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Keywords:

  • Arabidopsis thaliana;
  • Brassica napus;
  • cytoplasmic male sterility;
  • homeotic conversion

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary Material
  9. References
  10. Supporting Information

Homeotic conversions of anthers were found in cytoplasmic male sterile (CMS) plants of Brassica napus derived from somatic hybrids of B. napus and Arabidopsis thaliana. CMS line flowers displayed petals reduced in size and width and stamens replaced by carpelloid structures. In order to investigate when these developmental aberrations appeared, flower development was analysed histologically, ultrastructurally and molecularly. Disorganized cell divisions were detected in the floral meristems of the CMS lines at stage 4. As CMS is associated with mitochondrial aberrations, ultrastructural analysis of the mitochondria in the floral meristems was performed. Two mitochondrial populations were found in the CMS lines. One type had disrupted cristae, while the other resembled mitochondria typical of B. napus. Furthermore, expression patterns of genes expressed in particular floral whorls were determined. In spite of the aberrant development of the third whorl organs, BnAP3 was expressed as in B. napus during the first six stages of development. However, the levels of BnPI were reduced. At later developmental stages, the expression of both BnAP3 and BnPI was strongly reduced. Interestingly the expression levels of genes responsible for AP3 and PI activation such as LFY, UFO and ASK1 were higher in the CMS lines, which indicates that activation of B-genes in the CMS lines does not occur as in B. napus. Disrupted and dysfunctional mitochondria seem to be one of the first aberrations manifested in CMS which result in a retrograde influence of the expression levels of genes responsible for the second and third whorl organ differentiation.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary Material
  9. References
  10. Supporting Information

Floral organogenesis is influenced by nuclear–mitochondrial interactions as most notably demonstrated by cytoplasmic male sterility (CMS), which is a mitochondrially inherited trait manifested by failure to produce pollen (reviewed by Budar and Pelletier, 2001; Schnable and Wise, 1998). Besides the absence of viable pollen, abnormal development of anthers and other floral organs is often observed in CMS plants. For example, homeotic conversion of stamens into carpelloid structures has been described for CMS lines of rapeseed (Leino et al., 2003), tobacco (Farbos et al., 2001; Kofer et al., 1991), carrot (Linke et al., 2003) and wheat (Murai et al., 2002). These phenotypes resemble those of Arabidopsis floral homeotic gene mutants APETALA3 (AP3) and PISTILLATA (PI), in which stamens are replaced by carpelloid organs in the third whorl. However, in these mutants the petals are also converted to sepal-like organs in whorl 2 (Bowman et al., 1989; Jack et al., 1992, 1994). Although petals in CMS plants retain their identity, they can be reduced in size and changed in colour (Farbos et al., 2001; Leino et al., 2003). In this respect, flowers formed in most CMS plants resemble weak double mutant plants for the UNUSUAL FLORAL ORGANS gene (UFO) and one of the SCFUFO complex genes such as the Arabidopsis SKP1-LIKE (ASK1) gene or the AtCUL1 gene in which anthers are replaced by carpelloid or filamentous structures, and petals are often reduced in size (Ni et al., 2004; Wang et al., 2003; Zhao et al., 1999).

The identity of organs in whorl three is dependent upon the correct expression of AP3, PI and AGAMOUS (AG) (Bowman et al., 1991a; Coen and Meyerowitz, 1991). More recently, the action of other genes and co-factors was shown to be necessary for correct AP3 and PI activation (Jack, 2004; Lamb et al., 2002). The expression of AP3 and PI has two distinct phases. Initially, during the first six floral stages, AP3/PI activation is directly and indirectly dependent on the activity of the meristem identity genes LFY and AP1 (Lamb et al., 2002; Ng and Yanofsky, 2001; Weigel and Meyerowitz, 1993). After stage 6, when sepals completely enclose the floral bud (Smyth et al., 1990), AP3 and PI activation is facilitated by an autoregulatory pathway that is dependent on the relative concentrations of AP3 and PI proteins (Hill et al., 1998; Jack et al., 1992, 1994). In this later phase, when AP3 and PI mainly are involved in the regulation of organ growth in whorls 2 and 3, the expression of AP3 and PI is also regulated by UFO and ASK1 (Laufs et al., 2003; Samach et al., 1999; Zhao et al., 2001) by interacting genetically with LEAFY (LFY), a direct activator of AP3 and PI (Samach et al., 1999; Wang et al., 2003).

Precise control of cell division is important for normal floral organogenesis. Floral organ initiation results from patterned control of the numbers, loci, and planes of cell division, in accordance with the regulation of cell expansion (Meyerowitz, 1997). In the CMS lines of Brassica napus and Nicotiana tabacum, the second and third whorl organs are often smaller due to fewer cells (Farbos et al., 2001; Leino et al., 2003). In addition, modified cell division patterning in the third whorl organ primordia was reported (Farbos et al., 2001) similar to modifications in the size of the organ primordia (Linke et al., 2003). Organ identity genes are not required for the initiation of floral organogenesis. They rather alter the identity of organs but do not perturb the number or position of floral primordia within the whorl.

The CMS B. napus lines included in this study have the nuclear genome of B. napus but mitochondria with DNA derived mainly from Arabidopsis thaliana (Leino et al., 2003). These lines are characterized by homeotic conversions of stamens into carpelloid structures and smaller petals. These phenotypic aberrations suggest that the genes responsible for anther identity and formation are misregulated. Thus, genes known to be upstream of the B-genes AP3 and PI such as LFY, UFO and ASK1, as well as the B-genes and AG gene were analysed by determining their levels and patterns of expression. The results revealed that the expression patterns of all the genes in the CMS lines were analogous to those in B. napus, although the levels of AP3 and PI decreased drastically after stage 6 in the CMS lines. In contrast, the levels of LFY and UFO transcripts in the CMS lines increased at these later floral stages in comparison with B. napus. Structural alterations were observed in the mitochondria very early in flower development and seem to be one of the first phenotypic manifestations of the rearranged mtDNA between B. napus and A. thaliana.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary Material
  9. References
  10. Supporting Information

Floral development

All flowers in the CMS lines 4:19 and 41:17 investigated in this study were phenotypically similar to each other. For morphological overviews we refer to Leino et al. (2003). Stamens in B. napus were composed of a filament and an anther (Figure 1a). In contrast, third whorl floral organs in the CMS lines resembled an unfused carpel with ovule-like structures appearing at the internal margins of the organ (Figure 1b,c). The tip of these organs was covered with stigmatic-like cells (Figure 1b).

image

Figure 1. Phenotypic characterization of flower development in Brassica napus and the two CMS lines. (a) Fully developed B. napus stamen. (b) Third whorl organ of the CMS line 4:19. (c) Magnification of the ovule-like structure formed at the unfused margin of the carpel-like organ. (d–f) Inflorescences showing the phyllotaxis. Numbers relate the sequence of floral bud appearance in (d) B. napus, (e) CMS line 4:19, (f) CMS line 41:17. (g–i) Size of the floral meristem enclosed between the lateral sepals of (g) B. napus, (h) CMS line 4:19 and (i) CMS line 41:17. (j) Flower buds at stage 9. From left to right: B. napus, CMS line 4:19, CMS line 41:17. (k) Flower buds at stage 12 (petals are as long as the length of the lower stamen). From left to right: B. napus, CMS line 4:19, CMS line 41:17. (l) Flower buds at stage 12. Two sepals and two petals were removed. From left to right: B. napus, CMS line 4:19, CMS line 41:17. (m) Open flower of B. napus. (n) Open flower of the CMS line 4:19. (o) Open flower of the CMS line 41:17. Sepals were removed in (n) and (o). Nectaries are indicated by arrows in the base of the third whorl organs. SEM visualization of flower buds and petal epidermal surfaces. (p) Flower bud of B. napus at stages 9 and 10. At stage 12, petals completely cover the anthers, therefore, in order to visualize anthers, petals and sepals, flower buds at stage 10 were chosen for B. napus. (q) Flower bud of the CMS line 4:19 at stage 12. (r) Flower bud of the CMS line 41:17 at stage 12. Ovule-like structures appear at the margin of the third whorl organs (arrow). Detailed view of the base of the flower buds with the nectaries indicated by arrows: (s) B. napus, (t) CMS line 4:19 and (u) CMS line 41:17. Adaxial surfaces of petals: (v) B. napus and (w) CMS line 4:19. Abaxial surface in petal: (x) B. napus and (y) CMS line 4:19. Bar (b, j–l): 1 mm, (m–o) 2 mm (s–u): 100 μm. an, anther; cl, carpelloid structure; fl, filament; ls, lower sepal; ol, ovule-like structure; p, petal; pi, pistil; s, sepal; sl, stigmatic-like cell; S4, floral stage 4; us, upper sepal.

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The stages of floral development in B. napus that were determined by Polowick and Sawhney (1986) resembled the stages defined by Smyth et al. (1990) for A. thaliana. We used the stage definitions by Smyth et al. (1990).

By scanning electron microscopy (SEM) studies we found that floral bud phyllotaxis did not differ between the CMS plants and B. napus (Figure 1d–f). No differences were found between B. napus and the two CMS lines until stage 4 (Figure 1g–i). The size of the floral meristem enclosed between the lateral sepals was the same (98–100 μm) in both B. napus and the two CMS lines (Figure 1g–i). However, by stage 9, when all flower organs were established, including the final shape of the anthers, phenotypic differences between flowers of the CMS lines and B. napus were detected. Even though the length of the floral buds of B. napus and the two CMS lines was similar throughout flower development (Figure 1j–l), the petals of the CMS lines were narrower and shorter compared with those of B. napus (Figure 1j). By stage 9 when the anthers in B. napus reach the stigma, the CMS lines displayed carpelloid organs with ovule-like structures differentiating at the margins (Figure 1r), instead of anthers. Furthermore, the petals were narrower and shorter than in B. napus (Figure 1j) and at stage 12, the CMS petals had a curly appearance (Figure 1k,q,r). Even though whorl 2 and 3 organs were morphologically smaller and different in the CMS plants, the overall floral bud length was the same because both the pistil and the sepals were of the same size and shape as in B. napus (Figure 1j–l) (Leino et al., 2003).

Despite the fact that the carpelloid structures are not stamens, their arrangement in the flowers was the same as the arrangement of stamens in B. napus, that is four upper and two lower (Figure 1m–o,s–u) and nectaries were present at their bases. The spatial distribution of all other floral organs in flowers of the CMS lines was similar to that found in B. napus.

Additional phenotypic changes were found in CMS flowers arising later in the same inflorescence. The CMS line 4:19 developed 5.8 ± 0.5 (n = 30) carpelloid structures, while B. napus displayed a constant number of stamens (6.0 ± 0.0, n = 30). Furthermore, in CMS line 41:17, the carpelloid structures had a more curly feature (Figure 1o) than those formed in earlier flowers (Figure 1r).

Scanning electron microscopy analysis of B. napus petal surfaces revealed that the adaxial cells are conical in shape (Figure 1v), while the abaxial cells are more dome shaped (Figure 1x). Within each surface, all cells were uniform in size and shape and all revealed epicuticular ridges. In the CMS petals, on the contrary, the polarity between the adaxial and the abaxial side was not noticeable and instead, the two epidermal surfaces resembled each other with a tendency for abaxialization of the adaxial side (Figure 1w,y). On both surfaces of the CMS petals, cells of different sizes were found (Figure 1w,y). Petals in the CMS lines were smaller as a result of the reduced number of cells. In conclusion, the phenotypic analysis throughout flower development showed that CMS lines display floral alterations only in the second and third whorls.

Histological and ultrastructural analyses of the early floral meristem

In the two CMS lines investigated in this study, the second and third whorl organs were reduced in size, a feature also found in CMS lines of tobacco (Farbos et al., 2001) and in carrot CMS plants (Linke et al., 2003). To determine if this was due to aberrant cell division, we performed studies of cell division patterns in early floral meristems. Disorganization in the planes of cell division was found in young floral buds (stages 4 and 5) at the sites of the putative floral whorls 2 and 3 from which the petals and stamens will arise. Thus, the well established L1–L3 meristematic cell layers typical for B. napus (Figure 2a,b) were not truly maintained in the two CMS lines (Figure 2c–f). Another feature was the reduced cytoplasmic density due to higher numbers of small vacuoles (Figure 2a–f). These early alterations indicate that the CMS condition affects mechanisms that regulate the cell patterning in areas of the flower meristem that later on will give rise to second and third whorl organs.

image

Figure 2. Histological studies of flower development. Flower buds at floral stages 4 and 5 of (a) Brassica napus. (b) Magnification of the presumptive whorls 2 and 3. (c) Flower bud at stages 4 and 5 of the CMS line 4:19. (d) Magnification of the presumptive whorls 2 and 3. (e) Section of flower bud at stages 4 and 5 of the CMS line 41:17. (f) Magnification of the presumptive whorls 2 and 3. (g) Section of flower bud at stages 7 and 8 of B. napus. (h) Magnification of the anther primordium containing densely stained cells in the archesporial cells (star) surrounded by less stained cells that will give rise to the epidermis (arrow). (i) Thin section of flower bud at stages 7 and 8 of the CMS line 4:19. (j) Magnification of the third whorl organ primordium. (k) Thin section of flower bud at stage 9 of the CMS line 41:17. (l) Magnification of the third whorl organ with the ovule-like structure beginning to differentiate (arrow). Bar: (a, c and e) 20 μm; (b, d and f) 8 μm; (g, i and k) 100 μm; (h, j and l) 25 μm. anp, anther primordia; ca, carpel; clp, carpelloid primordium; cl, carpelloid structure; L1–3, meristematic cell layer.

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Observations of young developing anthers in thin cross sections revealed that in B. napus, anther compartments started to differentiate with archesporial cells acquiring their identity at stages 7 and 8 (Figure 2g,h), as shown by the density of the cytoplasm and the large nucleus present in the cells (Figure 2h). The archesporial cells that originated from the L2 layer were surrounded by more vacuolated cells that give rise to the epidermis of the anther (Figure 2h).

In contrast to true anther primordia, the carpelloid primordia formed in the CMS lines were not as wide and the cells were uniform throughout the organ (Figure 2i,j). Later in floral development, all different tissues that constitute the anthers in B. napus had differentiated and the two carpels were formed (data not shown). However, in the CMS lines, third whorl organs mainly elongated and the only further tissue differentiation was the formation of ovule-like structures at the margins of the organ followed later on by the development of stigmatic cells at the tip of the organs (Figures 1b and 2i–l). These histological studies reveal that CMS lines are incapable of sporogenous tissue differentiation followed by the homeotic conversion of male organs into carpelloid tissue. Such phenotypic alterations in the CMS lines are closely related to the fact that CMS-associated novel mitochondrial genes accumulate more in sporogenous initial cells as has been the case in CMS sunflower plants (Smart et al., 1994). In situ hybridization of B. napus (nap) CMS lines revealed that transcripts from the CMS-associated mitochondrial orf were preferentially localized to microsporangia (Geddy et al., 2005).

As the CMS trait is regulated by the mitochondria, it was of interest to investigate the mitochondrial structures in floral tissues. Ultrastructural studies revealed that mitochondria in B. napus flower buds at stages 4 and 6 had a large number of mitochondria that displayed well-developed cristae (Figure 3a,d). This is in contrast to the two types of mitochondria found in the CMS cells of the putative whorls 2 and 3 and of the carpelloid organ primordia. One type resembled B. napus mitochondria, although they were reduced in size and in number, while the other type lacked the cristae and the dense matrix (Figure 3b,c,e,f). In some of the mitochondria in the CMS lines, the outer membrane was ruptured (Figure 3e). The disrupted assembly of the inner membrane system present in the CMS mitochondria could reflect the presence of recombined mtDNA in the CMS lines (Leino et al., 2003).

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Figure 3. Comparison of mitochondria in Brassica napus and in the CMS lines. (a) Ultrastructural view of putative whorl 3 from a B. napus flower bud at stage 4. Mitochondria occupy a large portion of the cell. (b) Ultrastructural view of the putative whorl 3 from a CMS line 4:19 flower bud at stage 4. Mitochondria are small, lack cristae and most of the matrix content. (c) Ultra-thin section of putative whorl 3 from CMS line 41:17 flower bud at stage 4. (d) Ultra-thin section of stamen primordium from B. napus at stage 7. (e) Ultra-thin section of third whorl primordium from CMS line 4:19 at stage 6. (f) Ultra-thin section of third whorl primordia from CMS line 41:17 at stage 6 of flower development. mt, mitochondria; pl, plastid; v, vacuole. Bar: 1 μm.

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Transcript levels determined by real-time RT-PCR

Phenotypically, flower development in the CMS plants resembles that in its B. napus counterpart until stage 6/7 is reached but by the time third whorl organs begin to differentiate, homeotic modifications appear. We therefore determined the expression levels of several relevant genes such as BnLFY, BnUFO, BnASK, BnAG, BnAP3 and BnPI. All these genes are known for their involvement in petal and stamen formation (Bowman et al., 1991a; Coen and Meyerowitz, 1991; Zhao et al., 2001). Because of alterations related to cell division in second and third whorl floral organs (Figure 1) and cell patterning in flower meristems (Figure 2) of the CMS lines, transcription levels of BnTON1 and BnCycB1 were also analysed as both genes are mitosis-related (Capron et al., 2003; Torres-Ruiz and Jürgens, 1994). We performed expression level studies using two independent samples, one representing floral stages from 1 to 6 and one representing stages 7–9.

Stages 1–6

The genes BnLFY, BnUFO and BnASK formed group I, as they act upstream of the homeotic genes BnAG, BnAP3 and BnPI that formed group II. Their expression levels were analysed in the CMS lines in comparison with B. napus during the first six stages of flower development. ASK1 and UFO, together with other components, form the SCFUFO complex that interacts genetically with LFY and regulate AP3 and PI gene expression (Samach et al., 1999; Wang et al., 2003). Interestingly, the CMS line 4:19 revealed significantly higher transcript levels of these three genes than did B. napus flowers of stages 1–6 (Figure 4a).

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Figure 4. Results from real-time RT-PCR amplification of RNA isolated from Brassica napus, CMS lines 4:19 and 41:17 inflorescences containing floral buds from stages 1–6 and 7–9. Amplifications were performed with specific primers for: (a) BnASK1, BnUFO and BnLFY genes. (b) BnAG, BnAP3 and BnPI genes. (c) BnTON1 and BnCycB1 genes. Results were normalized to B. napus expression levels. Standard deviations levels are shown (n = 4). Letters indicate significant difference at the probability level P < 0.05 in comparison with B. napus.

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Of the group II genes, the expression levels of BnAG showed no significant difference between B. napus and the CMS lines (Figure 4b). This was also the case for BnAP3 in stages 1–6, while the expression was significantly reduced in floral bud stages 7–9 (Figure 4b). The expression levels of BnPI mRNA in the CMS lines were significantly reduced compared with the levels of B. napus floral buds in all stages (Figure 4b).

Histological studies of young flower meristems revealed alterations in the plane and order of cell divisions (Figure 2a–f). We therefore tested the transcription levels of two mitosis-related BnTON1 and BnCycB1 genes (group III). Their expression levels were higher in CMS line 4:19 compared with B. napus, while for line 41:17, the level was only slightly higher for BnTON1, whereas no statistical difference was found for either of the genes when compared with B. napus (Figure 4c).

Stages 7–9

In flower buds, the difference in expression levels of BnLFY and BnUFO between the CMS lines and B. napus was more accentuated (Figure 4a) during stages 7–9. However, no significant difference in BnASK expression level was noted between the CMS lines and B. napus at later stages of flower development (Figure 4a).

For the second group of genes, BnAP3, BnPI and BnAG, the expression levels were reduced in the CMS lines, especially for BnAP3 and BnPI (Figure 4b). Furthermore, the group III genes composed of BnTON1 and BnCycB1 exhibited reduced expression levels in the CMS lines (Figure 4c). In conclusion, the transcription levels of the B-genes in the CMS lines do not correspond to the levels of the transcripts of the genes directly responsible for BnAP3 and BnPI activation. As this activation does not occur properly, a response by upregulation of the upstream genes takes place. The exact reason for such blockage of the B-gene activation is, however, not known.

Spatial expression of LFY, AG, PI and AP3 genes during floral development

In order to determine whether homeotic conversions were associated only with differences in levels of gene expression or also with the pattern of gene expression, in situ hybridizations with BnLFY, BnAG, BnAP3 and BnPI were performed to visualize their expression patterns in flower meristems and young flower buds. In wild-type B. napus the mRNA of BnLFY was first detected in stage 2 during flower development (data not shown). Expression was present throughout flower development until stage 5. From this stage on, the BnLFY mRNA signal was excluded from the sepals but still present in petals, stamens and carpels (Figure 5a–c, data not shown). The expression pattern of BnLFY observed in the CMS lines was indistinguishable from B. napus (Figure 5a–c).

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Figure 5. Spatial mRNA expression using BnLFY, BnAG, BnPI, BnAP3, BnCycB1 and BnTON 1 Dig-labelled probes. BnLFY expression of (a) Brassica napus, (b) CMS line 4:19 and (c) CMS line 41:17. BnAG expression of (d) B. napus, (e) CMS line 4:19 and (f) CMS line 41:17. BnAP3 expression of (g) B. napus (arrowhead), (h) CMS line 4:19 (arrowhead) and (i) CMS line 41:17 (arrowhead). BnPI expression of (j) B. napus, (k) CMS line 4:19 and (l) CMS line 41:17. BnCycB1 expression of (m) B. napus, (n) CMS line 4:19 and (o) CMS line 41:17. BnTON1 expression of (p) B. napus, (q) CMS 4:19 and (r) CMS 41:17. BnTON1 expression in an inflorescence containing a stage 2 flower meristem: (s) B. napus and (t) CMS line 4:19. Flower stages are marked at the bottom of each figure. Bar: (a–r) 100 μm; (s,t) 20 μm. an, anther; cl, carpelloid structure; st, stamen primordium; w3–4, whorls 3 and 4.

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BnAG expression was detected in stages 2–4 in the centre of the meristem (data not shown). Later on, BnAG expression was confined to whorls 3 and 4 from which stamen and carpel primordia develop. No differences in the BnAG expression pattern in the CMS lines from the pattern in B. napus were found (Figure 5d–f).

BnAP3 was expressed as early as stage 3 (data not shown). Upon sepal differentiation (stage 4), transcripts were detected in the presumptive second and third whorls (Figure 5g–i) and from stage 5 onwards, BnAP3 was expressed throughout the developing petals and stamens in all three lines (data not shown). Until stamens and carpel-like organs were differentiated, the BnAP3 expression pattern in young CMS flower buds was similar to that of B. napus (Figure 5g–i). Later on, the accumulation of BnAP3 mRNA was similar to expression in the pistil. Signals were detected in the tip of the organ and in areas that resembled ovule-like structures (data not shown). This expression pattern is in accordance with the finding that the AP3 protein was expressed in the ovules of A. thaliana wild-type plants (Jack et al., 1994).

BnPI displayed an expression profile similar to BnAP3 but with an area more centred in the meristem (Figure 5j–l). As in the case of other genes, there was no difference in the expression pattern between B. napus and the two CMS lines.

Spatial expression of BnCycB1 and BnTON1 genes in young flower buds

Due to the disorganized plane of cell division formed mainly in presumptive whorls 2 and 3, in situ hybridizations were performed using probes for BnCycB1 and BnTON1. BnCycB1 mRNA was expressed in all mitotically active cells of flower tissues (Figure 5m–o). The expression level was high from stage 2 to stage 7. The carpel and stamen primordia showed a similar expression pattern in all the lines (Figure 5m–o). Later on in development, microsporogenous tissues and ovules showed the highest accumulation of transcripts in B. napus. The two CMS lines displayed the same pattern except for the microsporogenous tissue that was replaced by ovule-like structures (data not shown). As expected, BnTON1 expression resembled BnCycB1 expression (Figure 5p–r). A higher magnification image of the inflorescence area from B. napus and the CMS line 4:19 shows disorganized cell division in the CMS line (Figure 5s,t). In situ experiments showed that the formation of the homeotic organs in the CMS lines was not due to an incorrect expression of the homeotic genes in the floral whorls but rather due to differences in expression levels.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary Material
  9. References
  10. Supporting Information

Even though the CMS flower phenotypes resemble homeotic mutant phenotypes, homeotic genes in the CMS lines are not mutated. Rather, the aberrant phenotype is the result of the alloplasmic combination of a nucleus from one species with mitochondria from another species (Budar et al., 2003; Hanson and Bentolila, 2004). In those CMS systems that have been characterized molecularly, the different CMS-associated mitochondrial ORFs show very few similarities but still the products of the novel mtDNA regions result in similar effects (Hanson and Bentolila, 2004; Schnable and Wise, 1998). Furthermore, in most CMS systems, the new polypeptides encoded by CMS-induced mitochondrial genes are found in the mitochondrial membrane fractions or inside the mitochondria (Abad et al., 1995; Budar and Pelletier, 2001; Grelon et al., 1994; Song and Hedgcoth, 1994). Ultrastructural studies of the CMS lines revealed that the inner-membrane integrity of most mitochondria was disrupted in the flower tissues, while mitochondria in the fertile B. napus cultivar exhibited ordinary morphology. Farbos et al. (2001) reported similar results from studies of alloplasmic male-sterile lines of N. tabacum. Moreover, disrupted mitochondria were found in male-sterile tobacco plants obtained after transformation with an unedited copy of the mitochondrial atp9 gene (Hernould et al., 1998). Thus, once the structure of the mitochondria is disrupted in tissues that require a high energy level, male sterility can occur. Reduced ATP levels were found in the alloplasmic CMS lines included in this study (Teixeira et al., 2005) and in flower tissues of the alloplasmic CMS lines of tobacco (Bergman et al., 2000).

The homeotic conversion of anthers is correlated with decreased levels of AP3 and PI at later floral stages

A reduction in BnPI mRNA levels for the initial six stages was noted in CMS line 41:17 which has a more delayed flower development when compared with the CMS line 4:19. BnAG activation was not affected in the CMS plants during early flower development. At later stages, however, AG is only expressed in specific types of the ovule and stamen cells (Bowman et al., 1991b; Deyholos and Sieburth, 2000); this might account for the reduced BnAG levels we observed in the CMS lines after stage 6 as the CMS lines lack stamens which, thus, results in reduced numbers of cells accumulating BnAG mRNA.

Plants mutated in the SCFUFO complex genes resemble CMS plants in many aspects of vegetative and floral development by producing shorter plants and displaying conversion of anthers into carpelloid structures (Leino et al., 2003). Indeed, in such mutants, decreased levels of AP3 and PI expression were observed after stage 6 (Ni et al., 2004; Wang et al., 2003; Zhao et al., 1999), which was also found in our CMS system. ASK1 and UFO are components of the SCF complex which interacts genetically with LFY to facilitate the degradation of a negative regulator of B-class gene expression (Ni et al., 2004; Wang et al., 2003; Zhao et al., 2001). It is known from animal and yeast systems that association of F-box proteins with SCF complexes targets specific proteins for degradation (Patton et al., 1998). The levels of BnLFY and the two SCFUFO genes, BnUFO and BnASK1, were higher in the CMS lines but the accumulation of BnAP3 and BnPI mRNA was reduced. Reduction in the expression of these genes suggests that activation of the B-genes by the SCFUFO complex and LFY did not occur.

The apparent contradictory upregulated upstream genes and reduced expression levels of downstream genes might suggest that the phenotypes result from a blocked activation of BnAP3 and BnPI. Because anther development requires efficient mitochondrial activity (Mackenzie and McIntosh, 1999; Smart et al., 1994) during a short period of time, the reduced ATP levels formed in the flower tissues (Teixeira et al., 2005) might slow down the protein degradation necessary for the correct activation of AP3 and PI by the SCFUFO complex.

In turn, the BnAP3/PI autoregulation occurring after stage 6 of flower development (Goto and Meyerowitz, 1994; Jack et al., 1994; Samach et al., 1997) is not accomplished in CMS plants. At these later floral stages, BnLFY and BnUFO levels are higher in the CMS lines than in B. napus, suggesting that the role of these genes in activating BnAP3 and BnPI is affected. The reduced levels of BnPI, especially in the CMS line 41:17 at earlier floral stages, also reflect the hypothesized deficiency in the autoregulatory pathway, as the PI and AP3 genes are differentially activated (Honma and Goto, 2000; Tilly et al., 1998).

CycB1 proteins contain a motif that targets it for degradation through proteolysis (Capron et al., 2003). Tobacco plants expressing a non-degradable form of CycB1 showed mis-shaped cells, especially in mitotically active tissues like meristems. Accumulation of CycB1 mRNA was also associated with the mutant phenotype (Weingartner et al., 2004). Although the expression patterns revealed by in situ hybridizations of the two mitosis-related genes, BnCycB1 and BnTON1, showed no differences between the three lines, differences in expression levels were noticed for one of the CMS lines with real-time RT-PCR. BnCycB1 and BnTON1 mRNA concentration was higher in CMS line 4:19 compared with B. napus during the first floral stage.

The hypothesis that reduced energy levels produced by the mitochondria in CMS systems affect cell divisions in flower meristems and/or the general metabolism rate, leading to impairment of pollen production due to high energy demands during this process, was advanced by Farbos et al. (2001), Sabar et al. (2003) and Hanson and Bentolila (2004). We extend the hypothesis that the CMS phenotype in our Brassica lines is caused by reduced ATP levels in flower tissues (Teixeira et al., 2005) affecting a key mechanism responsible for proper cell cycling like the process of protein degradation driven by phosphorylation steps (Koepp et al., 1999).

Cytoplasmic male-sterile plants displaying a phenotype of abnormal anther formation found in the Brassica system reported here, in the alloplasmic lines of tobacco (Farbos et al., 2001) and in carrot (Linke et al., 2003) suggest that a common mechanism is involved. In all these CMS systems, the initial expression pattern of AP3 and PI homologous genes resembles their expression in the fertile parental lines whereas expression levels were reduced later in development. Among the homeotic genes, AP3 and PI genes have the most complex regulation in flower development involving numerous other genes and co-factors (Bowman et al., 1993; Honma and Goto, 2000; Lamb et al., 2002; Ng and Yanofsky, 2001). This complex regulation which must occur between stage 3 until third whorl organs are completely differentiated may not be achieved correctly in CMS systems, if they are hampered by reduced ATP levels. In this work we showed that the downregulation of BnAP3 and BnPI after floral stage 6 does not result from a downregulation of upstream genes. Rather, the regulation by BnLFY and the SCFUFO complex seems to be impaired.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary Material
  9. References
  10. Supporting Information

Plant material

Somatic hybrids between B. napus cv. Hanna and A. thaliana var. Landsberg erecta were produced by Forsberg et al. (1998) via protoplast fusion. The hybrids were backcrossed to B. napus cv. Hanna and the BC1 was screened for male sterility and aberrant flower phenotypes. In this study, two lines, 4:19 and 41:17, that displayed complete male sterility were selected. Each had been recurrently backcrossed to B. napus cv. Hanna as a pollinator to obtain a BC8 generation. Plants were grown in pots under controlled conditions in a climate chamber with 22/18°C day/night temperatures and a photoperiod of 16 h. Light intensity was 400 μmol m−2 sec−1 and the relative humidity was kept at 85%.

Light microscopy, transmission electron microscopy and SEM

Inflorescences from the B. napus cultivar and the two CMS lines were fixed with 2.5% glutaraldehyde in 0.05 m phosphate buffer, pH 6.8 (buffer A) at room temperature for 2 h, rinsed in buffer A, then post-fixed with 2% osmium tetroxide in buffer A for 2 h at room temperature. The material was rinsed three times in buffer A and dehydrated in a graded ethanol series (20, 40, 60, 80, 95 and 100%) and then gradually embedded in Spurr low-viscosity resin. Polymerization of the resin was carried out at 70°C for 12 h.

For light microscopy, 1 μm sections were cut with a glass knife on a Heidelberg HM 330 microtome (Microm, Heidelberg, Germany), floated on drops of distilled water on microscope slides and dried. Sections were stained with toluidine blue O, pH 4.5 in acetate buffer. All micrographs were taken with a Nikon Digital DS-5M L1 (Nikon Corporation, Tokyo, Japan).

For TEM studies, thin sections (90 nm) were stained with uranyl acetate and lead citrate and studied in a Philip CM 10 TEM (Phillips, Eindhoven, The Netherlands).

For SEM, whole inflorescences and different floral organs were fixed as described above until the dehydration step. The material was then critical point dried using CO2. The inflorescences were mounted on stubs and dissected using glass needles. After mounting the material was shadowed with gold and viewed with a JSM-6320F scanning microscope (JEOL, Tokyo, Japan).

In situ hybridization

All probes were obtained from B. napus cv. Hanna by PCR (primer sequences and PCR conditions are presented in Table S1) with subsequent cloning into pGEM-T easy vector. To prevent the possibility of cross-hybridization with other genes, the MADS box sequences from PI, AP3 and AG were avoided. For the LFY probe transcriptional factor-associated boxes were excluded. Fragments inserted into pGEM-T Easy Vector (Promega, Madison, WI, USA) were sequenced from both ends. Blast analysis revealed 95–97% identity with the A. thaliana genome for all the probes.

Inflorescences were fixed in 3.7% formaldehyde, 5% acetic acid and 50% ethanol. The material was then dehydrated with ethanol, cleared with histoclear and embedded in paraffin (Paraplast Plus; Sigma-Aldrich, Steinheim, Germany). Embedded tissues were sliced into serial 8 μm sections and attached to microscope slides that were coated with aminopropyltriethoxysilane (Sigma-Aldrich). Paraplast was removed by immersion in histoclear. Sections were rehydrated, incubated at 37°C for 25 min in a solution containing 1 μg ml−1 Proteinase K (Sigma), 100 mm Tris (pH 7.5), and 50 mm EDTA followed by two washes in H2O, 5 min each. After dehydration through an ethanol series, slides were air dried before application of the hybridization solution containing 75 ng per slide of DIG-labelled RNA probe, 50% formamide, 5% dextran sulphate, 1% blocking reagent and 200 μg ml−1 tRNA. After incubation in a humid box at 42°C overnight, slides were washed in 1x SSPE for 30 min at 45°C and incubated with 5 μg ml−1 RNase A for 30 min at 37°C and washed twice in RNAse buffer for 15 min at 37°C. Slides were then transferred to 0.5x SSPE for 5 min at 37°C and PBS for 5 min at room temperature. Samples were treated in 1% blocking reagent (Boehringer, Indianapolis, IN, USA) in maleic buffer (100 mm maleic acid, 150 mm NaCl, pH 7.5) for 45 min, followed by washing in BXT (1% BSA, 100 mm Tris, 150 mm NaCl and 0.3% Triton X-100, pH 7.5) for 45 min. The anti-digoxigenin-alkaline-phosphatase-couple antibody (Boehringer) was diluted 1:600 in BXT solution and a volume of 150 μl was applied to each slide. The sections were covered with a cover slip and incubated for 2 h, then washed (2 times, 20 min each) in BXT solution. Fresh colour-substrate solution (NBT and BCIP in 100 mm Tris, 50 mm MgCl2 and 100 mm NaCl, pH 9.5) was applied to each slide and incubated in the dark at room temperature. The signal was monitored starting at 2 h. Pictures were taken with a Nikon digital DS-5M L1 as soon as a signal was detected.

Real-time RT-PCR

RNA was extracted from inflorescences including flower buds from stage 1 to stage 6 (Smyth et al., 1990) and from flower buds from stage 7 to stage 9. Two biological samples were collected, each one containing large numbers of inflorescences from different plants. cDNA was synthesized using Superscript II RNase-Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Real-time RT-PCR reactions were carried out using an ABI Prism 7000 Sequence Detector (Applied Biosystems, Foster City, CA, USA) in MicroAmp Optical 96-well reaction plates with optical covers, according to the manufacturer's instructions. PCR reactions (final volume of 25 μl) were performed containing gene-specific primers and the passive reference dye ROX, in order to normalize fluorescence across the plate. Two templates using two independent biological RNA extractions were used. Reaction conditions were: 50°C for 2 min, 94°C for 10 min, followed by 40 cycles of 94°C for 15 sec, 60°C for 1 min. Primers were designed using Primer express software (Applied Biosystems) to flank introns so that genomic DNA contaminations would not amplify. Relative quantification values and standard deviations were calculated using the standard curve method according to the manufacturer's instructions (ABI Prism 7000 Sequence Detection System User Guide). Values were normalized to the B. napus line and results analysed with Microsoft Excel Software.

Data were analysed with one-way anova using the general linear model procedure in the MINITAB14 software. Pairwise comparisons were performed using the Bonferroni test. Each measurement was treated as an independent value.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary Material
  9. References
  10. Supporting Information

We are grateful to Professor Eva Sundberg and Dr Jens Sundström for helpful discussions, I. Eriksson for excellent laboratory assistance, A. Axén and at H. Ekwall for electron microscopy support and Sheila McCormick for editorial help. This work was supported by the Swedish Research Council (VR) and the Swedish research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS). R. T. Teixeira was supported by a fellowship from Fundação para a ciência e a Tecnologia – Ministério da Ciência e do Ensino Superior, Portugal.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary Material
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. Supplementary Material
  9. References
  10. Supporting Information

Table S1.  Primer sequences and PCR conditions.

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FilenameFormatSizeDescription
TPJ_2407_sm_tableS1.rtf12KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.