Post-pollination prefertilization drops affect germination rates of heterospecific pollen in larch and Douglas-fir.

Pollen of larch (Larix × marschlinsii) and Douglas-fir (Pseudotsuga menziesii) was used in homospecific and heterospecific crosses. Germination of heterospecific pollen in ovulo was reduced in post-pollination prefertilization drops. This provides evidence of selection against foreign pollen by open-pollinated exposed ovules in these two sister taxa, which share the same type of pollination mechanism. Of the other prezygotic stages in pollen-ovule interactions, uptake of pollen by stigmatic hairs did not show any selection. Pollen tube penetration of the nucellus was similar for hetero- and homospecific pollen tubes, but heterospecific tubes only delivered gametes in one cross. To test for differences in the post-pollination prefertilization drops of each species, drops were gathered and analysed. Glucose and fructose were present in similar amounts in Douglas-fir and larch, while sucrose was found in larch only. Other carbohydrates such as xylose and melezitose were species-specific. In P. menziesii, sucrose is absent due to its conversion to glucose and fructose by apoplastic invertases. In contrast, Larix × marschlinsii drops have sucrose because they lack apoplastic invertases. The presence of invertase activity shows that the composition of gymnosperm post-pollination prefertilization drops is not static but dynamic. Drops of these two species also differed in their calcium concentrations.

mRNA accumulation in the Cajal bodies of the diplotene larch microsporocyte.

In microsporocytes of the European larch, we demonstrated the presence of several mRNAs in spherical nuclear bodies. In the nuclei of microsporocytes, we observed up to 12 bodies, ranging from 0.5 to 6 µm in diameter, during the prophase of the first meiotic division. Our previous studies revealed the presence of polyadenylated RNA (poly(A) RNA) in these bodies, but did not confirm the presence of nascent transcripts or splicing factors of the SR family. The lack of these molecules precludes the bodies from being the sites of synthesis and early maturation of primary transcripts (Kolowerzo et al., Protoplasma 236:13-19, 2009). However, the bodies serve as sites for the accumulation of splicing machinery, including the Sm proteins and small nuclear RNAs. Characteristic ultrastructures and the molecular composition of the nuclear bodies, which contain poly(A) RNA, are indicative of Cajal bodies (CBs). Here, we demonstrated the presence of several housekeeping gene transcripts--α-tubulin, pectin methylesterase, peroxidase and catalase, ATPase, and inositol-3-phosphate synthase--in CBs. Additionally, we observed transcripts of the RNA polymerase II subunits RPB2 and RPB10 RNA pol II and the core spliceosome proteins mRNA SmD1, SmD2, and SmE. The co-localization of nascent transcripts and mRNAs indicates that mRNA accumulation/storage, particularly in CBs, occurs in the nucleus of microsporocytes.

Additional nucleoli and NOR activity during meiotic prophase I in larch (Larix decidua Mill.).

Transcriptional activity was investigated in successive stages of prophase I (male meiosis) of larch meiocytes. Br-UTP incorporated into RNA was detected by light and electron microscopy. Two peaks of RNA synthesis were identified in the nucleolus. The first occurred during the zygotene-pachytene stage and the second (not previously described in plant meiocytes) in the diplotene. These processes correlated with a considerable increase in nucleolus volume during these periods. At the end of the zygotene, several perinucleolar structures lying close to each other and containing rRNA, argyrophilic proteins, U3 small nucleolar RNA, and fibrillarin were observed. The occurrence of newly formed RNA was also observed in these structures. This suggests that the observed perinucleolar structures correspond to the additional nucleoli known from animals.

Larch (Larix kaempferi) xylem parenchyma cells respond to subfreezing temperature by deep supercooling.

In previous studies, xylem parenchyma cells (XPCs) in the boreal softwood species larch, which has thick and rigid walls similar to those of XPCs in boreal hardwood species, were shown to respond to subfreezing temperature by deep supercooling during summer but change their freezing behavior to extracellular freezing during winter. In this study, we re-examined freezing behavior of XPCs in larch by observation of deep etching of frozen samples as well as observation of re-warmed samples after freezing using a cryo-scanning electron microscope. The results showed that XPCs in larch adapts to subfreezing temperature by deep supercooling throughout all seasons. Such freezing behavior is the same as that of XPCs in boreal hardwood species.

[Characteristics of microsporogenesis in the Siberian larch growing under the conditions of technogenic load].

Studies of microsporogenesis in the Siberian larch growing in Krasnoyarsk and its suburbs have shown that meiosis starts in October. Microsporocytes winter at prophase I (leptoneme, diploneme). Reduction divisions in male generative buds are resumed and terminated in spring, in March. However, in the case of thaws during the autumn-winter period, meiotic division proceeds in the larch buds and this leads to the formation of degrading tetrads and pollen. Hence, the organic quiescence is absent in the larch in winter. It was shown that in the larch growing in the city, meiosis proceed more asynchronously than in the background tree stands. An increase of chromosomal aberrations during the reduction division was noted under the conditions of technogenic pollution.

New type of snRNP containing nuclear bodies in plant cells.

In larch (Larix decidua Mill.) microspores a new type of nuclear bodies has been found which are an element of the spatial organization of the splicing system in plant cell. These are bizonal bodies, ultrastructurally differentiated into a coiled part and a dense part. Using immunocytochemistry and in situ hybridization at the EM level, the coiled part of the bizonal body was found to contain snRNA including U2 snRNA, Sm proteins and nucleolar proteins of the agyrophilic type and fibrillarin. The dense part contains Sm proteins but lacks snRNA. Such a separation of macromolecules related to splicing occurring within the bizonal bodies microspore is striking by the similarity of these bodies to amphibian oocyte snurposomes. The occurrence in plant cells, beside widely known coiled bodies (CBs), also of other nuclear bodies related to splicing proves that in plants similarly as for animals the differentiation among domains containing elements of the splicing system occurs.