Callose Wall Formation Research Articles

Identification of Candidate Genes Involved in the Determinism of Pollen Grain Aperture Morphology by Comparative Transcriptome Analysis in Papaveraceae.

In the last decade, certain genes involved in pollen aperture formation have been discovered. However, those involved in pollen aperture shape remain largely unknown. In Arabidopsis, the interaction during the tetrad development stage of one member of the ELMOD protein family, ELMOD_E, with two others, MCR/ELMOD_B and ELMOD_A, can change the morphology of apertures from colpus (elongated) to pore (round). Here, comparative transcriptome analysis is used to identify candidate genes involved in the determination of pollen aperture morphology in Papaveraceae (order Ranunculales). Furthermore, the role of ELMOD genes in the genetic determinism of aperture shape was tested by comparative analysis of their expression levels using RNA-seq data and RT-qPCR. Two pairs of species belonging to two different subfamilies were used. Within each pair, one species has colpate pollen and the other porate (Fumarioideae-Dactylicapnos torulosa, 6-colpate, and Fumaria bracteosa, pantoporate; Papaveroideae-Eschsholzia californica, 5-7 colpate, and Roemeria refracta, 6-porate). The transcriptomes were obtained at the tetrad stage of pollen development. A total of 531 DEGs were found between the colpate and porate pollen species groups. The results from RNA-seq and RT-qPCR indicate that pollen aperture shape is not determined by the relative expression levels of ELMOD family genes in Papaveraceae. However, genes related to callose wall formation or cytoskeleton organisation were found, these processes being involved in pollen aperture formation. In addition, transcriptomes from anthers with pollen during the tetrad stage of three species (D. torulosa, R. refracta, and F. bracteosa) were obtained for the first time. These data will be available for further studies in the field of floral evolution and development.

Cytosolic phosphoglucose isomerase is essential for microsporogenesis and embryogenesis in Arabidopsis.

Phosphoglucose isomerase (PGI) catalyzes the interconversion of fructose-6-phosphate and glucose-6-phosphate, which impacts cell carbon metabolic flow. Arabidopsis (Arabidopsis thaliana) contains two nuclear PGI genes respectively encoding plastidial PGI1 and cytosolic PGI (cPGI). The loss of PGI1 impairs the conversion of F6P of the Calvin-Benson cycle to G6P for the synthesis of transitory starch in leaf chloroplasts. Since cpgi knockout mutants have not yet been obtained, they are thought to be lethal. The cpgi lethality can be rescued by expressing CaMV 35S promoter (p35S)-driven cPGI; however, the complemented line is completely sterile due to pollen degeneration. Here, we generated a cpgi mutant expressing p35S::cPGI-YFP in which YFP fluorescence in developing anthers was undetectable specifically in the tapetum and in pollen, which could be associated with male sterility. We also generated RNAi-cPGI knockdown lines with strong cPGI repression in floral buds that exhibited reduced male fertility due to the degeneration of most pollen. Histological analyses indicated that the synthesis of intersporal callose walls was impaired, causing microsporocytes to fail to separate haploid daughter nuclei to form tetrads, which might be responsible for subsequent pollen degeneration. We successfully isolated cpgi knockout mutants in the progeny of a heterozygous cpgi mutant floral-dipped with sugar solutions. The rescued cpgi mutants exhibited diminished young vegetative growth, reduced female fertility, and impaired intersporal callose wall formation in a meiocyte, and, thus, male sterility. Collectively, our data suggest that cPGI plays a vital role in carbohydrate partitioning, which is indispensable for microsporogenesis and early embryogenesis.

Cytological abnormalities during pollen development in interspecific hybrids of Nicotiana

Nicotiana alata is resistant to Tomato spotted wilt virus (TSWV) and of great value in breeding. However, hybrid sterility constrains the application of interspecific genetic resources. Previously, we obtained interspecific hybrids between a cytoplasmic male sterility (CMS) line of Nicotiana tabacum and N. alata, some of which were pollen sterile. In the present research, we studied the cytological abnormalities during pollen development in sterile hybrids (F1-D) by comparing pollen development with that in fertile hybrids (F1-S) from the same cross. Transmission electron microscopy and DiI staining showed that the membrane structures of microspores and pollen in F1-D sterile hybrids were impaired. Carbol fuchsin staining revealed that cytomixis, chromosome loss and asymmetric callose wall formation occurred with high frequency in the microsporocytes and microspores of the sterile hybrids. The cytoplasm and nucleus were lost in the microspores and pollen of sterile hybrids, leading to mature pollen grains that were vacuous and collapsed in the aperture region. In addition, delayed tapetum degradation was detected in the anther of sterile hybrids, and sporopollenin was deposited in the aperture region. Impaired membrane structures of microspores and pollen in F1-D sterile hybrids affected the integrity of the cells, and might be associated with chromosome, nuclear and cytoplasm loss, vacuous pollen, and sterility in F1-D hybrids. Abnormal tapetum degradation in the anther and irregular sporopollenin deposition in the pollen wall of the F1-D sterile hybrids might also be related to the pollen sterility. This study deepens our understanding of the cytological mechanisms of hybrid sterility, and may facilitate the application of TSWV-resistant resources in cultivated Nicotiana species through hybrid fertility restoration and backcross breeding.

Pollen Aperture Factor INP1 Acts Late in Aperture Formation by Excluding Specific Membrane Domains from Exine Deposition.

Accurate placement of extracellular materials is a critical part of cellular development. To study how cells achieve this accuracy, we use formation of pollen apertures as a model. In Arabidopsis (Arabidopsis thaliana), three regions on the pollen surface lack deposition of pollen wall exine and develop into apertures. In developing pollen, Arabidopsis INAPERTURATE POLLEN1 (INP1) protein acts as a marker for the preaperture domains, assembling there into three punctate lines. To understand the mechanism of aperture formation, we studied the dynamics of INP1 expression and localization and its relationship with the membrane domains at which it assembles. We found that INP1 assembly occurs after meiotic cytokinesis at the interface between the plasma membrane and the overlying callose wall, and requires the normal callose wall formation. Sites of INP1 localization coincide with positions of protruding membrane ridges in proximity to the callose wall. Our data suggest that INP1 is a late-acting factor involved in keeping specific membrane domains next to the callose wall to prevent formation of exine at these sites.

Anther and pollen development in the lodgepole pine dwarf mistletoe (Arceuthobium americanum) staminate flower

The lodgepole pine dwarf mistletoe, Arceuthobium americanum Nutt. ex Engelm., is a parasitic angiosperm that infects conifers in western Canadian forests. While production of viable pollen in anthers is critical to dwarf mistletoe reproduction, the few existing reports that examine staminate development in Arceuthobium are often incomplete or conflicting. The objective of this work was to investigate the developmental anatomy of anther and pollen of A. americanum using modern microscopy. We found that the microsporangium was toroidal from the outset and gave rise to a central peg-shaped sterile “columella” early in anther development. The endothecium was absent, the epidermis persisted as an “exothecium” fulfilling the role of the endothecium, and a primary parietal layer generated a secretory tapetum and middle layer. Thus, we suggest that a new category of anther wall development, the Arceuthobium type, be created. Microsporogenesis produced tetrahedral microspores via simultaneous cytokinesis and involved callose wall formation. Microgametogenesis resulted in round and atypical generative and vegetative nuclei. Additionally, the heterocolpate, echinate pollen grains, which were shed at the two-celled stage, were seen for the first time in their native state with environmental scanning electron microscopy. This work contributes to the understanding of A. americanum, the genus Arceuthobium, and angiosperms as a whole.

Genetic Regulation of Sporopollenin Synthesis and Pollen Exine Development

Pollen acts as a biological protector of male sperm and is covered by an outer cell wall polymer called the exine, which consists of durable sporopollenin. Despite the astonishingly divergent structure of the exine across taxa, the developmental processes of its formation surprisingly do not vary, which suggests the preservation of a common molecular mechanism. The precise molecular mechanisms underlying pollen exine patterning remain highly elusive, but they appear to be dependent on at least three major developmental processes: primexine formation, callose wall formation, and sporopollenin synthesis. Several lines of evidence suggest that the sporopollenin is built up via catalytic enzyme reactions in the tapetum, and both the primexine and callose wall provide an efficient substructure for sporopollenin deposition. Herein, we review the currently accepted understanding of the molecular regulation of sporopollenin biosynthesis and examine unanswered questions regarding the requirements underpinning proper exine pattern formation, as based on genetic evidence.

The influence of tetrad shape and intersporal callose wall formation on pollen aperture pattern ontogeny in two eudicot species

In flowering plants, microsporogenesis is accompanied by various types of cytoplasmic partitioning (cytokinesis). Patterns of male cytokinesis are suspected to play a role in the diversity of aperture patterns found in pollen grains of angiosperms. The relationships between intersporal wall formation, tetrad shape and pollen aperture pattern ontogeny are studied. A comparative analysis of meiosis and aperture distribution was performed within tetrads in two triporate eudicot species with contrasting aperture arrangements within their tetrads [Epilobium roseum (Onagraceae) and Paranomus reflexus (Proteaceae)]. Intersporal wall formation is a two-step process in both species. Cytokinesis is first achieved by the formation of naked centripetal cell plates. These naked cell plates are then covered by additional thick, localized callose deposits that differ in location between the two species. Apertures are finally formed in areas in which additional callose is deposited on the cell plates. The recorded variation in tetrad shape is correlated with variations in aperture pattern, demonstrating the role of cell partitioning in aperture pattern ontogeny.

Successive microsporogenesis in eudicots, with particular reference to Berberidaceae (Ranunculales)

The eudicot clade of angiosperms is characterised by simultaneous microsporogenesis and tricolpate pollen apertures. Successive microsporogenesis, where a distinct dyad stage occurs after the first meiotic division, is relatively rare in eudicots although it occurs in many early branching angiosperms including monocots. An extensive literature survey shows that successive microsporogenesis has arisen independently at least six times in eudicots, in five different orders, including Berberidaceae (Ranunculales). Microsporogenesis and pollen apertures were examined here using light and transmission electron microscopy in eleven species representing six genera of Berberidaceae. Successive microsporogenesis is a synapomorphy for the sister taxa Berberis and Mahonia (and possibly also Ranzania), the remaining genera are simultaneous. Callose wall formation in Berberis and Mahonia is achieved by centripetal furrowing, though centrifugal cell plates are more usual for this microsporogenesis type. This discrepancy could reflect the fact that the successive type in Berberidaceae is derived from the simultaneous type, and centripetal furrowing has been retained. Eudicots with successive microsporogenesis usually produce tetragonal or decussate tetrads, though occasional tetrahedral or irregular tetrads in Berberis and Mahonia indicate that the switch from simultaneous to successive division is incomplete or “leaky”. In contrast, linear tetrads produced by successive microsporogenesis in Asclepiadoideae (Apocynaceae s.l.) are the result of a highly specialised developmental pathway leading to the production of pollinia. Pollen in successive eudicots is dispersed as monads, dyads, tetrads, and as single grains in pollinia. Apertures are diverse, and patterns include spiraperturate, clypeate, irregular, monocolpate, diporate and inaperturate. It is possible that successive microsporogenesis, although rare, potentially occurs in other eudicots, for example, in species where pollen is inaperturate.

Fluorochromes for Detection of Callose in Meiocytes of Olive (Olea europaea L)

The callosic wall which covers microsporocyte mother cells during meiotic division has been studied using different fluorochromes as alternatives to the widely used aniline blue. We have confirmed that both acridine orange and 4′, 6′ diamidino-2-phenylindole (DAPI) produce a fluorescent response to callose which is comparable in specificity and intensity to that of aniline blue: therefore, they can be used to study callose wall formation. Staining properties of these fluorochromes, as well of those of curcumin and sirofluor, reported earlier as fluorescent stains for callose, are discussed. We also discuss the efficacy of the combined use of sirofluor and DAPI to study particular aspects of the deposition of callose.

Localization of Ca ++ ions and peroxidase activity in the zone of callose wall synthesis in dyads and tetrads of Larix decidua Mill

The localization of Ca ++ ions and peroxidase activity were studied cytochemically during succesive phases of callose synthesis in the cells of dyads and tetrads of Larix decidua. It has been found that at the time preceding the formation of callose wall separating the dyad and tetrad cells there occurs an increase in peroxidase activity and an accumulation of Ca ++ ions in zone of callose wall formation. These results suggest a physiological part of these factors in callose synthesis.

Differentiation of the tapetum in Avena. I. The cell surface.

The development of the tapetal cell surface and associated structures in Avena has been followed from cell formation to senescence. Plasmodesmata initially connect the tapetal cells to each other, the pollen mother cells, and the inner loculus wall cells. These connexions are subsequently severed, those to the sporogenous cells being broken first at the pollen mother cell surface during callose wall formation. Loss of cellulose from the tapetal walls was followed using the decline in the ability of the wall to bind the fluorescent brightener, Calcofluor White M2R New. Subplasma-membrane microtubules persist after loss of the cellulose wall. The tapetal plasma membrane facing the meiocytes then develops a series of depressions, or cups, over its surface, which are later the site of pro-orbicule formation. Sporopollenin is laid down over the pro-orbicules, to form orbicules, and over other tapetal cell surfaces. No morphological evidence was found for the intracytoplasmic formation of pro-orbicules or polymerized sporopollenin precursors. These observations on Avena are compared with those on other plants. The changes in the cell wall and associated structures, plasmodesmata and microtubules, are considered in detail, while the general significance of cell wall loss to the water relations of the tissue are assessed. Proposals that pro-orbicule formation results from non-specific accumulation of lipid at a free cell surface are rejected, instead this formation is considered to be related to the presence of a specially modified plasmamembrane surface.

Different patterns of callose wall formation during megasporogenesis in two species ofOenothera (Onagraceae)

The homozygousOenothera hookeri Torr. etGray shows the typical pattern ofOnagraceae with ± callose on the external walls of megaspore mother cells and tetrads. Megasporogenesis is heteropolar, and the micropylar megaspore is the mother-cell of the 4-celled embryo sac. The complex-heterozygousOenothera biennis L. during megasporogenesis generally has callose not on the external cell walls but only on the transversal walls of the tetrad. In 95% of the ovules both the external chalazal and the micropylar megaspores develop to embryo sac mother-cells. Megasporogenesis is homopolar, and competition between two developing embryo sacs for nutrition in the ovule occurs. The embryo sac with the “stronger” genotype wins the race against the other one. Polarity phenomena during ontogeny of the female gametophyte are related to nutritional supply and hormonal induction from the ovule. The introduction of a developmental-physiological point of view into the discussion about the evolution of the embryo sac inOnagraceae is therefore justified.

MORPHOLOGICAL AND QUANTITATIVE CHANGES IN THE POPULATION OF CELL ORGANELLES DURING MICROSPOROGENESIS OF PINUS SYLVESTRIS L. III. MORPHOLOGICAL CHANGES DURING THE TETRAD STAGE AND IN THE YOUNG MICROSPORE. A QUANTITATIVE APPROACH TO THE CHANGES IN THE POPUL

SUMMARY A description and discussion are given of the morphological changes of the cell organelles during the middle and late tetrad stage and the young microspore of Pinus sylvestris with special reference to the pollen wall formation. In the middle tetrad stage the template of the pollen wall pattern appears to be the result of Golgi material excretion and callose wall formation. The fine fibrillar material from the content of the Golgi vesicles, consisting partly of a polysaccharide, forms the primesexine. During the late tetrad stage the sporopollenin originates from the microspore and is deposited on membranes mainly outside the cell. It penetrates into the primesexine. The starch in the plastids disappears and the lipid granules become voluminous; both elements are related to the pollen wall formation. The swelling of the sacci may be an osmotic process. From the quantitative approach it appears, that the young microspore contains approximately one fourth of the number of cell organelles present in the cell during zygotene. The cell organelle population probably does not change in number during the meiotic stages. A general survey summarizes the whole process of microsporogenesis till the young microspore stage. The control of pollen wall formation is also discussed.

MORPHOLOGICAL AND QUANTITATIVE CHANGES IN THE POPULATION OF CELL ORGANELLES DURING MICROSPOROGENESIS OF PINUS SYLVESTRIS L. II. MORPHOLOGICAL CHANGES FROM PROMETAPHASE I UNTIL THE TETRAD STAGE

SUMMARY A morphological study was made with respect to the changes during microsporogenesis of Pinus sylvestris L. from prometaphase I until the tetrad stage. Described and discussed are the breakdown and rebuilding of the nuclear membrane, the mixing of karyoplasm and cytoplasm with the transfer of ribosomal material, the nuclear invaginations, the kinetochore and microtubuli. A decrease of starch in the plastids starts in interphase II. The mitochondria do not change. The “lipid-complex” appears in telophase II. Many vesicles are present in the cytoplasm during the cell divisions. Ribosomes are observed in all stages, polysomes become visible during interphase II and telophase II. Rough endoplasmic reticulum is absent. The Golgi body produces small and large vesicles and long flat cisternae, similar to smooth endoplasmic reticulum. These Golgi vesicles fuse and form the primary cell wall. The presence of the plasma membrane is postulated as necessary for the synthesis of callose. No difference has been found between the morphological events during callose wall formation and cellulose wall formation. A relation may exist between the callose wall formation and the polysomes and lipid granules and “lipid complex”, but a relation with smooth endoplasmic reticulum is not clear. During diplotene and early tetrad stage the formation of the callose wall proceeds in the same way.

MORPHOLOGICAL AND QUANTITATIVE CHANGES IN THE POPULATION OF CELL ORGANELLES DURING MICROSPOROGENESIS OF PINUS SYLVESTRIS L. I. MORPHOLOGICAL CHANGES FROM ZYGOTENE UNTIL PROMETAPHASE I

SUMMARY The membrane-like structures in the nucleus, the nuclear pore, the disappearance of the nuclear membrane, the synaptinemal complex, and changes in the nucleolus and karyoplasm are described and discussed. Plastids and mitochondria do not change during the microsporogenesis. Until diplotene the lipid granules with electron transparent vesicles are arranged into a “lipid complex”. During pachytene rough endoplasmic reticulum changes into smooth endoplasmic reticulum. During diplotene numerous Golgi bodies are present. Microtubules are present from zygotene and scattered in the cytoplasm. At diakinesis many vesicles appear. Ribosomes as well as polysomes are present. From diakinesis only ribosomes are observed. Callose wall formation starts in diplotene and is connected with the presence of many smooth endoplasmic reticulum cisternae and Golgi vesicles, probably also with polysomes and lipid granules.