5th—diffuse stages. <b>R</b>—Colocalisation between poly(A) RNA and newly formed transcripts. Arrows—contraction stages. <b>S—Level of both forms of RNA polymerase II: Pol II A and Pol II O during microsporocyte development</b> detected via immunostaining using the anti-phosph-serine5 CTD antibody (H14) and anti-phosph-serine2 CTD antibody (H5).</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