Normal Gametophytic Development Research Articles

Mitochondrial ribosomal protein S9M is involved in male gametogenesis and seed development in Arabidopsis.

Mitochondrial function is critical for cell vitality in all eukaryotes including plants. Although plant mitochondria contain many proteins, few have been studied in the context of plant development and physiology. We used knock-down mutant RPS9M to study its important role in male gametogenesis and seed development in Arabidopsis thaliana. Knock-down of RPS9M in the rps9m-3 mutant led to abnormal pollen development and impaired pollen tube growth. In addition, both embryo and endosperm development were affected. Phenotype analysis revealed that the rps9m-3 mutant contained a lower amount of endosperm and nuclear proteins, and both embryo cell division and embryo pattern were affected, resulting in an abnormal and defective embryo. Lowering the level of RPS9M in rps9m-3 affects mitochondrial ribosome biogenesis, energy metabolism and production of ROS. Our data revealed that RPS9M plays important roles in normal gametophyte development and seed formation, possibly by sustaining mitochondrial function.

A pair of nonspecific phospholipases C, NPC2 and NPC6, are involved in gametophyte development and glycerolipid metabolism in Arabidopsis.

Phospholipases play crucial roles in plant membrane lipid homeostasis. Nonspecific phospholipase C (NPCs) establish a unique class of phospholipases found only in plants and certain bacteria. Here, we show that two previously uncharacterized NPC isoforms, NPC2 and NPC6, are required for male and female gametophyte development in Arabidopsis. Double mutant plants of npc2-1 npc6-2 could not be retrieved because npc2-1 npc6-2 ovule and pollen development is affected. Genetic complementation, reciprocal crossing and microscope observation of npc2-1/- npc6-2/+ and npc2-1/+ npc6-2/- plants suggest that NPC2 and NPC6 are redundant and are required for normal gametophyte development. Both NPC2 and NPC6 proteins are localized to the plastids. Promoter-GUS assays in transgenic Arabidopsis revealed that NPC2 and NPC6 are preferentially expressed in floral organs rather than in leaves. In vitro enzyme assays showed that NPC2 and NPC6 hydrolyze phosphatidylcholine and phosphatidylethanolamine, but not phosphatidate, being consistent with the reported substrate selectivity of NPCs. The amounts of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol were increased in buds but not in flowers of npc2-1/- npc6-2/+ and npc2-1/+ npc6-2/- plants, presumably due to reduced phospholipid hydrolysis activity in developing flowers. Our results demonstrate that NPC2 and NPC6 play crucial roles in gametogenesis during flower development.

RWP-RK domain-containing transcription factors control cell differentiation during female gametophyte development in Arabidopsis.

The formation of gametes is a prerequisite for any sexually reproducing organism in order to complete its life cycle. In plants, female gametes are formed in a multicellular tissue, the female gametophyte or embryo sac. Although the events leading to the formation of the female gametophyte have been morphologically characterized, the molecular control of embryo sac development remains elusive. We used single and double mutants as well as cell-specific marker lines to characterize a novel class of gene regulators in Arabidopsis thaliana, the RWP-RK domain-containing (RKD) transcription factors. Morphological and histological analyses were conducted using confocal laser scanning and differential interference contrast microscopy. Gene expression and transcriptome analyses were performed using quantitative reverse transcription-PCR and RNA sequencing, respectively. Our results showed that RKD genes are expressed during distinct stages of embryo sac development. Morphological analysis of the mutants revealed severe distortions in gametophyte polarity and cell differentiation. Transcriptome analysis revealed changes in the expression of several gametophyte-specific gene families (RKD2 and RKD3) and ovule development-specific genes (RKD3), and identified pleiotropic effects on phytohormone pathways (RKD5). Our data provide novel insight into the regulatory control of female gametophyte development. RKDs are involved in the control of cell differentiation and are required for normal gametophytic development.

Characterization of ribulose-1, 5-bisphosphate carboxylase/oxygenase and transcriptional analysis of its related genes in Saccharina japonica (Laminariales, Phaeophyta)

Saccharina japonica is a common macroalga in sublittoral communities of cold seawater environments, and consequently may have highly efficient ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activity for carbon assimilation. In our study, we cloned the full-length Rubisco gene from S. japonica (SJ-rbc). It contained an open reading frame for a large subunit gene (SJ — rbcL) of 1 467 bp, a small subunit gene (SJ-rbcS) of 420 bp, and a SJ-rbcL/S intergenic spacer of 269 bp. The deduced peptides of SJ-rbcL and SJ-rbcS were 488 and 139 amino acids with theoretical molecular weights and isoelectric points of 53.97 kDa, 5.81 and 15.84 kDa, 4.71, respectively. After induction with 1 mmol/L isopropyl-β-D-thiogalactopyranoside for 5 h and purification by Ni2+ affinity chromatography, electrophoresis and western blot detection demonstrated successful expression of the 55 kDa SJ-rbcL protein. Real-time quantitative PCR showed that the mRNA levels of SJ-rbcL in gametophytes increased when transferred into normal growth conditions and exhibited diurnal variations: increased expression during the day but suppressed expression at night. This observation implied that Rubisco played a role in normal gametophytic growth and development. In juvenile sporophytes, mRNA levels of SJ-rbcL, carbonic anhydrase, Calvin-Benson-Bassham cycle-related enzyme, and chloroplast light-harvesting protein were remarkably increased under continuous light irradiance. Similarly, expression of these genes was up-regulated under blue light irradiance at 350 μmol/(m2·s). Our results indicate that long-term white light and short-term blue light irradiance enhances juvenile sporophytic growth by synergistic effects of various photosynthetic elements.

Stress treatments influence efficiency of microspore embryogenesis and green plant regeneration in hexaploid triticale (× Triticosecale Wittmack L.)

Doubled haploid (DH) production is a key technology in plant breeding and research. One emerging method of choice for DH production is microspore culture, which requires reprogramming of the microspores from their normal gametophytic development to a sporophytic development resulting in embryo formation. This commonly requires the application of stress such as cold, heat, or starvation. Here, we report the effect of different stress treatments on embryo formation and the proportion of green plants in triticale microspore culture. We observed different responses to the applied stress treatments among three studied genotypes. In general, a 3-wk cold stress treatment performed best with regard to the two criteria. For one genotype, the application of a 24- or 48-h heat stress gave similar or slightly better results and consequently may be an alternative for genotypes that are recalcitrant to the cold stress treatment.

Photosynthetic performance and fertility are repressed in GmAOX2b antisense soybean.

The alternative oxidase (AOX) is a cyanide-resistant oxidase that provides an alternative outlet for electrons from the respiratory electron transport chain embedded in the inner membrane of plant mitochondria. Examination of soybean (Glycine max) plants carrying a GmAOX2b antisense gene showed AOX to have a central role in reproductive development and fecundity. In three independently transformed antisense lines, seed set was reduced by 16% to 43%, whereas ovule abortion increased by 1.2- to 1.7-fold when compared with nontransgenic transformation control plants. Reduced fecundity was associated with reductions in whole leaf cyanide-resistant, salicylhydroxamic acid-sensitive respiration and net photosynthesis, but there was no change in total respiration in the dark. The frequency of potential fertilization events was reduced by at least one-third in the antisense plants as a likely consequence of prefertilization defects. Pistils of the antisense plants contained a higher proportion of immature-sized, nonfertile embryo sacs compared with nontransgenic control plants. Increased rates of pollen abortion in vivo and reduced rates of pollen germination in vitro suggested that the antisense gene compromised pollen development and function. Reciprocal crosses between antisense and nontransgenic plants revealed that pollen produced by antisense plants was less active in fertilization. Taken together, the results presented here indicate that AOX expression has an important role in determining normal gametophyte development and function.

Development of multicellular pollen of Eriobotrya japonica Lindl. through anther culture

Eriobotrya japonica Lindl. is a worldwide known tree important for its use as horticultural and ornamental plant, especially in sub-tropical and Mediterranean countries. Microspore embryogenesis through in vitro anther culture is a widely used method to generate genetic variability by obtaining gametic or somatic embryos with many applications for plant breeding. In this work, a protocol has been set up for anther culture in loquat which resulted in the formation of multicellular pollen as a first step to further attempt haploid–plant production via pollen-derived structures. The response of nine of the most widely grown loquat cultivars to anther culture has been evaluated, and four cultivars being selected due to their higher response. The occurrence of anther swelling and the development of calli were analyzed as typical morphological features and potential markers that accompany pollen induction and reprogramming in many systems. Microscopical analysis in responsive anthers and the comparison with the normal gametophytic pollen development was carried out to characterize the cellular changes promoted by the treatment in the anther. The presence of multicellular pollen in the in vitro system developed here indicated the switch of developmental programme which constitutes a crucial step in the design of protocols for the regeneration of microspore-derived embryos and plants. Monitoring structural analysis at different times of the culture revealed specific features of the early microspore embryogenic pathway as well as the cellular organization changes.

Microspore‐derived embryogenesis in pepper (Capsicum annuum L.): subcellular rearrangements through development

Background information. In vitro-cultured microspores, after an appropriate stress treatment, can switch towards an embryogenic pathway. This process, known as microspore embryogenesis, is an important tool in plant breeding. Basic studies on this process in economically interesting crops, especially in recalcitrant plants, are very limited and the sequence of events is poorly understood. In situ studies are very convenient for an appropriate dissection of microspore embryogenesis, a process in which a mixture of different cell populations (induced and non-induced) develop asynchronically.Results. In the present study, the occurrence of defined subcellular rearrangements has been investigated during early microspore embryogenesis in pepper, an horticultural crop of agronomic interest, in relation to proliferation and differentiation events. Haploid plants of Capsicum annuum L. (var. Yolo Wonder B) have been regenerated from in vitro anther cultures by a heat treatment at 35 degrees C for 8 days. Morphogenesis of microspore-derived embryos has been analysed, at both light and electron microscopy levels, using low-temperature-processed, well-preserved specimens. The comparison with the normal gametophytic development revealed changes in cell organization after embryogenesis induction, and permitted the characterization of the time sequence of a set of structural events, not previously defined in pepper, related to the activation of proliferative activity and differentiation. These changes mainly affected the plastids, the vacuolar compartment, the cell wall and the nucleus. Further differentiation processes mimicked that of the zygotic development.Conclusions. The reported changes can be considered as markers of the microspore embryogenesis. They have increased the understanding of the mechanisms controlling the switch and progression of the microspore embryogenesis, which could help to improve its efficiency and to direct strategies, especially in agronomically interesting crops.

Mitochondrial gene expression in developing male gametophytes of male-fertile and S male-sterile maize

Mitochondria play a critical role in the normal development of the plant male gametophyte and in the disruption of normal gametophyte development associated with cytoplasmically inherited male sterility (CMS). To investigate the role of mitochondria in these processes, the accumulation of mitochondrial gene transcripts and the accumulation of nuclear gene transcripts encoding mitochondrial proteins were investigated through male gametophyte development in normal maize and through the course of pollen abortion in CMS-S maize. Male gametophytes differing in developmental stage were isolated from male-fertile or male-sterile plants by sucrose density gradient centrifugation. Mature pollen was collected from dehiscent anthers of male-fertile plants. Aborted pollen, which collapsed during starch accumulation, was isolated from emergent tassels of CMS-S male-sterile plants. Microspores, developing pollen and mature pollen exhibited striking differences in mitochondrial transcript accumulation. Mature pollen lacked detectable mitochondrial transcripts. Aborted pollen of CMS-S plants contained abundant, intact transcripts of all mitochondrial genes studied, but prematurely degraded transcripts of several nuclear genes. Transcripts of the CMS-S associated mitochondrial open reading frames (orf355 and orf77) were detected from the early stages of microspore development through the aborted pollen stage. The implications of these findings are discussed in terms of the mitochondrial requirements for pollen function and the mechanism of pollen abortion in CMS-S maize.

Stress‐induced formation of haploid plants through anther culture in cork oak (Quercus suber)

Induction of haploid embryos and regeneration of plantlets have been obtained, for the first time, in cork oak (Quercus suber L.) by combining a starvation treatment in anther culture with a mild heat shock at 33°C for 5 days, followed by culture at 25°C in a simple agar medium without growth regulators. The same conditions had been shown previously to be optimal for embryogenic induction in isolated microspore cultures of several model species such as tobacco and wheat. These results support the notion that stress, particularly sucrose starvation, a heat shock or a combination of both treatments could be the major and general signal responsible for the inhibition of normal gametophytic development of the microspores and for the induction of the alternative embryogenic pathway. A similar approach may be used for the production of haploid and doubled haploids for plant breeding in other species that, like most forest trees, are still recalcitrant in anther culture.

Stress as the major signal controlling the developmental fate of tobacco microspores: towards a unified model of induction of microspore/pollen embryogenesis

Specific stress treatments (sucrose starvation, alone or combined with a heat shock) applied to isolated tobacco (Nicotiana tabacum L.) microspores irreversibly blocked normal gametophytic development and induced the formation of embryogenic cells, which developed subsequently into pollen-derived embryos by culture at 25°C in a sugar-containing medium. A cold shock at 4°C did not inhibit microspore maturation in vitro and did not induce cell division activity, even when combined with a starvation treatment. In the absence of sucrose, microspores isolated in the G1 phase of the cell cycle replicated their DNA and accumulated in G2. Late microspores underwent miotosis during the first day of culture which resulted in a mixed population of bicellular pollen grains and uninucleate microspores, both embryogenic. After the inductive stress treatments the origin of the first multicellular structures, formed in the sugar-containing medium, could be traced to divisions of the microspore cell or divisions of the vegetative cell of bicellular pollen, indicating that the symmetry of microspore mitosis in vitro is not important for embryogenic induction. These results represent a step forward towards a unified model of induction of embryogenesis from microspores/pollen which, within a relatively wide developmental window, are competent to deviate from normal gametophytic development and initiate the alternative sporophytic programme, in response to specific stress signals.

Maintenance of gametophytic development after symmetrical division in tobacco microspore culture

Isolated tobacco (Nicotiana tabacum L.) microspores maturing in vitro can be induced to undergo symmetrical divisions, instead of the normal asymmetrical first pollen mitosis, by addition of anther extracts to the culture medium. The two daughter cells in symmetrically divided pollen resemble vegetative pollen cells in cytological characteristics, nuclear size and chromatin condensation, are separated by a cell wall and remain viable during in vitro maturation. After transfer to a germination medium, only one of the two vegetativelike cells forms a pollen tube in vitro. Therefore, apparently normal gametophytic development can be maintained after symmetrical microspore division. These results are discussed in relation to current models for induction of microspore embryogenesis.

Nuclear DNA synthesis during the induction of embryogenesis in cultured microspores and pollen of Brassica napus L.

The dynamics of nuclear DNA synthesis were analysed in isolated microspores and pollen of Brassica napus that were induced to form embryos. DNA synthesis was visualized by the immunocytochemical labelling of incorporated Bromodeoxyuridine (BrdU), applied continuously or as a pulse during the first 24 h of culture under embryogenic (32 °C) and non-embryogenic (18 °C) conditions. Total DNA content of the nuclei was determined by microspectrophotometry. At the moment of isolation, microspore nuclei and nuclei of generative cells were at the G1, S or G2 phase. Vegetative nuclei of pollen were always in G1 at the onset of culture. When microspores were cultured at 18 °C, they followed the normal gametophytic development; when cultured at 32 °C, they divided symmetrically and became embryogenic or continued gametophytic development. Because the two nuclei of the symmetrically divided microspores were either both labelled with BrdU or not labelled at all, we concluded that microspores are inducible to form embryos from the G1 until the G2 phase. When bicellular pollen were cultured at 18 °C, they exhibited labelling exclusively in generative nuclei. This is comparable to the gametophytic development that occurs in vivo. Early bicellular pollen cultured at 32 °C, however, also exhibited replication in vegetative nuclei. The majority of vegetative nuclei re-entered the cell cycle after 12 h of culture. Replication in the vegetative cells preceded division of the vegetative cell, a prerequisite for pollen-derived embryogenesis.

Expression of myb-related genes in the moss, Physcomitrella patens.

Three cDNA clones encoding proteins containing a myb-related DNA binding domain have been isolated from a cDNA library prepared from protonemal tissue of the moss, Physcomitrella patens. The three cDNA clones between them encode two different classes of myb-like proteins, termed Pp1 and Pp2, that, outside of the myb domain, show no regions of significant homology. Acidic domains, capable of forming alpha-helical structures, are present in the carboxy-termini of the derived amino acid sequences from Pp1 and Pp2cDNAs suggesting that, like other myb genes, these proteins probably function as transcriptional activators. In contrast to other plants, where extensive myb-related gene families are present in the genome, a relatively small family is present in P. patens. Analyses of transcript levels during development of P. patens showed that maximum levels of transcription of the two genes occurred in young wild-type protonemal tissue that correlated with the time of maximum mitotic index. A decline in the expression of both genes occurs with increasing age of the wild-type tissue. Aberrant levels of expression of the two genes were observed in developmental mutants of P. patens which, as well as carrying specific morphological mutations, have greatly retarded protonemal growth rates. Transformation of wild-type P. patens with antisense constructs derived from Pp1 and Pp2 cDNA clones led to a dramatically reduced frequency of transformants when the expression of the reporter gene within the constructs was selected. Taken together, the data strongly suggest that expression of Pp1 and Pp2 is essential for cell growth during normal gametophytic development of P. patens.

Derepression of the cell cycle by starvation is involved in the induction of tobacco pollen embryogenesis

Microspectrophotometry following Feulgen staining and autoradiography following (3H)-thymidine labelling were used to study cell-cycle events during pollen development in tobacco (Nicotiana tabacum L.). During normal gametophytic pollen development in the anther and in vitro the generative nucleus passes through the S phase to the G2 phase soon after microspore mitosis, while the vegetative nucleus remains arrested in G1 (=G0). During embryogenie induction by an in vitro starvation treatment of immature pollen ongoing DNA replication in the generative nucleus is completed and followed by DNA replication in the vegetative cell in a large fraction of the pollen grains. Addition of the DNA replication inhibitor hydroxyurea to the starvation medium postpones S phase entry until the pollen is transferred to a rich medium and does not affect embryo formation. These results demonstrate that one of the crucial events of embryogenic induction is the derepression of the G1 arrest in the cell cycle of the vegetative cell.

THE PHYSICAL AND CHEMICAL ENVIRONMENT OF THE DEVELOPING EMBRYO OF PINUS RESINOSA

The relationship between changes in soluble protein, hexose sugar, total lipid concentration, and osmotic potential occurring in gametophytic supernatant of Pinus resinosa Ait. during in vivo embryogenesis was measured. The effects of varying sucrose levels of culture medium on in vitro embryo and gametophyte development were examined. Increases in embryo volume, and fresh and dry weight of the female gametophyte during in vivo embryogenesis coincide with increasing levels of soluble protein, hexose sugar, and total lipid in the gametophytic supernatant. In contrast, osmotic potential of the supernatant increased only slightly between the zygote and proembryo stages of embryo development, and remained constant thereafter. Gametophytes plus embryos grown in vitro achieved dry weights approaching those of in ovulo gametophytes on media containing levels of sucrose up to 21%. Gametophytes on media with sucrose concentrations up to 21% also resembled normal in ovulo gametophytes in appearance. However, embryo development appeared to be suspended on treatment media containing from 9% to 21% sucrose, while embryos degenerated on media with constant sucrose levels of 3% and 6%. A treatment medium containing approximately 12% sucrose would provide an osmotic environment that duplicates that found in ovulo. While greater sucrose levels promoted more normal gametophyte development in Pinus resinosa, we failed to achieve complete development of the embryo in vitro. Conclusions and implications drawn from these results are discussed.

Male gametophyte development in monosomics of maize

The development of male gametophytes in diploid and monosomic-1, -2, -3, -4, -6, -7, -8, -9, and -10 maize plants was characterized. Developmental differences due to nullisomy in the gametophyte were evaluated by comparing the development of haploid and nullisomic microspores formed by monosomic plants, while differences due to gene dosage in the sporophyte were evaluated by comparing the development of haploid microspores in monosomic plants with those in diploids. These analyses show that (i) male gametophytes nullisomic for the chromosomes analyzed are developmentally delayed and eventually abort; (ii) male gametophytes nullisomic for chromosome 2 or 6 can reach the first mitosis, but those nullisomic for chromosomes 1, 3, 4, 7, 8, 9, or 10 do not reach the first division; and (iii) monosomy of chromosome 1, 2, 3, 4, 6, 7, 8, or 9 interferes with normal male gametophyte development, and monosomic-2 and -9 plants specifically cannot support pollen maturation.Key words: aneuploidy, monosome, nullisome, microspore, r-X1, deficiency.

Growth regulation and development in Physcomitrella patens: an insight into growth regulation and development of bryophytes

Observations made on the effects of auxin, of cytokinin and of various light treatments on cultures of wild-type Physcomitrella patens, grown either on solid medium or in continuously-replaced liquid medium, have allowed us to suggest probable roles for these factors during the early stages of normal gametophyte development. Further data obtained by studying mutants affected in their responses to one or both types of hormones or to light have supported our model of gametophyte development and enabled us to elaborate it.

An Ultrastructural and Stereological Analysis of Pollen Grains of Hyoscyamus niger During Normal Ontogeny and Induced Embryogenic Development

Selected nuclear and cytoplasmic changes of pollen grains of Hyoscyamus niger during normal gametophytic development and embryogenic development, induced by anther culture, were analyzed and compared ultrastructurally using stereological methods. Potentially embryogenic, uninucleate pollen could be identified within 6 hr of culture by an increased ratio of the volume density of the nucleolar granular zone to the volume density of the fibrillar zone and an increased ratio of dispersed to condensed chromatin in the nucleoplasm. Nonembryogenic pollen in vitro and in vivo possessed prominent nucleolar fibrillar zones and low ratios of dispersed to condensed chromatin. These differences may reflect changes in nuclear activity in potentially embryogenic pollen grains during early stages of culture. Following the first haploid mitosis, in potentially embryogenic pollen the generative cell maintained its large granular nucleolus and high ratio of dispersed to condensed chromatin through its first division to form a proembryoid. The volume fraction of the cytoplasm occupied by mitochondria and plastids and the area fraction occupied by RER and Golgi cisternae differed in the generative cells of potentially embryogenic and nonembryogenic pollen. Those changes only detected in generative cells of potentially embryogenic pollen include: increased area and complexity of cytoplasmic membranes, increased mitochondrial volume, and the presence of plastids at all stages of development. These results support the idea that embryogenic induction of H. niger takes place at the uninucleate stage of development and that subsequent nuclear and cytoplasmic changes are essential for continued sporophytic development.

AN ULTRASTRUCTURAL AND STEREOLOGICAL ANALYSIS OF POLLEN GRAINS OF HYOSCYAMUS NIGER DURING NORMAL ONTOGENY AND INDUCED EMBRYOGENIC DEVELOPMENT

Selected nuclear and cytoplasmic changes of pollen grains of Hyoscyamus niger during normal gametophytic development and embryogenic development, induced by anther culture, were analyzed and compared ultrastructurally using stereological methods. Potentially embryogenic, uninucleate pollen could be identified within 6 hr of culture by an increased ratio of the volume density of the nucleolar granular zone to the volume density of the fibrillar zone and an increased ratio of dispersed to condensed chromatin in the nucleoplasm. Nonembryogenic pollen in vitro and in vivo possessed prominent nucleolar fibrillar zones and low ratios of dispersed to condensed chromatin. These differences may reflect changes in nuclear activity in potentially embryogenic pollen grains during early stages of culture. Following the first haploid mitosis, in potentially embryogenic pollen the generative cell maintained its large granular nucleolus and high ratio of dispersed to condensed chromatin through its first division to form a proembryoid. The volume fraction of the cytoplasm occupied by mitochondria and plastids and the area fraction occupied by RER and Golgi cisternae differed in the generative cells of potentially embryogenic and nonembryogenic pollen. Those changes only detected in generative cells of potentially embryogenic pollen include: increased area and complexity of cytoplasmic membranes, increased mitochondrial volume, and the presence of plastids at all stages of development. These results support the idea that embryogenic induction of H. niger takes place at the uninucleate stage of development and that subsequent nuclear and cytoplasmic changes are essential for continued sporophytic development.