1 Running Title: Abolition of tapetum PCD ruins microsporogenesis *Corresponding author

20 Pages·2006·0.64 MB·English

Plant Cell Physiol. 47(6): 784–787 (2006)
doi:10.1093/pcp/pcj039, available online at www.pcp.oupjournals.org
JSPP © 2006
Short Communication
Abolition of the Tapetum Suicide Program Ruins Microsporogenesis
Takahiro Kawanabe 1, Tohru Ariizumi 1, Maki Kawai-Yamada 2, Hirofumi Uchimiya 2 and
Kinya Toriyama 1, *
1 Laboratory of Environmental Biotechnology, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555 Japan
2 Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, 113-0032 Japan
; D
Microsporogenesis in angiosperms takes places within have also reported that the nuclei of tapetal cells and the tissues o
w
the anther. Microspores are surrounded by a layer of cells, of the anther wall were TUNEL (terminal deoxynucleotidyl- nlo
the tapetum, which degenerates during the later stages of transferase-mediated dUTP nick end-labeling) positive, which ad
e
pollen development with cytological features characteristic indicated that the fragmentation of DNA was detected in situ d
of programmed cell death (PCD). We report herein that the through the incorporation of fluorescein 12-dUTP at the free fro
m
expression of AtBI-1, which suppresses Bax-induced cell 3′-OH ends of the fragmented DNA. Similar features of PCD h
death, in the tapetum at the tetrad stage inhibits tapetum together with the release of cytochrome c, which is characteris- ttps
degeneration and subsequently results in pollen abortion, tic of animal apoptosis, have been also reported in sunflower in ://a
c
while activation of AtBI-1 at the later stage does not. Our conjunction with the study of cytoplasmic male sterility (Balk ad
e
results demonstrate that the PCD signal commences at the and Leaver 2001). The role of PCD and an onset signal for m
tetrad stage and that the proper timing of PCD in the tape- PCD in the tapetum, however, have not yet been elucidated. ic.o
u
tum is essential for normal microsporogenesis. In animal systems, studies of apoptosis have revealed p
.c
pathways where proteins of the Bcl-2 family play key roles. om
Keywords: Arabidopsis thaliana — Microsporogenesis — The Bcl-2 family includes pro-apoptotic (e.g. Bax, Bak and /p
c
Pollen development — Programmed cell death — Tapetum. Bid) and anti-apoptotic (e.g. Bcl-2, Bcl-xl and Ced-9) members p/a
that appear to control the initiation of apoptosis through mito- rtic
Abbreviations: AtBI-1, Arabidopsis thaliana ortholog of Bax chondria (Gross et al. 1999). When Bax is translocated from le-a
inhibitor-1; BI-1, Bax inhibitor-1; ER, endoplasmic reticulum; PCD, b
cytosol to the outer membrane of mitochondria, it induces the s
pferroagsrea-mmmedeida tecde dllU TdePa nthic; k TeUndN-lEaLbe, litnegr.minal deoxynucleotidyltrans- rceolnesaesqe uoefn ptlryo tteriigngs,e rssu cahp oaps tcoysitso c(hLrioum eet ca,l .i n1t9o9 6th, eJ ucyrgtoensosml, eainedr tract/4
7
et al. 1998). A Bax gene has been shown to induce PCD in /6
/7
plant cells (Lacomme and Cruz 1999, Kawai-Yamada et al. 84
The development of pollen grains (microsporogenesis) has 2001), although comparative genomics has revealed that Bcl-2 /18
2
family-like proteins are absent in plants (Aravind et al. 1999). 6
long interested plant biologists. Microspore and pollen devel- 3
1
opment takes place within the anthers of angiosperm flowers A suppressor of Bax-induced cell death has been identified in 6 b
(reviewed by Mascarenhas 1989, McCormick 1993, Scott plants as well as in humans. Bax inhibitor-1 (BI-1) is such an y g
1994). Microspores are surrounded by a layer of cells, i.e. the anti-apoptoic protein that localizes to the ER membrane, and is ue
tapetum. The tapetum is known to provide nutrition to develop- conserved in both animal and plant species (Chae et al. 2003). st o
ing microspores and pollen grains, as well as exine, the main Overexpression of AtBI-1, a homolog of mammalian BI-1 n 1
structural components of the pollen wall. The tapetum degener- identified in Arabidopsis thaliana, has been shown to inhibit 1 A
ates during the later stages of pollen development. B20a0x1-i)n.duced cell death in A. thaliana (Kawai-Yamada et al. pril 2
It has been speculated that tapetum degeneration is a pro- 0
1
grammed cell death (PCD) event (for a review, see Wu and To determine the critical developmental stage when the 9
Cheung 2000). Based on ultrastructural observation of tapetal PCD signal commences in the tapetum and to elucidate the role
cells in two angiosperms (Lobivia rauschii and Tillandsia alb- of PCD of the tapetum for microsporogenesis, we employed
ida), Papini et al. (1999) have reported that the degradation of Bax and AtBI-1 genes to alter PCD in the tapetum.
tapetal cells shows cytological features characteristic of PCD. A mouse Bax gene or the AtBI-1 gene of A. thaliana was
These include cell shrinkage, condensation of chromatin, connected to the tapetum-specific promoter of the Osg6B gene
swelling of the endoplasmic reticulum (ER) and the persist- or the LTP12 gene (Tsuchiya et al. 1994, Ariizumi et al. 2002).
ence of mitochondria. Wang et al. (1999) have reported that The commencement of promoter activation is different between
toward the end of the unicellular stage of pollen development them. The LTP12 promoter becomes active starting in the
in barley, oligonucleosomal DNA cleavage was observed. They uninucleate microspore stage (Ariizumi et al. 2002), while
*Corresponding author: E-mail, [email protected]; Fax, [phone].
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Abolition of tapetum PCD ruins microsporogenesis 785
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Fig. 1 Tapetal cells are killed by Bax /78
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or sustained by AtBI-1, whereas PCD /1
induces degeneration of tapetum in the 82
6
wild type. Light microscopy images of 3
1
anther sections at the mature stage 6
b
(A–D), transmission electron micro- y
scopy (TEM) images of tapetum at the gu
e
uninucleate microspore stage (E–H), s
TEM images of microspores at the t o
n
uninucleate microspore stage (I–L) in 1
1
wild type (A, E, I), Osg6B::AtBI-1 (B, A
p
FL,T PJ1),2 ::OBsagx6 B(:D:B, aHx , (LC),. GBa, , Kb)a cualnad; ril 2
0
Msp, microspore; P, plastid; Ta, tape- 19
tum; Tc, tectum; Va, vacuole. Bar =
20µm (A–D), 1.5µm (E–H) and
0.5µm (I–L).
Osg6B becomes active at an earlier stage, the tetrad stage, and gene is expected to cause cell death resulting in pollen abor-
continues until the bicellular pollen stage, as determined by tion. Expression of the AtBI-1 gene is expected to cause the
promoter–β-glucuronidase (GUS) fusion experiments in trans- inhibition of PCD of the tapetum, and the surviving tapetum
genic A. thaliana (T. Kawanabe, T. Ariizumi and K. Toriyama, would affect pollen development.
unpublished data). In the current study, four chimeric con- Transgenic plants with LTP12::Bax, Osg6B::Bax and
structs, LTP12::Bax, LTP12::AtBI-1, Osg6B::Bax and Osg6B:: Osg6B::AtBI-1 were male-sterile. In contrast, transgenic plants
AtBI-1, were introduced into A. thaliana. Expression of the Bax with LTP12::AtBI-1 were fertile and set seeds normally (Table
786 Abolition of tapetum PCD ruins microsporogenesis
Table 1 Sterile and fertile plants were obtained depending on al. 2002). It is likely that PCD signaling commences at the
the active stage of the promoter tetrad stage.
Why did AtBI-1 cause pollen abortion under the regulation
Promoter (active stage in anther) BAX AtBI-1
of the Osg6B promoter, but not under the LTP12 promoter?
LTP12 (uninucleate to bicellular) 6/6 0/15 It is considered that the signal pathway of PCD was inhibited
Osg6B (tetrad to bicellular) 4/4 8/11 by AtBI-1 at the tetrad stage and that PCD did not proceed
further in the Osg6B::AtBI-1 plants. Inhibition of PCD in the
The number of male-sterile plants/total number of kanamycin-resistant
transgenic A. thaliana with LTP12::Bax, LTP12::AtBI-1, Osg6B::BAX tapetal cells consequently affects normal pollen development,
and Osg6B::AtBI-1 are presented. arresting the supply of nutrients to the microspores, which
results in male sterility. The appearance of some fertile plants
with Osg6B::AtBI-1 (Table 1) might indicate that the function D
o
1). Tapetum and microspore development were observed by of AtBI-1 is not enough to suppress cell death completely. w
n
light and electron microscopy in at least three transgenic lines In contrast, when the LTP12 promoter starts to drive AtBI-1 at lo
a
d
for each construct. In the mature anther of the wild type, the the uninucleate microspore stage, PCD becomes irreversible e
d
complete disappearance of the tapetum was clearly observed and AtBI-1 no longer inhibits PCD. In the LTP12::AtBI-1 fro
(Fig. 1A). In contrast, in the tapetal cells of Osg6B::AtBI-1 plants, normal PCD takes place, resulting in production of m
h
plants, tapetum degeneration was not observed even at the fertile pollen. ttp
s
mature stage (Fig. 1B). The tapetal cells began to enlarge with There have been several reports on the use of genetic cell ://a
large vacuoles at the uninucleate microspore stage (Fig. 1F). ablation for investigation of the role of the anther tapetum dur- ca
d
Electron-dense and organelle-rich cytoplasm and vacant vacu- ing microspore development. Using the promoters of tapetum- e
m
oles were well contrasted in the enlarged tapetal cells (Fig. 1F). specific genes to express cytotoxic molecules in transgenic ic
.o
Exine formation was highly defective in the Osg6B::AtBI-1 plants, ablation of tapetal cells in transformed anthers has been u
p
plants (Fig. 1J), as shown by the fact that the bacula were demonstrated to result in microspore abortion and complete .co
clearly shorter than those of the wild type (Fig. 1I) and almost male sterility (Mariani et al. 1990, Paul et al. 1992, Hird et al. m/p
no tectum was formed. The microspores collapsed and were 1993). In contrast to the killing of tapetum cells, however, an cp
finally crushed by the enlarged tapetum (Fig. 1B). approach to alter the PCD program so as to allow the tapetal /artic
In the case of the Osg6B::Bax plants, vacuolation of the cells to live longer has not been reported so far. Our results le
tapetum at the mature stage was also observed as in the Osg6B:: demonstrated, for the first time, that the PCD signal com- -ab
s
AtBI-1 (Fig. 1C). The tapetum was faintly and uniformly mences at the tetrad stage and that the proper timing of PCD in tra
c
stained, and no cytoplasmic components were observed at the the tapetum is essential for normal microsporogenesis. t/4
uninucleate microspore stage (Fig. 1G), indicating that the tap- 7/6
etal cells were completely destroyed by the action of Bax. The /7
Materials and Methods 8
4
organelles in the microspores were not evident and the micro- /1
8
spores were almost empty of contents (Fig. 1K). 2
cDNAs of mouse BAX (accession no. L22472) and AtBI-1 6
3
LTP12::Bax was shown to cause male sterility similarly to (accession no. AB025927) were cloned into pGEM-3Zf(–) (Promega). 16
the case of the Osg6B::Bax. Pollen development, however, was A fragment of the Osg6B promoter (Tsuchiya et al. 1994) or the LTP12 b
y
less severely affected than that in the Osg6B::Bax plants. For- promoter (Ariizumi et al. 2002) was ligated to the HindIII–SpeI site or g
u
mation of exine with bacula and tectum was observed in HindIII–XbaI site. The HindIII–SacI fragment was then ligated to the es
LTP12::Bax (Fig. 1D, L), although only poorly developed bac- same site of pBI101. The resulting binary vectors were transferred into t on
Agrobacterium tumefaciens strain C58. Transformation of A. thaliana 1
ula were observed in the Osg6B::Bax (Fig. 1K), indicating that ecotype Columbia used floral dip methods (Clough and Bent 1998), 1 A
exine formation is arrested at a later stage in the LTP12::Bax and transformants were selected using 20mg liter–1 kanamycin. For p
plants than in the Osg6B::Bax plants. The tapetum of the sectioning, the wild-type and transgenic flowers at different stages ril 2
0
LTP12::Bax plants at the uninucleate microspore stage lacked were fixed overnight in 3% glutaraldehyde in 100mM phosphatase 1
9
buffer (pH7.0) and dehyderated in a graded ethanol series. The subse-
plastids with relatively electron-transparent vesicles, plastglob-
quent procedures were carried out as previously described (Ariizumi et
uli, which were observed in the wild type (Fig. 1E, H). The
al. 2003, Ariizumi et al. 2004)
tapetum development appeared to be arrested. These features
were probably caused by the fact that in contrast to the Osg6B
Acknowledgments
promoter, the LTP12 promoter became active in the later devel-
opmental stages and thus had less effect on the tapetum and
This research was supported by a Research for the Future grant
microspore development.
and a Grant-in-Aid from the Japan Society for the Promotion of
In A. thaliana, complete disappearance of the tapetum cell Science.
layer is observed after the second pollen mitosis at the tricellu-
lar pollen stage. Loss of the cell walls of the tapetum begins as
early as the tetrad stage (Owen and Makaroff 1995, Zhang et
Abolition of tapetum PCD ruins microsporogenesis 787
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(Received January 11, 2006; Accepted March 17, 2006) /6
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