Exine Wall Research Articles

Pollen wall degradation in the Brassicaceae permits cell emergence after pollination.

Despite attempts to degrade the sporopollenin in pollen walls, this material has withstood a hundred years of experimental treatments and thousands of years of environmental attack in insects and soil. We present evidence that sporopollenin, nonetheless, locally degrades only minutes after pollination in Arabidopsis thaliana flowers, and describe here a two-part pollen germination mechanism in A. thaliana involving both chemical weakening of the exine wall and swelling of the underlying intine. We explored naturally occurring components from pollen and stigma surfaces and found a tripartite mix of hydrogen peroxide, peroxidase and catalase enzymes (all at high levels at the pollination interface) to be experimentally sufficient to degrade the sporopollenin of some Brassicaceae family members. At pollination, factors carried on the pollen surface may mix with factors on the stigma surface in a reaction that locally oxidizes the exine pollen wall. Hydrogen peroxide, catalases, and peroxidases are biologically present at the right time and place and, when mixed experimentally, are sufficient to degrade the walls of susceptible pollen. Our work on native biochemistry for breaching sporopollenin suggests new research directions in pollen aperture evolution and could aid efforts to analyze sporopollenin's composition, needed for application of this corrosion-resistant, but long-intractable material.

A Ploidy-Sensitive Mechanism Regulates Aperture Formation on the Arabidopsis Pollen Surface and Guides Localization of the Aperture Factor INP1.

Pollen presents a powerful model for studying mechanisms of precise formation and deposition of extracellular structures. Deposition of the pollen wall exine leads to the generation of species-specific patterns on pollen surface. In most species, exine does not develop uniformly across the pollen surface, resulting in the formation of apertures–openings in the exine that are species-specific in number, morphology and location. A long time ago, it was proposed that number and positions of apertures might be determined by the geometry of tetrads of microspores–the precursors of pollen grains arising via meiotic cytokinesis, and by the number of last-contact points between sister microspores. We have tested this model by characterizing Arabidopsis mutants with ectopic apertures and/or abnormal geometry of meiotic products. Here we demonstrate that contact points per se do not act as aperture number determinants and that a correct geometric conformation of a tetrad is neither necessary nor sufficient to generate a correct number of apertures. A mechanism sensitive to pollen ploidy, however, is very important for aperture number and positions and for guiding the aperture factor INP1 to future aperture sites. In the mutants with ectopic apertures, the number and positions of INP1 localization sites change depending on ploidy or ploidy-related cell size and not on INP1 levels, suggesting that sites for aperture formation are specified before INP1 is brought to them.

Proteome Profiling of Maize Pollen Coats Reveals Novel Protein Components

The pollen coat, which covers the exine wall of pollen, is essential for initial sexual contact and thus successful fertilization in flowering plants. Pollen coat proteins (PCPs) not only mediate species specificity but are also needed for pollen–stigma recognition and pollen germination on the stigma. Maize (Zea mays L.) is one of the most common cereal crops in the world. To date, only a few PCPs from maize have been identified and characterized. In the present study, we extracted the pollen coat fraction from maize inbred line B73 with chloroform and purified the PCPs via a phenol-based protocol prior to two-dimensional gel electrophoresis (2-DE). By proteomic analysis, 26 protein spots were successfully identified and classified into 12 unique proteins. The protein composition of the maize pollen coat is distinctly different from those of the dicot plants Arabidopsis and rapeseed. Of the proteins identified in this study, eight (including profilins, caleosin, Zea m 2, β-expansin-10, exopolygalacturonase, Rho GDP-dissociation inhibitor 1, Ras-related protein Rab-2-A, and putative subtilase) had not previously been observed in the maize pollen coat. Bioinformatic analysis showed that nine of the PCPs were secreted proteins and that most of them were extracellular. These PCPs are potentially involved in pollen germination and tube growth. The current study extracted and identified the pollen coat proteins of maize, one of the most important crops throughout the world. Proteome profiling of the maize pollen coat revealed many novel protein components potentially involved in pollen–stigma interactions and pollen germination. Our results provide basic knowledge and further the functional characterization of PCPs in wind-pollinated species such as maize.

Cytochemistry of pollen development in Brachypodium distachyon

Brachypodium distachyon is a widely recognized model plant belonging to subfamily Pooideae with a sequenced genome. To gain a better understanding of the male reproductive development in B. distachyon we examined pollen morphology and cytochemical changes of microspore cytoplasm from pollen mother cell stage to mature pollen using light, fluorescent and scanning electron microscopy. Our results show that B. distachyon exhibits a typical monocot-type pollen ontogeny. Meiosis in the pollen mother cells is accomplished by successive cytokinesis generating isobilateral tetrads. Cytochemical examination indicated that microspore cytoplasm contains variable amounts of insoluble carbohydrates and proteins at different developmental stages. Deposition of starch in the cytoplasm of microspores starts at the bicellular stage and continues till the mature pollen stage. The formation of the exine wall progresses by the deposition of sporopollenin from the tapetum layer of the anther. The mature pollen is trinucleate, spheroidal in shape and possesses a single pore with an annulus and operculum. The exine pattern is smooth and of granular type.

Co-regulation of exine wall patterning, pollen fertility and anther dehiscence by Arabidopsis apyrases 6 and 7

An NCBI nucleotide blast keyed to apyrase (ATP-diphosphohydrolases, EC 3.6.1.5) conserved regions revealed five apyrases, AtAPYs (3-7), in addition to the previously identified AtAPY1 and 2. Here we report the functional analyses of two of the newly defined apyrases, AtAPY6 and AtAPY7. We analyzed tissue specificity of AtAPY6 and 7 expression by qRT-PCR and promoter:GUS fusion assays. We characterized the phenotypes of single and double knockout mutants for AtAPY6 and 7 in anther and pollen by light microscopy and electron microscopy. The transcripts of both AtAPY6 and 7 are expressed in mature pollen grains. Single knockout mutants of AtAPY6 and 7 displayed a minor change in pollen exine pattern under scanning electron microscopy without obvious change in fertility. Double knockout mutants of AtAPY6 and 7 (apy6apy7) displayed severe defects in pollen exine pattern, deformed pollen shape and reduced male fertility. An analysis of pollen from heterozygous apy6apy7 plants suggests that the defects in pollen exine wall are determined by the diploid genome. Our findings demonstrate that AtAPY6 and AtAPY7 are enzymes that play an important role in exine development of pollen grains, possibly through regulating the production of key polysaccharides needed for proper assembly of the exine layer.

Cytological investigation of anther development in DGMS line Shaan-GMS in Brassica napus L.

The cytological mechanism of male sterility of Shaan-GMS, a natural mutant dominant genic male sterile (DGMS) line in Brassica napus L., is not well studied. Cytological observation was made on different-size buds of DGMS line 0A30A derived from Shaan-GMS line. The pollen mother cells (PMCs) of DGMS line 0A30A were degenerating at the beginning of meiosis and could not pass the anaphase I stage, with no dyads or tetrads formed, suggesting that the DNA damage checkpoint and spindle assembly checkpoint were activated in sterile anthers. During the meiosis process of sterile anthers in the sterile plants, several kinds of abnormal meiotic cells could be observed: nuclei condensed PMCs, cells with micronuclei, collapsed cells, plasmolysis cells, cells connected with nucleoplasmic bridge, and microspore analogue developed from PMCs without meiosis but enclosed by the exine wall. The results suggested Shaan-GMS to be a new type of DGMS line in B. napus.

Soybean Pollen Anatomy, Viability and Pod Set under High Temperature Stress

AbstractHigh temperature (HT) stress is one of the major environmental factors influencing yield of soybean (Glycine max L. Merr.) in the semi‐arid regions. Experiments were conducted in controlled environments to study the effects of HT stress on anatomical changes of pollen and their relationship to pollen function in soybean genotype K 03‐2897. Objectives of this study were to (a) quantify the effect of HT stress during flowering on pollen function and pod set and (b) observe the anatomical changes in pollen grains of soybean plants grown under HT stress. Plants were exposed to HT (38/28 °C) or optimum temperature (OT, 28/18 °C) for 14 days at flowering stage. HT stress significantly decreased in vitro pollen germination by 22.7 % compared to OT. Pollen from HT stress was deformed; it had a thicker exine wall and a disintegrated tapetum layer. HT stress decreased pod set percentage (35.2 %) compared to OT. This study showed that decreases in pollen in vitro germination by HT stress were caused by anatomical changes in pollen, leading to decreased pod set percentage under HT stress.

Study of Radiation Effects on Upland Cotton (Gossypium hirsutum L.) Pollen Grain Irradiated by 60Co-gamma Ray

This paper studied the effects of 60Co-gamma ray on the pollen grains of upland cotton. The irradiation effects on pollen grains were tested in terms of the ultrastructural changes in the exine and interior of pollen grains, their germination rate, the single primer amplification reaction polymorphism of ovule developed after the pistils were pollinated by the pollen grains which had been irradiated with 60Co-gamma ray, and the law of genetic variation of their M1, M2 progeny. The results showed that 60Co-gamma ray had no effects on the exine wall of the pollen grains. The interior structure of pollen grain were destroyed significiantly. The interior wall became thin and irregular, and part of it concavitied to the inner. The endoplasmic reticulum depolymerized. The amount and the density of pollen grain inclusions increased. The number of pollen tubes in style decreased by 38%, compared with the control group (natural pollen grain). The single primer amplification reaction polymorphism of ovule increased. The germination percentage of M1 progeny was decreased by 41.03%. And with the cotton plants of M1 progeny, the length of taproot, longest lateral root, average lateral root, the number of lateral root, and the height of seedling decreased by 22.24%, 18.93%, 11.80%, 28.02%, 23.05%, respectively, compared with the control group. The percentage of sterility plants was 56.7%. The coefficients of variations of boll number, lint percentage, perimeter of stem, seed index, fruit branch number, longissimus fruit branch and plant height increased by 103.206%, 74.588%, 75.96%, 69.83%, 33.25% and 29.624%, 11.843%, respectively., compared with the control group. With the cotton plants of M2 progeny, the percentage of sterility plants was 56.7%. And the coefficients of variations of boll number, seed cotton yield, fruit branch number, plant height, boll height, and lint percentage increased by 21.944%, 16.261%, 3.827%, 3.986%, 7.25% and 0.497%, respectively., compared with the control group. The coefficients of variations and change range of agronomic traits in M2 progeny were less than them in M1 progeny.

Alpha-Particles and 60Co y-Rays Have Different Biological Effects on Upland Cotton (Gossypium hirsutum L.) Pollen Grains

To compared the irradiation effects by y-rays and alpha-particles on the structure and function of upland cotton pollen grains during pollination, pollen grains from Gossypium hirsutum L. cultivar “Lumian 21” were irradiated by alpha-particles (1, 10, 30, 40 and 100 Gy) and y-rays (20, 30 and 40 Gy). We looked for changes in the ultrastructure of the exine and interior walls of the pollen grains. After germinating the irradiated pollen grains in liquid medium, we looked for changes in germination rate, the number of pollen tubes in styles after pollination and F-actin distribution in the pollen tubes. Alpha-particles penetrated and etched the exine and interior walls of the pollen grains and also damaged the organelles in the pollen grains. More pollen grains were destroyed as the alpha-particles dose increased. y-rays had no effects on the exine walls of the pollen grains but had stronger effects on the interior structure of the pollen grains than those induced by alpha-particle irradiation. The pollen grain germination rate, the number of pollen tubes in the styles and the average length of the pollen tubes in pollen grains treated with y-ray irradiation were decreased and shortened with increased y-rays irradiation dose, compared with that of control. However, under alpha-particle irradiation, these features initially increased at a low level of irradiation (1 Gy) and then decreased as the dose was increased. There was evident damage to F-actin structures in pollen tubes induced by the two kinds of mutagens. The mechanisms underlying the biological effects induced by alpha-particles and y-rays were different. The function and structure of pollen tubes in pollination and fertilization may be affected by the ultrastructure of pollen grains.

Pollen ontogeny in Magnolia liliflora Desr.

Pollen ontogeny contributes significantly to the evolutionary analysis and the understanding of the reproductive biology of seed plants. Although much research on basal angiosperms is being carried out there are still many important features about which little is known in these taxa, such as the sporophytic structures related to pollen development and morphology. In this study, pollen development of Magnolia liliflora was analyzed by optical microscopy and transmission electron microscopy. The aim of this paper was to supply data that will help characterize basal angiosperms. Microsporogenesis is of the successive type, so that tetrads are decussate or isobilateral. The callosic walls form by the centripetal growth of furrows. The secretory tapetum develops orbicules, which start to form in the microspore tetrad stage. Pollen grains are shed at the bicellular stage. The exine wall has a granular infratectum. Ultrastructural changes observed in the cytoplasm of microspores and tapetal cells are related to the development of the pollen grain wall and orbicules. Centrifugal cell plates are more usual for the successive type of microsporogenesis. The presence of the successive type of microsporogenesis with callosic walls formed by the centripetal growth of furrows could reflect the fact that the successive type in Magnoliaceae is derived from the simultaneous type. The granular infratectum of the ectexine and the presence of orbicules could indicate that this species is one of the most evolved of the genus.

Microsporogenesis and pollen formation in cassava

This article describes the complete microsporogenesis and pollen formation in cassava (Manihot esculenta Crantz) at the various developmental stages (pollen mother cell, meiosis, tetrads, early free spore, mid uninucleate, late uninucleate, binucleate and mature pollen grain). Light microscopy, transmission electron microscopy and confocal laser scanning microscopy were used for the study. Floral bud size and other inflorescence characteristics were correlated with specific stages of the microspore development. This association allows a rapid selection of floral buds with similar microspore developmental stages, useful when a large number of homogeneous cells are needed for analysis and for in vitro induction of androgenesis. This article also compares methods for digestion the exine wall in microspores.

Anther, pollen and tapetum development in safflower, Carthamus tinctorius L

In safflower, the anther wall at maturity consists of a single epidermis, an endothecium, a middle layer and the tapetum. The tapetum consists mainly of a single layer of cells. However, this single-layer appearance is punctuated by loci having 'two-celled' groupings due to additional periclinal divisions in some tapetal cells. Meiotic division in microsporocytes gives rise to tetrads of microspores. The primexine is formed around the protoplasts of microspores while they are still enveloped within the callose wall. Just prior to microgametogenesis, the microspores enlarge through the process of vacuolation, and the exine wall pattern becomes established. Microgametogenesis results in the formation of 3-celled pollen grains. The two elongated sperm cells appear to be connected. The exine wall is highly sculptured with a distinct tectum, columellae, a foot layer, an endexine and a thin intine. Similar to other members of the Asteraceae family, the tapetum is of the invasive type. The most novel finding of this study is that in addition to the presence of invasive tapetal cells, a small population of 'non-invasive' tapetal cells is also present. The tapetal cells next to the anther locules in direct contact with the microspores become invasive and start to grow into the space between developing microspores. These tapetal cells synthesize tryphine and eventually degenerate at the time of gametogenesis releasing their content into the anther locules. A smaller population of non-invasive tapetal cells is formed as a result of periclinal divisions at the time of tapetum differentiation. These cells are not exposed to the anther locules until the degeneration of the invasive tapetal cells. The non-invasive tapetal cells have a different cell fate as they synthesize pollenkitt. This material is responsible for allowing some pollen grains to adhere to each other and to the anther wall after anther dehiscence. This observation explains the out-crossing ability of Carthamus species and varieties in nature.

UV and visible light screening by individual sporopollenin exines derived from Lycopodium clavatum (club moss) and Ambrosia trifida (giant ragweed)

We have investigated the UV–visible light transmission of three types of micrometre-sized sporopollenin exine shells, two derived from Lycopodium clavatum (club moss) spores and one from Ambrosia trifida (giant ragweed) pollen. We have used spectrophotometer measurements of partial monolayers of exines and microscope absorbance imaging to derive the light transmission properties of individual exines. Measurements have been made for exines in air when light transmission losses are due to a combination of absorption, reflection and scattering processes and for exines dispersed in a liquid for which the refractive index (RI) is approximately equal to the RI of the exine such that reflection and scattering effects are negligible. Overall, it found that the light transmission of a single exine wall is approximately 50%. This value of the transmission is due mainly to light absorption, is similar for the three exines studied here and varies only slightly with light wavelength over the range 200–900 nm.

ATP-Binding Cassette Transporter G26 Is Required for Male Fertility and Pollen Exine Formation in Arabidopsis

The highly resistant biopolymer, sporopollenin, gives the outer wall (exine) of spores and pollen grains their unparalleled strength, shielding these structures from terrestrial stresses. Despite a limited understanding of the composition of sporopollenin, it appears that the synthesis of sporopollenin occurs in the tapetum and requires the transport of one or more sporopollenin constituents to the surface of developing microspores. Here, we describe ABCG26, a member of the ATP-binding cassette (ABC) transporter superfamily, which is required for pollen exine formation in Arabidopsis (Arabidopsis thaliana). abcg26 mutants are severely reduced in fertility, with most siliques failing to produce seeds by self-fertilization and mature anthers failing to release pollen. Transmission electron microscopy analyses revealed an absence of an exine wall on abcg26-1 mutant microspores. Phenotypic abnormalities in pollen wall formation were first apparent in early uninucleate microspores as a lack of exine formation and sporopollenin deposition. Additionally, the highest levels of ABCG26 mRNA were in the tapetum, during early pollen wall formation, sporopollenin biosynthesis, and sporopollenin deposition. Accumulations resembling the trilamellar lipidic coils in the abcg11 and abcg12 mutants defective in cuticular wax export were observed in the anther locules of abcg26 mutants. A yellow fluorescent protein-ABCG26 protein was localized to the endoplasmic reticulum and plasma membrane. Our results show that ABCG26 plays a critical role in exine formation and pollen development and are consistent with a model by which ABCG26 transports sporopollenin precursors across the tapetum plasma membrane into the locule for polymerization on developing microspore walls.

Molecular cloning and characterization of BcMYBogu, a novel member of the MYB family involved in OguCMS in Brassica campestris ssp. chinensis

In the attempt to elucidate the molecular mechanism of CMS. Ogura cytoplasmic male sterile (OguCMS) lines were obtained in Chinese cabbage after interspecific hybridization between Brassica. napus L. OguCMS and B. campestris ssp. chinensis followed by recurrent backcross with B. campestris ssp. chinensis as the pollen donor. The CMS lines were significantly characterized by the whitish anther and indehiscence of anther. The tapetal hypertrophy with excess vacuola-tion was the first observed defective soon after the tetrad stage, subsequently the microspores defected in pollen wall forma-tion, and later the cytoplasm detached from the exine wall and underwent degeneration. With aid of cDNA-AFLP and RACE approaches, we cloned the BcMYBogu(GenBank accession No: EF127861) in Chinese cabbage, which is premature expressed in early and middle stage floral buds of OguCMS lines, and predicted to encode a novel protein with a DNA binding domain: SH[AL]QKY[RF] motif at the N-terminus. Phylogenetic comparison revealed that the BcMYBogu was clustered with AtMYB32, AtMYB26 and AtMYB4, which were indicated to be involved in male sterility in Arabidopsis thaliana. The BcMYBogu transcript was detected in rosette leaves, floral buds and stems by RT-PCR analysis. Compared with the maintainer, the expression level of BcMYBogu was increased in these organs, especially in floral buds of OguCMS lines. Our investigation suggests that BcMYBogu is a new member of the MYB family involved in male sterility in Chinese cabbage.

Morphology and anatomy of pollen grains from male-fertile and male-sterile cultivars of chestnut (Castanea sativa Mill.)

SummaryPollen morphology and ultrastructure are described for four male-fertile cultivars (‘Firdola’, ‘Karamehmet’, ‘Sarıașlama’, and ‘Hacıömer’) and two male-sterile cultivars (‘OsmanogȈlu’ and ‘Vakit Kestanesi’) of chestnut (Castanea sativa Mill.) using light microscopy and both scanning and transmission electron microscopy. Pollen grains of both male-fertile and male-sterile chestnut cultivars are tricolporate, the germinal furrow extending almost the full length of the grain axis. Pollen grains have a slightly reticulate exine. Pollen grain length varied from 13.33 – 21.30 µm, and decreased significantly in the male-sterile cultivars. Three different pollen shapes were observed among the cultivars: prolate, perprolate, and sub-prolate. The ultrastructure of the pollen grains did not differ between male-fertile and male-sterile cultivars. Intine, exine and total wall thickness (exine + intine) of pollen grains were determined as: 83.2 – 153.1 nm, 432.8 – 520.0 nm, and 516.0 – 651.6 nm, respectively; and variations were significant (P ≤ 0.05) among cultivars. The percentages of in vitro germination of pollen grains of male-fertile cultivars were between 11 – 78%, and the variations among cultivars were significant (P ≤ 0.05). The percentages of empty pollen grains observed among cultivars ranged from 3 – 32%. The correlation coefficient between the percentage of normal pollen and the germination rate was r = 0.898 (P ≤ 0.01).

Study of male sterility in Taiwania cryptomerioides Hayata (Taxodiaceae)

A study of male sterility over a period of three consecutive years on a conifer species endemic to Taiwan, Taiwania cryptomerioides Hayata (Taxodiaceae), was done for this article. With the aids of fluorescence and electron microscopic observations, the ontogenic processes in the fertile and sterile microsporangia are compared, using samples collected from Chitou Experimental Forest and Yeou-Shoei-Keng Clonal Orchard of the National Taiwan University, Nantou, Taiwan. The development of male strobili occurred from August to the end of March. Microsporogenesis starts with the formation of the archesporium and ends with the maturation of 2-celled pollen grains within the dehiscing microsporangium. Before meiosis, there was no significant difference in ultrastructure between the fertile and sterile microsporangia. Asynchronous pollen development with various tetrad forms may occur in the same microsporangium of either fertile or sterile strobili. However, a callose wall was observable in the fertile dyad and tetrad, but not in the sterile one. After dissolution of the callose wall, the fertile microspores were released into the locule, while some sterile microspores still retained as tetrads or dyads with intertwining of exine walls in the proximal faces. As a result, there was no well developed lamellated endexine and no granulate ectexine or intine in the sterile microspores. Eventually, the intracellular structures in sterile microspores were dramatically collapsed before anthesis. The present study shows that the abortion in pollen development is possibly attributed to the absence of the callose wall. The importance of this structure to the male sterility of T. cryptomerioides is discussed.

Callose synthase (CalS5) is required for exine formation during microgametogenesis and for pollen viability in Arabidopsis

Callose (beta-1,3-glucan) is produced at different locations in response to biotic and abiotic cues. Arabidopsis contains 12 genes encoding callose synthase (CalS). We demonstrate that one of these genes, CalS5, encodes a callose synthase which is responsible for the synthesis of callose deposited at the primary cell wall of meiocytes, tetrads and microspores, and the expression of this gene is essential for exine formation in pollen wall. CalS5 encodes a transmembrane protein of 1923 amino acid residues with a molecular mass of 220 kDa. Knockout mutations of the CalS5 gene by T-DNA insertion resulted in a severe reduction in fertility. The reduced fertility in the cals5 mutants is attributed to the degeneration of microspores. However, megagametogenesis is not affected and the female gametes are completely fertile in cals5 mutants. The CalS5 gene is also expressed in other organs with the highest expression in meiocytes, tetrads, microspores and mature pollen. Callose deposition in the cals5 mutant was nearly completely lacking, suggesting that this gene is essential for the synthesis of callose in these tissues. As a result, the pollen exine wall was not formed properly, affecting the baculae and tectum structure and tryphine was deposited randomly as globular structures. These data suggest that callose synthesis has a vital function in building a properly sculpted exine, the integrity of which is essential for pollen viability.

Programmed cell death during the transition from multicellular structures to globular embryos in barley androgenesis

Androgenesis represents one of the most fascinating examples of cell differentiation in plants. In barley, the conversion of stressed uninucleate microspores into embryo-like structures is highly efficient. One of the bottlenecks in this process is the successful release of embryo-like structures out of the exine wall of microspores. In the present work, morphological and biochemical studies were performed during the transition from multicellular structures to globular embryos. Exine wall rupture and subsequent globular embryo formation were observed only in microspores that divided asymmetrically. Independent divisions of the generative and the vegetative nuclei gave rise to heterogeneous multicellular structures, which were composed of two different cellular domains: small cells with condensed chromatin structure and large cells with normal chromatin structure. During exine wall rupture, the small cells died and their death marked the site of exine wall rupture. Cell death in the small cell domain showed typical features of plant programmed cell death. Chromatin condensation and DNA degradation preceded cell detachment and cytoplasm dismantling, a process that was characterized by the formation of vesicles and vacuoles that contained cytoplasmic material. This morphotype of programmed cell death was accompanied by an increase in the activity of caspase-3-like proteases. The orchestration of such a death program culminated in the elimination of the small generative domain, and further embryogenesis was carried out by the large vegetative domain. To date, this is the first report to show evidence that programmed cell death takes part in the development of microspore-derived embryos.

Time-lapse tracking of barley androgenesis reveals position-determined cell death within pro-embryos.

Following abiotic stress to induce barley (Hordeum vulgare L.) androgenesis, the development of 794 enlarged microspores in culture was monitored by time-lapse tracking. In total, 11% of the microspores tracked developed into embryo-like structures (type-I pathway), 36% formed multicellular structures (type-II pathway) and 53% of the microspores followed gametophytic divisions, accumulated starch and died in the first days of tracking (type-III pathway). Despite the microspore fate, enlarged microspores showed similar morphologies directly after stress treatment. Ultrastructural analysis, however, revealed two morphologically distinct cell types. Cells with a thin intine layer and an undifferentiated cytoplasm after stress treatment were associated with type-I and type-II pathways, whereas the presence of differentiated amyloplasts and a thick intine layer were associated with the type-III pathway. Tracking revealed that the first morphological change associated with embryogenic potential was a star-like morphology, which was a transitory stage between uninucleate vacuolated microspores after stress and the initiation of cell division. The difference between type-I and type-II pathways was observed during the time they displayed the star-like morphology. During the transition phase, embryo-like structures in the type-I pathway were always released out of the exine wall at the opposite side of the pollen germ pore, whereas in the type-II pathway multicellular structures were unable to break the exine and to release embryo-like structures. Moreover, by combining viability studies with cell tracking, we show that release of embryo-like structures was preceded by a decrease in viability of the cells positioned at the site of exine wall rupture. These cells were also positively stained by Sytox orange, a cell death indicator. Thereby, we demonstrate, for the first time, that a position-determined cell death process marks the transition from a multicellular structure into an embryo-like structure during barley androgenesis.