Michał Świdziński

Transcriptional state and distribution of poly(A) RNA and RNA polymerase II in differentiating Hyacinthus orientalis L. pollen grains

Spatial distribution of poly(A) RNA, hypophosphorylated Pol IIA, and hyperphosphorylated Pol IIO form of polymerase RNA II was characterized using immunofluorescence, immunogold and fluorescence in situ hybridization (FISH) techniques in relationship to transcriptional activity in the microspore and developing pollen of H. orientalis. During the course of pollen development our results reflected much higher transcriptional activity in the vegetative cell than in the generative cell. The highest levels of transcription in pollen cells were observed in young pollen grains, successively decreasing during pollen maturation, reaching a minimum just before anthesis. Levels of poly(A) RNA were higher in the vegetative cell than in the generative cell during all observed stages of pollen development. Accompanying physiological inhibition of the RNA synthesis in mature pollen cells was a strong accumulation of poly(A) RNA in the cytoplasm, especially in the vegetative cell. Alterations in transcriptional activity of differentiating pollen cells were accompanied by changes in the level and localization pattern of both forms of Pol II. During high transcriptional activity in the pollen nuclei, both forms of RNA Pol II occurred at the periphery of chromatin masses, as well as in the areas between them. A strong decrease in Pol IIO levels was observed in generative and vegetative nuclei as transcriptional activity of pollen cells apparently became inhibited. Finally, just before anthesis, an almost complete lack of the Pol IIO was observed in both pollen nuclei. In contrast, the level of Pol IIA significantly increased during the later stages of pollen development, in spite of apparent transcriptional inhibition in both pollen cells. This rich pool of the hypophosphorylated form of Pol II was located mainly over the central areas of condensed chromatin clumps, which was especially visible in the generative nucleus. Spatial and temporal aspects of RNA synthesis, including poly(A) RNA, as well as organization of transcriptional machinery appear to be closely related in developing pollen cells.

Transcriptional activity and distribution of splicing machinery elements during Hyacinthus orientalis pollen tube growth

Summary.The localization of newly formed transcripts and molecules participating in pre-mRNA splicing, i.e., small nuclear ribonucleoproteins (snRNPs) and SC35 protein, in growing pollen tubes of Hyacinthus orientalis L. were analyzed in vitro and in vivo. The results indicated that the restart of RNA synthesis occurred first in the vegetative and then in the generative nucleus of both in vitro and in vivo growing pollen tubes. Changes in RNA synthesis were accompanied by the redistribution of splicing machinery elements in both vegetative and generative nuclei of the growing pollen tube. At stages of pollen tube growth when the vegetative and generative nuclei were transcriptionally active, clear differences in the distribution pattern of the splicing system components were observed in both pollen nuclei. While both small nuclear RNA with a trimethylguanosine cap on the 5′ end and SC35 protein were diffusely distributed in the nucleoplasm in the vegetative nucleus, the studied antigens were only present in the areas between condensed chromatin in the generative nucleus. When the transcriptional activity of both pollen nuclei could no longer be observed at later stages of pollen tube growth, snRNPs and SC35 protein were still present in the vegetative nuclei but not in the generative nuclei. We, therefore, investigated potential differences in the spatial organization of splicing system elements during pollen tube growth. They clearly reflect differences in gene expression patterns in the vegetative and the generative cells, which may be determined by the different biological roles of angiosperm male gametophyte cells.

Intracellular organization of the pre-mRNA splicing machinery during Hyacinthus orientalis L. pollen development

Spatial organization of splicing machinery elements in metabolically and functionally different pollen cells during Hyacinthus orientalis pollen grain development was examined by localization of trimethylguanosine (TMG) snRNA and Sm proteins, representing splicing small nuclear ribonucleoproteins, as well as SR splicing factors was investigated. In young pollen grains the level of all labeled antigens was the highest displaying essentially uniform distribution in the vegetative and generative nucleus. In the polarized microspore, as well as in the vegetative cell of the young pollen grain, both TMG snRNA and Sm proteins were also found highly concentrated in Cajal bodies. After detachment of the generative cell from the sporoderm, the redistribution of splicing machinery elements into speckled-shape clusters was observed in both nuclei in the pollen. In the mature pollen grain, labeled antigens were still present, both in the vegetative and the generative nucleus. The results reflected that in differentiating H. orientalis pollen cells, the intracellular organization pre-mRNA splicing machinery undergoes significant and characteristic changes during the course of pollen grain development. Changes in the distribution of spliceosomal components relate to the transcriptional activity of both pollen cells during their maturation.

Homogalacturonan deesterification during pollen–ovule interaction in Larix decidua Mill.: an immunocytochemical study

Studies on angiosperm plants have shown that homogalacturonan present in the extracellular matrix of pistils plays an important role in the interaction with the male gametophyte. However, in gymnosperms, knowledge on the participation of HG in the pollen–ovule interaction is limited, and only a few studies on male gametophytes have been reported. Thus, the aim of this study was to determine the distribution of HG in male gametophytes and ovules during their interaction in Larix decidua Mill. The distribution of HG in pollen grains and unpollinated and pollinated ovules was investigated by immunofluorescence techniques using monoclonal antibodies that recognise high methyl-esterified HG (JIM7), low methyl-esterified HG (JIM5) and calcium cross-linked HG (2F4). All studied categories of HG were detected in the ovule. Highly methyl-esterified HG was present in the cell walls of all cells throughout the interaction; however, the distribution of low methyl-esterified and calcium cross-linked HG changed during the course of interaction. Both of these categories of HG appeared only in the apoplast and the extracellular matrix of the ovule tissues, which interact with the male gametophyte. This finding suggests that in L. decidua, low methyl-esterified and calcium cross-linked HG play an important role in pollen–ovule interaction. The last category of HG is most likely involved in adhesion between the pollen and the ovule and might provide an optimal calcium environment for pollen grain germination and pollen tube growth.

The influence of abscisic acid on the ethylene biosynthesis pathway in the functioning of the flower abscission zone in Lupinus luteus.

Flower abscission is a highly regulated developmental process activated in response to exogenous (e.g. changing environmental conditions) and endogenous stimuli (e.g. phytohormones). Ethylene (ET) and abscisic acid (ABA) are very effective stimulators of flower abortion in Lupinus luteus, which is a widely cultivated species in Poland, Australia and Mediterranean countries. In this paper, we show that artificial activation of abscission by flower removal caused an accumulation of ABA in the abscission zone (AZ). Moreover, the blocking of that phytohormones biosynthesis by NDGA (nordihydroguaiaretic acid) decreased the number of abscised flowers. However, the application of NBD - an inhibitor of ET action - reversed the stimulatory effect of ABA on flower abscission, indicating that ABA itself is not sufficient to turn on the organ separation. Our analysis revealed that exogenous ABA significantly accelerated the transcriptional activity of the ET biosynthesis genes ACC synthase (LlACS) and oxidase (LlACO), and moreover, strongly increased the level of 1-aminocyclopropane-1-carboxylic acid (ACC) - ET precursor, which was specifically localized within AZ cells. We cannot exclude the possibility that ABA mediates flower abscission processes by enhancing the ET biosynthesis rate. The findings of our study will contribute to the overall basic knowledge on the phytohormone-regulated generative organs abscission in L. luteus.

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

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.

Methyl jasmonate-dependent senescence of cotyledons in Ipomoea nil

Jasmonic acid methyl ester (JAMe) has been recently shown to play a crucial role in many physiological processes. In this paper, we focused on cotyledon senescence in Ipomoea nil and revealed that JAMe and darkness are the main factors stimulating the process examined. What is more, we showed that mefenamic acid (a jasmonate biosynthesis inhibitor) reverses the stimulatory effect of darkness on senescence. In plants growing under dark conditions, stimulation of JASMONIC ACID CARBOXYL METHYLTRANSFERASE (InJMT) expression and, consequently, an increase in JAMe content, have been observed. In turn, the level of jasmonic acid (JA) gradually decreased. Moreover, dark-grown seedlings demonstrated a lower PSII functional activity and a reduced chlorophyll content and autofluorescence. All of these data suggest that JAMe is a signal molecule controlling the senescence of cotyledons in I. nil.

Photoperiodic flower induction in Ipomoea nil is accompanied by decreasing content of gibberellins

The involvement of gibberellins (GAs) in the control of flower induction in the short-day plant Ipomoea nil has been investigated. To clarify the molecular basis of this process, we identified the full-length cDNAs of the InGA20ox3 and InGA2ox1 genes, which encode enzymes responsible for GA biosynthesis and catabolism, respectively. We studied the expression patterns of both genes and determined the tissue and cellular immunolocalisation of gibberellic acid (GA3) in the cotyledons of 5-day-old seedlings growing under inductive and non-inductive photoperiodic conditions. In the second half of the inductive night, which is crucial for flower induction in I. nil, InGA20ox3 expression decreased, whereas InGA2ox1 mRNA accumulated, which indicates that photoperiod regulates the activity of both genes. Furthermore, these changes are correlated with GA3 level. Thus, our results support the thesis that the proper balance between the expression of the InGA20ox3 and InGA2ox1 genes and low GA3 content correlate with photoperiodic flower induction in I. nil.

Gibberellic acid affects the functioning of the flower abscission zone in Lupinus luteus via cooperation with the ethylene precursor independently of abscisic acid

The abscission of plant organs is a phytohormone-controlled process. Our study provides new insight into the involvement of gibberellic acid (GA3) in the functioning of the flower abscission zone (AZ) in yellow lupine (Lupinus luteus L.). Physiological studies demonstrated that GA3 stimulated flower abortion. Additionally, this phytohormone was abundantly presented in the AZ cells of naturally abscised flowers, especially in vascular bundles. Interesting interactions among GA3 and other modulators of flower separation were also investigated. GA3 accumulated after treatment with the ethylene (ET) precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Abscisic acid (ABA) treatment did not cause such an effect. Furthermore, the expression of the newly identified LlGA20ox1 and LlGA2ox1 genes encoding 2-oxoglutarate-dependent dioxygenases fluctuated after ACC or ABA treatment which confirmed the existence of regulatory crosstalk. GA3 appears to cooperate with the ET precursor in the regulation of AZ function in L. luteus flowers; however, the presented mechanism is ABA-independent.