© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
A light and electron microscopy analysis of the events leading to male sterility in Ogu-INRA CMS of rapeseed (Brassica napus)
1Centro de Biotecnología y Genómica de Plantas, UPM-INIA, ETSI Agrónomos, Ciudad Universitaria s/n, E-28040 Madrid, Spain
2Station de Génétique et d'Amélioration des Plantes, IJPB, INRA UR254, Route de Saint-Cyr, F-78026 Versailles Cedex, France
3Instituto de Recursos Naturales, Centro de Ciencias Medioambientales, CSIC, Serrano 115-bis, E-28006 Madrid, Spain
* To whom correspondence should be addressed: E-mail: mlucas{at}ccma.csic.es
Received 30 September 2007; Revised 11 December 2007 Accepted 20 December 2007
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Ogura cytoplasmic male sterility (CMS) occurs naturally in radish and has been introduced into rapeseed (Brassica napus) by protoplast fusion. As with all CMS systems, it involves a constitutively expressed mitochondrial gene which induces male sterility to otherwise hermaphroditic plants (so they become females) and a nuclear gene named restorer of fertility that restores pollen production in plants carrying a sterility-inducing cytoplasm. A correlative approach using light and electron microscopy was applied to define what stages throughout development were affected and the subcellular events leading to the abortion of the developing pollen grains upon the expression of the mitochondrial protein. Three central stages of development (tetrad, mid-microspore and vacuolate microspore) were compared between fertile, restored, and sterile plants. At each stage observed, the pollen in fertile and restored plants had similar cellular structures and organization. The deleterious effect of the sterility protein expression started as early as the tetrad stage. No typical mitochondria were identified in the tapetum at any developmental stage and in the vacuolate microspores of the sterile plants. In addition, some striking ultrastructural alterations of the cell's organization were also observed compared with the normal pattern of development. The results showed that Ogu-INRA CMS was due to premature cell death events of the tapetal cells, presumably by an autolysis process rather than a normal PCD, which impairs pollen development at the vacuolate microspore stage, in the absence of functional mitochondria.
Key words: Brassica napus, cell death, light and electron microscopy, mitochondria, plastids, pollen development, Ogu-INRA cytoplasmic male sterility, transgenic-restored plants, tapetum
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Cytoplasmic male sterility (CMS) is a double genetic system that controls sex determinism in many Angiosperms. It involves the maternally inherited mitochondrial genome that induces male sterility (absence of viable pollen) and nuclear, Mendelian gene(s) called Rf for restorers of fertility, thus allowing pollen production even in the presence of the sterility-inducing cytoplasm (for reviews, see Hanson and Bentolila, 2004; Chase, 2007). CMS has been widely used in crops for the large-scale production of hybrid seeds (Havey, 2004). In addition, it exemplifies the importance of mitochondrial function in pollen development.
Pollen development is a well-characterized process (Bedinger, 1992; McCormick, 2004; Scott et al., 2004; Ma, 2005), which starts at the end of meiosis with the formation of a tetrad of microspores and ends at the dehiscence of anthers when mature pollen is released. It relies on the concomitant differentiation and activity of cells from both the sporophyte (the mother plant) and the gametophyte (the pollen). The tapetum is the innermost sporophytic cell layer that is in direct contact with the developing pollen in the anther locule. It is a highly active secretory tissue that provides a large part of the pollen exine wall components, necessary for normal development and germination of pollen. In most species, including Brassicaceae, the tapetum enters programmed cell death (PCD) before anthesis, normally around the time of the first pollen mitosis. The important role of the tapetum cell layer is demonstrated by the amount of male sterility mutations that directly affect tapetum functions (reviewed in Ma, 2005). Moreover, the timeliness of tapetum breakdown is crucial for the viability of pollen: premature degeneracy, or its delay, both lead to pollen abortion (Kawanabe et al., 2006; Li et al., 2006; Vizcay-Barrena and Wilson, 2006).
In many CMS systems, abnormal tapetum development parallels, and sometimes precedes, the collapse of pollen. This has been observed for instance in the Texas-CMS of maize, the petiolaris CMS in sunflower, and the Ogura CMS in radish and rapeseed. In petiolaris CMS of sunflower, the early degeneracy of the tapetum has been suggested to result from the premature triggering of programmed cell death (PCD), that normally occurs in this tissue at the end of pollen development (Balk and Leaver, 2001).
The Ogura CMS was first described in a Japanese radish cultivar (Ogura, 1968). It is present in gynodioecious populations of wild radish in Japan (Murayama et al., 2004). Ogura (1968) reported an early breakdown of the tapetum in anthers of male sterile radish plants. The Ogura CMS has been introduced into Brassica crops by sexual crosses that result in alloplasmic Ogura Brassica lines (Bannerot et al., 1974). These lines had several undesirable traits due to their alloplasmic state (Bannerot et al., 1977). Brassica cybrids were obtained from protoplast fusion, among which were the cybrids carrying the so-called Ogu-INRA CMS, which leads to complete abortion of pollen development in anthers of normal morphology (Gourret et al., 1992; Pelletier et al., 1983). The Ogu-INRA CMS in rapeseed exactly corresponds to the Ogura CMS in radish: both result from the expression of the orf138 mitochondrial gene, originated from Ogura radish (Bonhomme et al., 1992; Krishnasamy and Makaroff, 1993) and the same restorer gene, Rfo, is necessary and sufficient to restore fertility in both species (Brown et al., 2003; Desloire et al., 2003; Koizuka et al., 2003; Uyttewaal, 2007). In addition, Ogu-INRA rapeseed plants show premature breakdown of the tapetal cell layer as occurs in Ogura radish plants (Ogura, 1968; Gourret et al., 1992).
The aim of this work was to define precisely when, during development, pollen abortion occurred in Ogu-INRA male sterile rapeseed plants. To achieve this goal, a correlative structural approach at the light and electron microscopy levels of resolution at different developmental stages in fertile, sterile and restored plants was carried out. In addition, this work aimed to check whether the restoration of fertility by Rfo reverted completely the cellular events that could be observed in the fertile plants.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant material
The rapeseed (Brassica napus) plants used in this work have the same nuclear genomic background, cv. Pactol, a commercial variety from Cargill. Fertile plants (F) designated the Pactol cultivar; they have a normal B. napus cytoplasm. Sterile plants (S) were obtained from Cargill and have the Ogu-INRA cytoplasm in the Pactol nuclear background. Restored plants (R) were obtained by introducing the Rfo gene from radish into an S plant via Agrobacterium transformation (Uyttewaal, 2007).
Plants were grown in the greenhouse at 20–25 °C under a 16/8 h light/dark cycle.
Specimen processing for microscopy
Flower buds of different sizes were collected from plants of all three genotypes (F, S, R). The specimens were classified in groups according to their length: less than 1 mm, between 1–2 mm, 2–3 mm, and 3–4 mm. The anthers were carefully excised under a binocular microscope in a humid chamber and immediately fixed by submersion in a solution of 4% formaldehyde and 5% glutaraldehyde in 0.02 M sodium cacodylate buffer, pH 7.2, at room temperature under vacuum. After 3 h, the fixative solution was replaced by a fresh one and fixation continued overnight at 4 °C. Next day, the specimens were washed in a cacodylate buffer (0.05 M sodium cacodylate, 1% sucrose), three times for 30 min each at 4 °C, and post-fixed with a solution of 1% osmium tetroxide in the above cacodylate buffer, for 5 h at 4 °C. After three washes, 30 min each, at 4 °C with the same cacodylate buffer, the anthers were dehydrated in a series of increased concentration of ethanol in water: 30%, 50%, for 30 min each, and 70%, overnight, at the same temperature. Dehydration continued at 4 °C with 1% uranyl acetate in 70% ethanol for 2 h, 90% ethanol for 10 min, 96% ethanol for 30 min, and 100% ethanol for 2 h with one change after the first hour. Infiltration in London Resin White was carried out with mixtures of resin:ethanol (v:v) of 1:3, 1:1, and 3:1 for 3 h, overnight, and 3 h, respectively, then in pure resin for 24 h at 4 °C with shaking. After two changes of resin, for 3 h and overnight at 4 °C with shaking, the specimens were placed into gelatine capsules, filled up with resin, and allowed to polymerize for 24 h at 60 °C.
Sectioning and observation
Semi-thin sections (1–2 µm) were cut from the polymerized blocks in a Reichert Ultracut S ultramicrotome (Leica), stained with 1% toluidine blue O, observed on a Leitz photomicroscope under bright field and photographed using an Olympus DC10 digital camera. Small trapezoid areas containing different pollen developmental stages surrounded by the tapetal cells, as identified on the sections observed on the light microscope, were trimmed down by hand on the surface of the blocks. Thin sections (70 nm) were cut, collected on 600 mesh formvar-coated copper grids, stained with a solution of 2% lead citrate, and examined using a STEM LEO 910 electron microscope equipped with a Gatan Bioscan 792 digital camera.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The screening of the sections, cut from randomly selected blocks, under the light microscope permitted a relative parallelism between anther size and the pollen developmental stages to be established. Anthers from buds less than 1 mm in length only contained sporophytic tissues. Phases of meiosis were observed in anthers from buds between 1 and 2 mm. Tetrads of microspores were mostly seen in 2–3 mm buds, occasionally in 1–2 mm specimens. Buds with 2–3 mm of length also contained further developmental stages up to the mid-microspore, characterized by small cytoplasmic vacuoles and a central nucleus. Vacuolate microspores, with a large cytoplasmic vacuole and a polarized nucleus, and young bicellular pollen grains, only in specimens from fertile and restored plants, were found in 3–4 mm long buds. Approximately 10 blocks containing anthers between 1 mm and 4 mm were cut for each genotype (F, S, R). Comparable central stages of pollen development (tetrad, mid-microspore, and vacuolate microspore) of all three genotypes were analysed under the light and the electron microscopes.
The general structure of the tissues from the three genotypes was analysed in semi-thin sections stained with toluidine blue O (Fig. 1). Pollen development was paralleled in fertile and restored plants. Most of the structural features displayed in the fertile plants were also found in the restored plants, although in the tapetal cells from the restored plants, some vacuoles were larger than in the fertile tapetum cells, at the vacuolate microspore stage (Fig. 1A, C, D, F, G, I). Nevertheless, the pollen developmental process in restored plants seems to be similar to that in the fertile plants (at least in the stages covered in this study) and yielded identically viable pollen: 92% of stained pollen grains after Alexander (1969) staining (data not shown). By contrast, pollen development in the sterile plants was strikingly impaired. At the tetrad stage, the tapetal cells of the sterile plants developed a large vacuole (Fig. 1B). Then, at the mid-microspore stage, the tapetal cells underwent an evident swelling (Fig. 1E). The exine was thinner than in the mid-microspores from the fertile and restored (compare Fig. 1E with D and F). At the vacuolate microspore stage, toluidine blue staining, especially in the tapetal area, was paler than its fertile and restored counterparts; no evident tapetal tissue organization could be observed in the sterile specimens (compare Fig. 1H with G and I). The vacuolate microspores stuck together, with deformed shapes, in the central part of the locule (Fig. 1H).
|
For a detailed subcellular characterization of the developmentally sequential changes of maturing pollen and tapetum, ultrathin sections from specimens of all three genotypes were observed on a transmission electron microscope. Pollen development comprises sequentially ordered and strictly regulated physiological events that are mirrored by defined morphological changes throughout the different stages undergone by the pollen and tapetal cells. The major ultrastructural features observed in the all three genotypes under study are summarized in Table I. A number of organelles such as the cell nucleus, vacuoles, vesicles, dictyosomes of the Golgi apparatus, endoplasmic reticulum, plastids, and mitochondria could be identified in the developing pollen and tapetal cells. Their relative abundance, subcellular arrangement and ultrastructural changes can be considered as hallmarks to define the different developmental stages. Thus, deviations from the normal pattern in the sterile plants could be described. The so-called normal development of the fertile/restored plants is illustrated in Figs 2
–4 with examples from fertile anthers and in Fig. 6 with snapshots from restored specimens.
|
|
|
|
|
|
The subcellular organization of the microspores at the tetrad stage looked alike in the fertile/restored and sterile plants, with a dense cytoplasm rich in ribosomes and organelles such as plastids and mitochondria (Fig. 2A–D). The tapetal cells of the fertile and restored plants at this stage showed numerous dictyosomes and associated vesicles with a polarized localization towards a characteristic wavy plasma membrane (Fig. 2A, E), indicative of a high secretion activity. The plastids were organized around the nuclei, with accumulation of lipids (plastoglobuli) inside (Fig. 2E). From this pattern, two major differences were observed in the sterile tapetal cells: a large cytoplasmic vacuole and the absence of typical mitochondria (Fig. 2B, F, H). Instead, some pale, round or oval-shaped organelles with an outer double membrane and an inner membraneous system were seen (Fig. 2F, H). The size of these structures was clearly larger than the tapetal mitochondria in fertile/restored plants.
Mid-microspores from sterile anthers showed a less-developed exine than their fertile/restored counterparts, with no other noticeable differences (Fig. 3A, B, C, E). Mitochondria were clearly recognized (Fig. 3D, E). At this stage of pollen development, the tapetal cells in the fertile/restored plants showed a still intense secretion activity reflected by a wavy plasma membrane and the release of a densely stained material (Fig. 3A, H). The plastids, located around the cell nucleus, showed some low-electron-dense areas, presumably plastoglobuli, coexisting with the denser inclusions also seen in the previously reported stage (Fig. 3F, H). The subcellular arrangement of the sterile tapetum looked substantially different (Fig. 3B, G, I). The cytoplasm was less electron-dense and the only well-defined organelles were the cell nucleus and the surrounding plastids with almost no low-electron dense inclusions (Fig. 3G). Chromatin was highly condensed (as opposed to the dispersed pattern in the fertile/restored). Numerous vesicles and rough endoplasmic reticulum (RER)-coated areas were observed in the cytoplasm (Fig. 3I). Doubled-membrane organelles similar to those found in the tetrad stage were also seen, but no typical active mitochondria were identified (Fig. 3I).
The most striking ultrastructural differences between the fertile/restored pattern and that of the sterile plants were observed at the vacuolate microspore stage (Figs 4, 5). The subcellular organization of the vacuolate microspores developed within sterile anthers was greatly affected as compared to the normal pattern; this did not occur in the previously compared developmental stages. Long strands of RER bud off from the outer membrane of the nuclear envelope (Figs 4D, 5A–C). Pale, doubled-membrane organelles with an internal membraneous system were observed, but typical mitochondria were not found (Figs 4D and 5A–C versus Fig. 4C). The microspore nucleus displayed some large masses of condensed chromatin (Figs 4D, 5A). The tapetal cells were plasmolysed (compare Figs 4B, G with E and F). Their remnants were seen as a disorganized mixture of organelles that occupied a large area in the locule, clustering together the microspores in the central part (Figs 4G, 5D–F). Examples of the total disintegration of the cell nucleus are shown in Fig. 5D and F: a patch of condensed chromatin attached to some of the nuclear envelope and an isolated nucleolus could be observed. These appeared mixed up with plastids with little or no low-electron dense deposits, vesicles of different sizes with or without organelles inside and atypical double-membrane organelles (Fig. 5E). No typical mitochondria were observed.
Fertile and restored microspores underwent pollen mitosis I and pollen grains, with vegetative and generative cells, were formed (Fig. 6A, B). Further stages than the vacuolate microspore were not observed in the sterile plants. The ultrastructural study of the restored specimens (Fig. 6C–F) showed a still intact tapetum with the characteristic organelles of the tapetal cells in Brassicaceae species (Wu et al., 1997): the tapetosomes and plastids containing an intense accumulation of osmiophylic material, the so-called elaioplasts. Typical, well-preserved mitochondria in both the bicellular pollen (Fig. 6E) and tapetum (Fig. 6F) were found.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Previous reports of thorough microscopy analyses of cellular events upon CMS are scarce. For the Ogura CMS, the original paper from Ogura (Ogura, 1968) contains a light microscopy study, reporting a decrease in tapetal cell stainability and progressive vacuolization of the tapetum as the first observable signs of abnormal development in sterile anthers of radish. In Brassica napus, some early reports concerned alloplasmic genotypes carrying the unmodified Ogura radish cytoplasm (Polowick and Sawhney, 1990, 1991), with severe consequences on floral morphology (Pelletier et al., 1983; Gourret et al., 1992). To our knowledge, the only microscopy analysis reported until now on Ogu-INRA Brassica genotypes is that from Gourret et al. (1992). In this previous paper, B. napus with different types of cytoplasms were analysed, including the so-called Ogu-INRA cytoplasm (cybrid 58 in Pelletier et al., 1983; Gourret et al., 1992). The present report gives a much more complete and detailed analysis of the CMS. Moreover, observations of restored plants which were not available in 1992, have been added.
Fixation is critical to achieve an optimal structural preservation, thus minimizing possible artefacts that would lead to misinterpretation. In this work, the conventionally applied protocols for the fixation of anther tissues, with a mixture of 4% formaldehyde and 5% glutaraldehyde, was implemented by adding 1% sucrose to the buffer used to wash out the fixative (Fedorova et al., 1999). This modification prevented shrinkage of the cytoplasm of the microspores and separation between adjacent tapetal cells, very sensitive to changes of the osmotic pressure. The protocol of fixation and post-fixation with osmium tetroxide provided an excellent ultrastructural preservation of all tissues and developmental stages studied. Specimens susceptible to fixation artefacts, such as the vacuolate microspore (Platt et al., 1998), were efficiently well preserved. The protocol applied also permitted the unequivocal visualization of the membrane systems and subcellular organelles. In particular the inner and outer membranes of the mitochondria were clearly recognized in all tissues and stages analysed from fertile and restored specimens. However, typical mitochondria were not observed in the tapetal cells of sterile anthers, at all stages. It should be noted, that on the same sections, except those of the vacuolate microspore stage, well-preserved typical mitochondria were found in developing microspores, indicating that the fixation and staining of these samples were also efficient. Tapetal mitochondrial alterations were also reported in male-sterile anthers of tobacco H2.11 line (Hernould et al., 1998), Beta vulgaris (Majewska-Sawka et al., 1993), Petunia hybrida cv. Blue Bedder (Bino, 1985), and sunflower (Horner, 1977).
By using a transgenic plant carrying the Rfo restorer gene from radish as the restored sample, misleading observations that could have resulted from the analysis of restored plants with a large introgression of radish chromosome were prevented (Delourme et al., 1998; Giancola et al., 2003). Indeed, restored B. napus genotypes bred from Raphanobrassica interspecific hybrids produced more aborted pollen grains than the corresponding fertile maintainer plants (our unpublished results). By contrast, the transgenic restored plant used in this study is perfectly fertile, and the accumulation of the sterility protein ORF138 was completely abolished in its anthers (Uyttewaal, 2007). No differences in pollen development between the fertile and restored samples were observed both at the light and electron microscopy resolutions. Similarly, in general terms, the structure and organization of the tapetal cells were similar in the two genotypes, although sometimes a slightly lower stainability, and more vacuolization of the tapetum of restored plants was observed in light microscopy. These light differences can therefore be considered as insignificant for pollen development and fertility. In any case, they are not related to the Ogu-INRA CMS.
At early developmental stages, the ultrastructural study in the tapetal cells from fertile and restored plants revealed a typical high secretory activity, known to be necessary for callose digestion, the nutrition of developing pollen and exine formation (Bedinger, 1992; Vizcay-Barrena and Wilson, 2006; Wu and Cheung, 2000). In sterile specimens, secretion did not appear to be as much as in the corresponding stages from fertile and restored plants. This may be related to the low development of the exine observed in mid- and vacuolate microspores of sterile plants, as occurs in other cytoplasmic male sterility systems (Audran and Bouillot, 1981; Horner, 1977; Majewska-Sawka et al., 1993).
Tapetum degeneration is a crucial step in pollen development and results from PCD (Bedinger, 1992; Papini et al., 1999; Wu and Cheung, 2000). This is characterized by a progressive disintegration of the cell's organization, with cell shrinkage, chromatin condensation at the periphery of the nucleus, expansion of the RER cisternae to confine portions of the cytoplasm, rupture of the tonoplast (Papini et al., 1999; Wu and Cheung, 2000) and DNA fragmentation, as detected by TUNEL staining (Vizcay-Barrena and Wilson, 2006). In Brassica napus and some other angiosperms, mitochondria persist until the last stages of tapetal degeneration (Papini et al., 1999; Platt et al., 1998). Alterations in the right timing (premature or retarded) of tapetum degeneration lead to pollen abortion (Ku et al., 2003; Li et al., 2006; Luo et al., 2006). A previous ultrastructural study on the sequential changes throughout microsporogenesis and tapetum development in B. napus L. has described that the final phase of tapetum degeneration occurred at the time of the second pollen mitosis (mid-bicellular pollen grain stage) (Platt et al., 1998). Here, we report that, in the Ogu-INRA sterile anthers, total tapetum disintegration takes place much earlier, at the vacuolate microspore stage. The first signs of subcellular alterations, related with normal tapetum PCD, were observed at the mid-microspore phase and consisted of some chromatin condensation. However, mitochondria degenerated much earlier, at the tetrad phase. In male-sterile anthers of Beta vulgaris, the earliest irregularities in the cell ultrastructure were also found in the tapetum, at the tetrad stage, affecting the mitochondria (Majewska-Sawka et al., 1993). It is likely, therefore, that the energy necessary to sustain the nutritive and secretory functions of the tapetum cannot be satisfactorily provided in the sterile plants from the early pollen developmental stages. This could lead to a severe cell fatigue, which would prematurely trigger the signals of PCD as previously suggested for the PET1 CMS in sunflower (Balk and Leaver, 2001; Sabar et al., 2003).
Nevertheless, as opposed to PET1-CMS in sunflower, the subsequent events in the sterile tapetum of Ogu-INRA CMS plants did not follow the normal, programmed tapetum degeneration pattern described in this species by Platt et al. (1998). In the former, the initial persistence of mitochondria has been described during the tapetum PCD; while in the latter case, the ultrastructure of the mitochondria was dramatically altered before other morphological signals of cell death were appreciated. In tapetal cells of Ogu-INRA CMS plants, instead of a progressive cell disorganization and the intense accumulation and final release of lipidic and proteinaceous reserves, the whole content of the cells was extruded into the anther locule in an uncontrolled cell burst. At the end of the process, the former tapetal area and even the space in between the microspores was filled with a mixture of vesicles, organelle-containing cytoplasm portions, and remnants of the cell nucleus and apparently unaltered plastids. Therefore plastids persisted until the last stages of tapetum degeneration in sterile plants. This is also true for fertile plants, when normal tapetum PCD occurs. These observations support the idea that plastids may play an important role during the degeneration (or senescence) of plant cells or tissues (non-photosynthetic), as suggested for legume nodules (Lucas et al., 1998).
Interestingly, several male-sterile mutants have been previously described that share some characteristics with the Ogu-INRA CMS. In the Arabidopsis mutant ms1, whose microspores abort after release from the tetrads, tapetal cell breakdown does not occur by the normal process of PCD and follows an alternative cell death process, with tapetal cells becoming highly vacuolated in the early stages and degenerating prematurely (Vizcay-Barrena and Wilson, 2006). The results presented in this work and those described by Vizcay-Barrena and Wilson (2006) in Arabidopsis support the idea that, if active mitochondria are necessary when tapetum degeneration occurs during PCD, a more passive process of tapetum cell death might have no need for active mitochondria. In the ms1 mutant, as well as in male-sterile plants where the function of the AtMYB103 gene was blocked (Li et al., 2007), pollen wall formation is aberrant with intine and deposition of exine incomplete, reminding us of our observations on the poor exine deposition on mid-microspores of Ogu-INRA sterile plants. In Allium, an abnormal tapetum behaviour causing male sterility was characterized by extremely early degeneration and tapetum hypertrophy and autolysation (Holford et al., 1991). Similarly, in the (Capsicum annuum L.) cytoplasmic male-sterile line CMS 21A, an abnormally swollen tapetum pressed against the uni-nucleate microspores and the absence of sporopollenine were reported (Luo et al., 2006).
The vacuolate microspores in Ogu-INRA CMS showed ultrastructural markers of PCD: chromatin condensation at the periphery of the nucleus, expansion of the RER cisternae to confine portions of the cytoplasm, and rupture of the tonoplast. Degenerating microspore mother cells of cytoplasmic triazine-resistant male sterile (ctr) lines of B. napus appeared to develop numerous endoplasmic reticulum-derived vesiculated structures, which might be involved in the lysis of organelles (Grant et al., 1986). The tapetal dysfunction in the Arabidopsis mutant ms1 may subsequently induce PCD in the microspores, blocking the process of pollen development (Vizcay-Barrena and Wilson, 2006).
The observations presented in this work therefore indicate that the deleterious effect of the orf138 gene expression starts as early as the tetrad stage, and affects the structure of mitochondria in tapetal cells, but not in microspores, although immunolocalization of the ORF138 protein in sterile anthers showed that the sterility protein accumulates in microspores (Uyttewaal, 2007). It leads to an uncontrolled and premature cell death process of the tapetum, in turn affecting the viability of the developing pollen, which finally aborts at the stage of vacuolate microspore.
|
Acknowledgements |
|---|
We thank Alfred Martin-Canadell for growing and looking after the plants; Maria Carmen Risueño (CIB-CSIC) for giving us access to the photomicroscope equipped with a digital camera; Fernando Pinto and Sara Paniagua (Electron Microscopy Service of the CCMA-CSIC) for their technical support. PGM was funded by the programme ‘Ramón y Cajal’ from the Spanish Ministry of Education and Science; MU was funded by the Plant Biology and Plant Genetics and Breeding Research Departments of INRA; JRHM was funded by the VERT Marie Curie Early Training Site. This work was partially funded by the French–Spanish bilateral action Picasso project (French side) and Acción Integrada HF2004-0240 (Spanish side), with the support of the French Ministry of Foreign Affairs (EGIDE) and the Spanish Ministry of Education and Science, respectively.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Alexander MP. Differential staining of aborted and non aborted pollen. Stain Technology (1969) 44:117–122.[Web of Science][Medline]
Audran JC, Bouillot J. Sporoderm infrastructural and cytochemical modifications in cytoplasmic male-sterile broad-bean (Vicia faba L.). Acta Societatis Botanicorum Poloniae (1981) 50:223–226.[Web of Science]
Balk J, Leaver CJ. The PET1-CMS mitochondrial mutation in sunflower is associated with premature programmed cell death and cytochrome c release. The Plant Cell (2001) 13:1803–1818.
Bannerot H, Boulidard L, Cauderon Y, Tempé J. Transfer of cytoplasmic male sterility from Raphanus sativus to Brassica oleracea. Proceedings EUCARPIA Crop Section, Cruciferae (1974) 25:52–54.
Bannerot H, Boulidard L, Chupeau Y. Unexpected difficulties met with the radish cytoplasm in Brassica oleracea. Eucarpia Cruciferae Newsletter (1977) 2:16.
Bedinger P. The remarkable biology of pollen. The Plant Cell (1992) 4:879–887.
Bino RJ. Ultrastructural aspects of cytoplasmic male sterility in Petunia hybrida. Protoplasma (1985) 127:230–240.[CrossRef][Web of Science]
Bonhomme S, Budar F, Lancelin D, Small I, Defrance MC, Pelletier G. Sequence and transcript analysis of the Nco2.5 Ogura-specific fragment correlated with cytoplasmic male sterility in Brassica cybrids. Molecular and General Genetics (1992) 235:340–348.[CrossRef]
Brown GG, Formanova N, Jin H, Wargachuk R, Dendy C, Patil P, Laforest M, Zhang J, Cheung WY, Landry BS. The radish Rfo restorer gene of Ogura cytoplasmic male sterility encodes a protein with multiple pentatricopeptide repeats. The Plant Journal (2003) 35:262–272.[CrossRef][Web of Science][Medline]
Chase CD. Cytoplasmic male sterility: a window to the world of plant mitochondrial–nuclear interactions. Trends in Genetics (2007) 23:81–90.[CrossRef][Web of Science][Medline]
Delourme R, Foisset N, Horvais R, Barret P, Champagne G, Cheung WY, Landry BS, Renard M. Characterisation of the radish introgression carrying the Rfo restorer gene for the Ogu-INRA cytoplasmic male sterility in rapeseed (Brassica napus L.). Theoretical and Applied Genetics (1998) 97:129–134.[CrossRef][Web of Science]
Desloire S, Gherbi H, Laloui W, et al. Identification of the fertility restoration locus, Rfo, in radish, as a member of the pentatricopeptide-repeat protein family. EMBO Reports (2003) 4:588–594.[CrossRef][Web of Science][Medline]
Fedorova E, Thomson R, Whitehead LF, Maudoux O, Udvardi MK, Day DA. Localization of H+-ATPase in soybean root nodules. Planta (1999) 209:25–32.[CrossRef][Web of Science][Medline]
Giancola S, Marhadour S, Desloire S, et al. Characterization of a radish introgression carrying the Ogura fertility restorer gene Rfo in rapeseed, using the Arabidopsis genome sequence and radish genetic mapping. Theoretical and Applied Genetics (2003) 107:1442–1451.[CrossRef][Web of Science][Medline]
Gourret J, Delourme R, Renard M. Expression of ogu cytoplasmic male sterility in cybrids of Brassica napus. Theoretical and Applied Genetics (1992) 83:549–556.[Web of Science]
Grant I, Beversdorf WD, Peterson RL. A comparative light and electron microscopic study of microspore and tapetal development in male fertile and cytoplasmic male sterile oilseed rape (Brassica napus). Canadian Journal of Botany (1986) 64:1055–1068.
Hanson MR, Bentolila S. Interactions of mitochondrial and nuclear genes that affect male gametophyte development. The Plant Cell. (2004) 16(Supplement):S154–S169.
Havey MJ. The use of cytoplasmic male sterility for hybrid seed production. In: Molecular biology and biotechnology of plant organelles—Daniell H, Chase C, eds. (2004) Springer. 623–634.
Hernould M, Suharsono, Zabaleta E, Carde JP, Litvak S, Araya A, Mouras A. Impairment of tapetum and mitochondria in engineered male-sterile tobacco plants. Plant Molecular Biology (1998) 36:499–508.[CrossRef][Web of Science][Medline]
Holford P, Croft J, Newbury HJ. Structural studies of microsporogenesis in fertile and male-sterile onions (Allium cepa L.) containing the cms-S cytoplasm. Theoretical and Applied Genetics (1991) 82:745–755.[Web of Science]
Horner HT. A comparative light- and electron-microscopic study of microsporogenesis in male-fertile and cytoplasmic male-sterile sunflower (Helianthus annuus). American Journal of Botany (1977) 64:745–759.[CrossRef][Web of Science]
Kawanabe T, Ariizumi T, Kawai-Yamada M, Uchimiya H, Toriyama K. Abolition of the tapetum suicide program ruins microsporogenesis. Plant Cell Physiology (2006) 47:784–787.
Koizuka N, Imai R, Fujimoto H, Hayakawa T, Kimura Y, Kohno-Murase J, Sakai T, Kawasaki S, Imamura J. Genetic characterization of a pentatricopeptide repeat protein gene, orf687, that restores fertility in the cytoplasmic male-sterile Kosena radish. The Plant Journal (2003) 34:407–415.[CrossRef][Web of Science][Medline]
Krishnasamy S, Makaroff CA. Characterization of the radish mitochondrial orfB locus: possible relationship with male sterility in Ogura radish. Current Genetics (1993) 24:156–163.[CrossRef][Web of Science][Medline]
Ku S, Yoon H, Suh HS, Chung Y-Y. Male-sterility of thermosensitive genic male-sterile rice is associated with premature programmed cell death of the tapetum. Planta (2003) 217:559–565.[CrossRef][Web of Science][Medline]
Li SF, Iacuone S, Parish RW. Suppression and restoration of male fertility using a transcription factor. Plant Biotechnology Journal (2007) 5:297–312.[CrossRef][Web of Science][Medline]
Li N, Zhang DS, Liu HS, et al. The rice tapetum degeneration retardation gene is required for tapetum degradation and anther development. The Plant Cell (2006) 18:2999–3014.
Lucas MM, Van de Sype G, Hérouart D, Hernández MJ, Puppo A, de Felipe MR. Immunolocalization of ferritin in determinate and indeterminate legume root nodules. Protoplasma (1998) 204:61–70.[CrossRef][Web of Science]
Luo X-D, Dai1 L-F, Wang S-B, Wolukau JN, Jahn M, Chen J-F. Male gamete development and early tapetal degeneration in cytoplasmic male-sterile pepper investigated by meiotic, anatomical and ultrastructural analyses. Plant Breeding (2006) 125:395–399.[CrossRef][Web of Science]
Ma H. Molecular genetic analyses of microsporogenesis and microgametogenesis in flowering plants. Annual Review Plant Biology (2005) 56:393–434.[CrossRef]
Majewska-Sawka A, Rodriguez-Garcia MI, Nakshima H, Jassen B. Ultrastructural expression of cytoplasmic male sterility in sugar bett (Beta vulgaris L.). Sexual Plant Reproduction (1993) 6:22–32.[Web of Science]
McCormick S. Control of male gametophyte development. The Plant Cell. (2004) 16(Supplement):S142–S153.
Murayama K, Yahara T, Terachi T. Variation of female frequency and cytoplasmic male-sterility gene frequency among natural gynodioecious populations of wild radish (Raphanus sativus L.). Molecular Ecology (2004) 13:2459–2464.[CrossRef][Medline]
Ogura H. Studies on the new male sterility in japanese radish, with special references to utilization of this sterility towards the practical raising of hybrid seeds. Memoirs of the Faculty of Agriculture of Kagoshima University (1968) 6:39–78.
Papini A, Mosti S, Brighigna L. Programmed cell-death events during tapetum development of angiosperms. Protoplasma (1999) 207:213–221.[CrossRef][Web of Science]
Pelletier G, Primard C, Vedel F, Chétrit P, Rémy R, Rousselle P, Renard M. Intergeneric cytoplasmic hybridization in Cruciferae by protoplast fusion. Molecular and General Genetics (1983) 191:244–250.[CrossRef]
Platt KA, Huang AHC, Thomson WW. Ultrastructural study of lipid accumulation in tapetal cells of Brassica napus L. cv. Westar during microsporogenesis. International Journal of Plant Science (1998) 159:724–737.[CrossRef][Web of Science]
Polowick PL, Sawhney VK. Microsporogenesis in a normal line and the ogu cytoplasmic male-sterile line of Brassica napus. I. The influence of high temperatures. Sexual Plant Reproduction (1990) 3:263–276.[Web of Science]
Polowick PL, Sawhney VK. Microsporogenesis in a normal line and the ogu cytoplasmic male-sterile line of Brassica napus. II. The influence of intermediate and low temperatures. Sexual Plant Reproduction (1991) 4:22–27.[Web of Science]
Sabar M, Gagliardi G, Balk J, Leaver CJ. ORFB is a subunit of F(1)F(O)-ATP synthase: insight into the basis of cytoplasmic male sterility in sunflower. EMBO Reports (2003) 4:1–6.[Medline]
Scott RJ, Spielman M, Dickinson HG. Stamen structure and function. The Plant Cell. (2004) 16(Supplement):S46–S60.
Uyttewaal M. Caractérisation biochimique de protéines de la famille Pentatricopeptide repeat chez Arabidopsis thaliana et le colza. (2007) Thesis, University Paris-Sud XI, Orsay, France.
Vizcay-Barrena G, Wilson ZA. Altered tapetal PCD and pollen wall development in the Arabidopsis ms1 mutant. Journal of Experimental Botany (2006) 57:2709–2717.
Wu H-M, Cheung AY. Programmed cell death in plant reproduction. Plant Molecular Biology (2000) 44:267–281.[CrossRef][Web of Science][Medline]
Wu SSH, Platt KA, Ratnayake C, Wang TW, Ting JTL, Huang AHC. Isolation and characterization of novel neutral-lipid-containing organelles and globuli-filled plastids from Brassica napus tapetum. Proceedings of the National Academy of Sciences, USA (1997) 94:12711–12716.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  N. Szydlowski, P. Ragel, S. Raynaud, M. M. Lucas, I. Roldan, M. Montero, F. J. Munoz, M. Ovecka, A. Bahaji, V. Planchot, et al. Starch Granule Initiation in Arabidopsis Requires the Presence of Either Class IV or Class III Starch Synthases PLANT CELL, August 1, 2009; 21(8): 2443 - 2457. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Uyttewaal, N. Arnal, M. Quadrado, A. Martin-Canadell, N. Vrielynck, S. Hiard, H. Gherbi, A. Bendahmane, F. Budar, and H. Mireau Characterization of Raphanus sativus Pentatricopeptide Repeat Proteins Encoded by the Fertility Restorer Locus for Ogura Cytoplasmic Male Sterility PLANT CELL, December 1, 2008; 20(12): 3331 - 3345. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||