Mitogen-activated protein kinases are developmentally regulated during stress-induced microspore embryogenesis in Brassica napus L

Plant mitogen-activated protein kinase (MAPK) cascades are involved in extracellular stress signalling pathways, leading to different cellular responses. Stress-induced microspore embryogenesis involves the internalization of an extracellular stress signal, generating a number of cellular responses where MAPK cascades might be involved. These responses include a change of the developmental programme, the entry into an early proliferative stage and, subsequently, into differentiation stages during haploid embryogenesis. In this work we studied the expression during microspore embryogenesis of several kinases, to assess their putative role in these events. The known Brassica napus MAP kinase kinase kinases (MAP3Ks BnMAP3K1, BnMAP3K1 and BnMAP3K, the BnBSK kinase and B. napus extracellular signal-regulated kinase (ERK) homologues were analysed by electron microscope (EM) in situ hybridization, immuno-gold labelling, immunofluorescence and western blotting. The differential in situ expression of these kinases suggests a role for them during embryogenesis. Two different expression patterns were observed, indicating a different regulation. BnMAP3K1, BnMAP3K, and the ERKs showed a pattern consistent with a role mainly in proliferative events. Conversely, BnMAP3K1 and BnBSK, presented a pattern that suggested an involvement in differentiation stages. In addition, ERK homologues migrate to the nucleus immediately after induction, being found in a phosphorylated state in a larger amount.     

Cytoskeletal changes and induction of embryogenesis in microspore and pollen cultures of Brassica napus L.

Microspores and pollen of Brassica napus were cultured under conditions leading to embryo formation. Concomitant changes in cytoskeletal configurations were analysed. The microfilamental cytoskeleton exhibited a loss of polarity in embryogenic cells but cytochalasin treatment revealed that microfilaments do not influence embryogenesis. Two embryogenic pathways started from microspores and were either characterized by turned division planes or by division when the nucleus was in the cell centre. In both cases microtubules clearly exhibited new arrangements and likely played a major role in newly induced symmetrical division. In pollen, embryogenic development started in the vegetative cell provided the generative cell was arrested near the pollen wall. The concomitant disappearance of defined microtubular arrays is likely to be responsible for the positioning of the cell.     

Intra- and extracellular lipid composition and associated gene expression patterns during pollen development in Brassica napus

Pollen development in angiosperms is regulated by the interaction of products contributed by both the gametophytic (haploid) and sporophytic (diploid) genomes. In entomophilous species, lipids are major products of both sporophytic and gametophytic metabolism during pollen development. Mature pollen grains of Brassica napus are shown to contain three major acyl lipid pools as follows: (i) the extracellular tryphine mainly consisting of medium-chain neutral esters; (ii) the intracellular membranes, particularly endoplasmic reticulum, mainly containing phospholipids; and (iii) the intracellular storage lipids, which are mostly triacylglycerols. This paper reports on the kinetics of accumulation of these lipid classes during pollen maturation and the expression patterns of several lipid biosynthetic genes and their protein products that are differentially regulated in developing microspores/ pollen grains (gametophyte) and tapetal cells (sporophyte) of B. napus. Detailed analysis of three members of the stearoyl-ACP desaturase (sad) gene family by Northern blotting, in situ hybridization and RT-PCR showed that the same individual genes were expressed both in gametophytic and sporophytic tissues, although under different temporal regulation. In the tapetum, maximal expression of two marker genes for lipid biosynthesis (sad and ear) occurred at a bud length of 2–3 mm, and the corresponding gene products SAD and EAR were detected by Western blotting in 3–4 mm buds, coinciding with the maximal rates of tapetal lipid accumulation. These lipids are released following tapetal cell disintegration and are relocated to form the major structural component of the extracellular tryphine layer that coats the mature pollen grain. In contrast, in developing microspores/pollen grains, maximal expression of the lipid marker genes sad, ear, acp and cyb5 was at the 3–5 mm bud stages, with the SAD and EAR gene products detected in 4–7 mm buds. This pattern of expression coincided with accumulation of the intracellular storage and membrane lipid components of pollen. These results suggest that, although the same genes may be expressed in the sporophytic tapetal cells and in gametophytic tissues, they are regulated differentially leading to the production of the various contrasting lipidic structures that are assembled together to give rise to a viable, fertile pollen grain.     

Nuclear pore dynamics during pollen development and androgenesis in Brassica napus

Changes in nuclear pore complex (NPC) densities, NPCs/nucleus and NPCs/μm³, are described using freeze-fractured Brassica napus microspores and pollen in vivo and in vitro. Early stages of microspore- and pollen-derived embryogenic cells were also analysed. The results of in vivo and in vitro pollen development indicate an increase in activity of the vegetative nucleus during maturation of the pollen. At the onset of microspore and pollen culture, NPC density decreased from 15 NPCs/μm² at the stage of isolation to 9 NPCs/μm², under both embryogenic and non-embryogenic conditions. This implies that the drop in NPC density might be a result of culturing the microspores and pollen rather than an indication for microspore and pollen embryogenesis in Brassica napus. However, after 1 day in culture under embryogenic conditions, the NPC density increased again and stabilised around 13 NPCs/μm², whereas under non-embryogenic conditions the NPC density remained about 9 NPCs/μm². This low density of 9 NPCs/μm² was also found in the nuclei of sperm cells, in contrast to the 19 NPCs/μm² found in the vegetative nucleus. It means that, although both the vegetative and sperm nuclei are believed to be metabolically rather inactive in mature pollen, the NPC density of vegetative nucleus is twice as high as the NPC density of the sperm nuclei. In a few cases, embryos formed suspensor-like structures with a NPC density of 9 NPCs/μm², indicating a lower nucleocytoplasmic exchange of the nuclei of the suspensor cells than with the nuclei in the embryo proper. In addition, observations on NPCs and other organelles, obtained by high resolution cryo-scanning microscopy, are presented. Received: 29 December 1999 / Revision accepted: 3 March 2000     

Induction of microspore embryogenesis in Brassica napus L. is accompanied by specific changes in protein synthesis

Culture temperature determines the developmental fate of isolated microspores from Brassica napus L. At 18°C, tricellular pollen develops, whereas culture at 32°C for 8 h leads to the quantitative and synchronous induction of embryogenesis, and ultimately to the formation of embryos. We investigated the changes in protein synthesis that are associated with this 8-h inductive period by using in-situ [³⁵S]methionine labeling, followed by two-dimensional (2-D) gel electrophoretic analysis of the radiolabeled proteins. Qualitative and quantitative computer analyses of 2-D [³⁵S]methionine protein patterns showed six polypeptides specifically labeled under embryogenic culture conditions. Eighteen polypeptides incorporated [³⁵S]methionine at a statistically significant higher rate under embryogenic culture conditions (32°C) than in the controls (18°C), whereas one protein was preferentially labeled under non-embryogenic culture conditions (18°C). These results indicate that only a limited number of proteins detectable in the 2-D gels of microspore extracts are associated with the early induction of embryogenesis. The reproducible identification of the differentially radiolabeled proteins in the 2-D gels allow the sequencing of representative peptides and the isolation of the corresponding cDNAs. This may lead to the identification and characterization of proteins associated with the very first stages of plant embryogenesis.Abbreviations 2-D two-dimensional We would like to thank Dr. H. Van Steeg (Rijks Instituut voor Milieubeheer (RIVM), Bilthoven, The Netherlands) for use of the PhosphorImager apparatus. This research was carried out as part of the EC-Bridge project Regulation of the inductive phase of microspore embryogenesis and EC-Science project The role of mitotic and cytoskeletal genes in the induction of plant cell division.     

Cold pretreatment enhances microspore embryogenesis in oilseed rape (Brassica napus L.)

Stress is an essential component during embryogenesis induction in microspore culture. Cold pretreatment has been used in cereal microspore culture but very seldom attempted in Brassica microspore culture. The effect of cold pretreatment of flower buds subjected to a liquid medium on microspore embryogenesis was investigated in spring and winter Brassica napus, as well as in B. rapa and B. oleracea. Cold pretreatment significantly enhanced microspore embryogenesis (by 1–7 fold) compared to commonly used microspore culture protocol in B. napus, while it was less effective in B. rapa or even negative in B. oleracea. The appropriate duration of cold pretreatment was found to be 2–4 days, which stimulated the best microspore embryogenesis. Cold pretreatment was also able to promote embryo development including the improvement of embryo quality and acceleration of embryogenesis. When incorporating with medium refreshing, cold pretreatment could initiate the most microspore embryogenesis than any other treatment used. With further improvement cold pretreatment method may have a positive potential in Brassica breeding programmes.     

Brassica napus pollen development during generative cell and sperm cell formation

Summary Brassica napus pollen development during the formation of the generative cell and sperm cells is analysed with light and electron microscopy. The generative cell is formed as a small lenticular cell attached to the intine, as a result of the unequal first mitosis. After detaching itself from the intine, the generative cell becomes spherical, and its wall morphology changes. Simultaneously, the vegetative nucleus enlarges, becomes euchromatic and forms a large nucleolus. In addition, the cytoplasm of the vegetative cell develops a complex ultrastructure that is characterized by an extensive RER organized in stacks, numerous dictyosomes and Golgi vesicles and a large quantity of lipid bodies. Microbodies, which are present at the mature stage, are not yet formed. The generative cell undergoes an equal division which results in two spindle-shaped sperm cells. This cell division occurs through the concerted action of cell constriction and cell plate formation. The two sperm cells remain enveloped within one continuous vegetative plasma membrane. One sperm cell becomes anchored onto the vegetative nucleus by a long extension enclosed within a deep invagination of the vegetative nucleus. Plastid inheritance appears to be strictly maternal since the sperm cells do not contain plastids; plastids are excluded from the generative cell even in the first mitosis.     

Characterization of polarity development through 2- and 3-D imaging during the initial phase of microspore embryogenesis in Brassica napus L

Isolated microspores of B. napus in culture change their developmental pathway from gametophytic to sporophytic and form embryo-like structures (ELS) upon prolonged heat shock treatment (5 days at 32 °C). ELS express polarity during the initial days of endosporic development. In this study, we focussed on the analysis of polarity development of ELS without suspensor. Fluorescence microscopy and 3-D confocal laser scanning microscopy (CLSM) without tissue interfering enabled us to get a good insight in the distribution of nuclei, mitochondria and endoplasmic reticulum (ER), the architecture of microtubular (MT) cytoskeleton and the places of 5-bromo-2′-deoxy-uridine (BrdU) incorporation in successive stages of microspore embryogenesis. Scanning electron microscopy (SEM) analysis revealed, for the first time, the appearance of a fibrillar extracellular matrix-like structure (ECM-like structure) in androgenic embryos without suspensor. Two types of endosporic development were distinguished based upon the initial location of the microspore nucleus. The polarity of dividing and growing cells was recognized by the differential distributions of organelles, by the organization of the MT cytoskeleton and by the visualization of DNA synthesis in the cell cycle. The directional location of nuclei, ER, mitochondria and starch grains in relation to the MTs configurations were early polarity indicators. Both exine rupture and ECM-like structure on the outer surfaces of ELS are supposed to stabilize ELS's morphological polarity. As the role of cell polarity during early endosporic microspore embryogenesis in apical–basal cell fate determination remains unclear, microspore culture system provides a powerful in vitro tool for studying the developmental processes that take place during the earliest stages of plant embryogenesis.     

Significance of preprophase bands of microtubules in the induction of microspore embryogenesis of Brassica napus

Microspores of Brassica napus L. cv. Topas, undergo embryogenesis when cultured at 32.5 °C for the first 18–24 h and then at 25 °C. The first division in heat-treated microspores is a symmetric division in contrast to the asymmetric division found after the first pollen mitosis in-planta or in microspores cultured continuously at 25 °C. This asymmetric division is unique in higher plants as it results in daughter cells separated by a non-consolidated wall. The cytoskeleton has an important role in such morphological changes. We examined microtubule (MT) organization during the first 24 h of heat induction in the embryogenic B. napus cv. Topas and the non-embryogenic B. napus breeding line 0025. Preprophase bands (PPBs) of MTs appeared in cv. Topas microspores in late uninucleate microspores and in prophase figures after 4–8 h of heat treatment. However, more than 60% of the PPBs were not continuous bands. In contrast, PPBs were never observed in pollen mitosis; MT strands radiated from the surface of the nuclear envelope throughout microspore maturation to the end of prophase of pollen mitosis I, during in-planta development and in microspores cultured at 25 °C. Following 24 h of heat treatment, over 95% of the microspores appeared to have divided symmetrically as indicated by the similar size of the daughter nuclei, but only 7–16% of the microspores eventually formed embryos. Discontinuous walls were observed in more than 50% of the divisions and it is probable that the discontinuous PPBs gave rise to such wall abnormalities which may then obstruct embryo development. Preprophase bands were not formed in heat-treated microspores of the non-embryogenic line 0025 and the ensuing divisions showed discontinuous walls. It is concluded that the appearance of PPBs in heat-induced microspores marks sporophytic development and that continuous PPBs are required for cell wall consolidation and embryogenesis. It follows that induced structures with two equally condensed nuclei, do not necessarily denote symmetric divisions. Received: 22 October 1998 / Accepted: 28 November 1998     

Initiation and development of microspore embryogenesis and plant regeneration of Brassica nigra

Brassica nigra is generally regarded as a recalcitrant species for microspore culture among Brassica crops. Conditions for reliable induction of microspore embryogenesis of B. nigra were studied in this context. Flower bud length and microspore developmental stage were correlated with further embryogenesis. The optimal bud size range was 2.0–2.5 mm for the highest proportion of totipotent, late uninucleate microspore and the highest frequency of microspore embryogenesis. Treatment of a short heat shock by incubating the microspore culture at 32°C for 24 h was suitable for the microspore survival, sustained cell divisions, and further induced embryogenesis. Subsequently, the use of NLN medium with the addition of 13% sucrose and 0.1% activated charcoal (AC) provided the optimal conditions for the development of microspore-derived embryos (MDEs). The early cotyledonary (EC) stage embryos cultured on MS medium fortified with 4.6 μM zeatin (ZT) and 0.12 μM indole-3-acetic acid (IAA) resulted in the most efficient rates of plantlet regeneration. The ploidy levels of regenerated plants of B. nigra were determined by flow cytometry, revealing that 50.6% were diploid. The results enable the advancement of breeding programs and genetic studies in B. nigra.     

Uptake and distribution of sinigrin in microspore derived embryos of Brassica napus L

In Brassica napus, glucosinolates are transported from all parts of the plant into the embryo during seed development. In this study we describe the uptake of the alkenyl glucosinolate sinigrin by microspore derived embryos from high and low glucosinolate genotypes. Microspore derived embryos develop completely isolated from maternal tissues unlike zygotic embryos, which contains glucosinolates transported into the embryo synthesised in the vegetative tissues. The sinigrin in the culture medium was almost completely absorbed by the embryos after three days of culture. The embryos of high and low glucosinolate genotypes were equally capable of absorbing sinigrin from the medium. A significant increase in different alkenyl glucosinolates following feeding of sinigrin suggests induction of biosynthetic enzymes in the embryos. Following excess feeding of sinigrin, we found a strong uptake against a concentration gradient and stable accumulation by the embryos. The glucosinolate was detected in single dissected cotyledons by a photometric test and by HPLC. This test could potentially be useful for screening mutants defective in glucosinolate uptake into the embryo.     

Differential development of plastids during microspore embryogenesis in barley

Summary In order to understand and limit albino plantlet formation during pollen embryogenesis in barley (Hordeum vulgare L. cv. Igri), plastid feature was followed during pollen embryogenesis under two anther culture conditions and compared to plastid development in the zygotic embryo. The first condition was characterized by cold pretreatment and maltose in the induction medium. Both embryos and calli were then obtained. During pollen embryo development, up to 30% of plastids had abnormal features. Disruptions mainly affected the plastid size, the feature of plastid envelopes, thylakoid and granum organization, as well as starch accumulation. In pollen calli, superficial cells had meristematic features. Up to 50% of plastids exhibited the above mentioned abnormalities. Internal cells were highly vacuolated with amyloplast-like plastids; envelopes had normal features but no internal membrane was detected. Pollen embryo-derived plantlets had a green-to-albino ratio (G/A) being equal to 1.0, whereas calli-derived embryos only formed albino plantlets. The second condition was characterized by mannitol pretreatment and the presence of both maltose and mannitol in the induction medium. No callus was formed but most of microspore-derived structures developed haploid embryos and then the green plantlets (200 plantlets per 100 responding anthers, G/A=9.4). In this case, plastid development in zygotic and pollen embryos were similar and almost no albino plantlets were formed.     

Temperature controls both gametophytic and sporophytic development in microspore cultures of Brassica napus

Summary Temperature controls the developmental fate of isolated Brassica napus microspores in vitro. Culture at 32.5°C leads to sporophytic development and the formation of embryos. Here we show that culture at 17.5°C leads to gametophytic development, and the formation of pollen-like structures at high frequencies (up to 80% after 7 days in culture). Early stages of both developmental pathways are observed in culture at 25.0°C, and embryos are produced at low frequencies (0.7%) at that temperature. Culturing B. napus microspores at 32.5°C versus 17.5°C brings the switch from gametophytic to sporophytic development under simple experimental control and provides a convenient tool for investigating the cellular and molecular mechanisms controlling this developmental switch.     

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.     

Hormones in zygotic and microspore embryos of Brassica napus

A number of studies have used microspore-derived embryos (MDEs) as amodel for examining a range of processes, including hormonal regulation ofembryo development. We examined the hormonal physiology of MDEs with theprimaryobjective of testing the validity of using the MDE system as a model forhormonally-regulated development in zygotic embryos, through late stages. To dothis we identified and quantified endogenous levels of abscisic acid (ABA),indole-3-acetic acid (IAA) and a number of gibberellins (GAs), includingGA₁₉, GA₂₀, GA₁ and GA₈ in bothMDEsand zygotic embryos. The presence of GA₁₉, together with itsC₁₉ metabolites indicates that the early-13 hydroxylation pathway isoperative in both embryo systems. Gibberellins A₄ and GA₉were also identified, thereby confirming the presence of the earlynon-hydroxylation pathway in B. napus MDEs and zygoticembryos. In general, the pattern of change of hormone (ABA, IAA, GA₁and GA₂₀) content per embryo through embryogenesis was similar forMDEs and zygotic embryos. Indole-3-acetic acid and GA₁ increased toamaximum at day 30 after culture (DAC) before decreasing. Abscisicacid levels increased to a maximum at day 35, and declined in zygoticembryos but not in MDEs. GA₂₀ increased to the final harvest atmaturity, or day 40. The absolute content (g/embryo) of each hormone, howeverwas appreciably lower (5- to 15- fold) in the MDEs. This was not the result ofdilution into surrounding medium for ABA or IAA; GA₁, however, didaccumulate in the medium. Although there were absolute quantitative differencesin the levels of IAA and ABA found in the two embryo systems, the similaritiesin the pattern of hormone changes suggests that the MDE system can serve as auseful model for examining the physiological roles of hormones duringembryogenesis.     

Effects of ascorbic acid,alpha-tocopherol,and glutathione on microspore embryogenesis in Brassica napus L.

The impact of culture conditions and addition of antioxidants to media on microspore embryogenesis in rapeseed (Brassica napus cv. ‘PF704’) was investigated. Different concentrations of ascorbic acid (0, 5, 10, 20, 50, 100, and 200 mg l^?1) and alpha (α)-tocopherol (0, 5, 10, 20, 50, 100, and 200 mg l^?1) were evaluated along with two temperature pretreatments (18 d at 30°C; 2 d at 32.5°C followed by 16 d at 30°C). In addition, combinations of reduced glutathione (0, 10, 50, and 100 mg l^?1) and ascorbic acid (5 and 10 mg l^?1) were tested. Microspore embryogenesis was significantly enhanced using 10 mg l^?1 ascorbic acid (334 embryos per Petri dish) compared with untreated cultures (184 embryos per Petri dish) at 30°C. α-Tocopherol (5 and 10 mg l^?1) enhanced (312 and 314 embryos per Petri dish, respectively) microspore embryogenesis relative to untreated cultures (213 embryos per Petri dish) at 30°C, although there were no significant differences among cultures treated with 5–50 mg l^?1 α-tocopherol. When 50 mg l^?1 α-tocopherol was combined with 5 or 10 mg l^?1 ascorbic acid, embryogenesis was significantly enhanced (308 and 328 embryos per Petri dish, respectively) relative to other ascorbic acid levels. Moreover, 10 mg l^?1 of reduced glutathione and 5 mg l^?l ascorbic acid enhanced microspore embryogenesis (335 embryos per Petri dish) compared to cultures without reduced glutathione (275 embryos per Petri dish). Microspore embryogenesis could be improved by adding ascorbic acid, α-tocopherol, and reduced glutathione when the appropriate combination and temperature pretreatment were selected.     

Improved microspore embryogenesis induction and plantlet regeneration using putrescine,cefotaxime and vancomycin in Brassica napus L.

The effects of three periods of exposure (12, 24 and 48 h) to different levels of putrescine (0, 0.2, 0.5, 1.0, 2.0 and 5.0 mg l^?1), as well as three incubation periods (24, 48 and 72 h) to different levels of cefotaxime and vancomycin (0, 50, 100, 200 and 500 mg l^?1) on microspore embryogenesis of rapeseed cv. ‘Hyola 401’ were assessed. Microspore embryogenesis was enhanced about threefold compared with untreated culture following 48 h treatment with 0.2 mg l^?1 putrescine. Putrescine treatment at 0.5 mg l^?1 for 48 h effectively induced root formation and increased normal plantlet regeneration by 92 % when microspore-derived embryos (MDEs) were transferred to regeneration medium. The highest embryo yield (184.2 embryos Petri dish^?1) was possible when induction medium was supplemented with 50 mg l^?1 cefotaxime for 24 h and the highest normal regeneration was observed in cultures exposed to 50 and 100 mg l^?1 at all durations tested. More abnormal MDEs (76 and 82 %) were observed when microspores treated with 200 and 500 mg l^?1 cefotaxime many of which failed to regenerate normally and resulted in callusing. Vancomycin at 100 mg l^?1 during the 48 h exposure increased the number of MDEs (181.6 embryos Petri dish^?1) in contrast to untreated cultures (93.6 embryos Petri dish^?1) but, normal plantlet regeneration decreased as vancomycin level increased and high callusing (84 and 90 %) was observed with 200 and 500 mg l^?1 for 72 h. Microspore embryogenesis and plant regeneration could be improved by putrescine, cefotaxime and vancomycin when appropriate levels and durations of incubation were selected.