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Arbidopsis Okee
Arbidopsis Okee
Environmental stresses frequently decrease plant fertility. In Arabidopsis, the effect of salt stress on reproduction was
examined using plants grown in hydroponic medium. Salt stress inhibited microsporogenesis and stamen filament elongation.
Because plants grown in hydroponic media can be rapidly and transiently stressed, the minimum inductive treatment to cause
ovule abortion could be determined. Nearly 90% of the ovules aborted when roots were incubated for 12 h in a hydroponic
medium supplemented with 200 mM NaCl. The anatomical effects of salt stress on maternal organs were distinct from those in
the gametophyte. A fraction of cells in the chalaza and integuments underwent DNA fragmentation and programmed cell
death. While three-fourths of the gametophytes aborted prior to fertilization, DNA fragmentation was not detected in these
cells. Those gametophytes that survived were fertilized and formed embryos. However, very few of these developing embryos
formed seeds; most senesced during seed development. Thus, during seed formation, there were multiple points where stress
could prematurely terminate plant reproduction. These decreases in fecundity are discussed with respect to the hypothesis of
serial adjustment of maternal investment.
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Plant Physiology, August 2004, Vol. 135, pp. 23582367, www.plantphysiol.org 2004 American Society of Plant Biologists
RESULTS
Salt Stress Reduces Seed Production
Sun et al.
Figure 4. Salt stress disrupted the normal development of Arabidopsis pollen grains, ovules, and embryos. A, In healthy pollen
grains, cytoplasm was dispersed throughout the cell. Arrowheads identify pollen nuclei. B, Prior to pollen abortion, cells became
highly vacuolated. C, Many of these vacuolated pollen cells lysed and collapsed. D, A normal stage-12 flower, with the tips of the
petals just emerging from behind the sepals. Flowers at this stage were stressed and the ovules within monitored over time for
developmental defects. E, After 24 h, the flower shown in D opened and the stamens and petals had markedly increased in size.
As stamen filaments lengthened, anthers (a) coated the stigma (st) with pollen. F, A stage-12 flower that was salt stressed. Eighteen
hours later pollen dehisced, but filaments were too short to pollinate the stigma. G, A stage-12 flower that was salt stressed.
Twenty-four hours later, the filaments elongated sufficiently to pollinate the stigma. H, Within a normal stage-12 flower, the
gametophyte (g) is fully developed and competent to be fertilized. I, One day after salt stress, starch granules (arrows)
accumulated in the gametophyte. J and K, Subsequently, these starch grains were mobilized from ovules, and the cells in the
gametophyte become vacuolated. L, In the gametophyte, subcellular components were lysed and condensed. M, In some cells,
condensation of debris left little evidence that a gametophyte had occupied this space. N, Healthy suspensor (s) and embryo (e).
O, Following salt stress, an embryo aborted at the globular stage of embryogenesis. P, Before they senesced, the embryo and
suspensor became highly vacuolated.
Plant Physiol. Vol. 135, 2004
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Sun et al.
Figure 6. Salt stress derailed gametophyte development. Ovules from healthy (AC) and stressed (D and E) plants were marked at
stage 2-IV, and ovule development was monitored as a function of time. In terms of developmental time, the stressed ovules
shown in D and E correspond to similar developmental times for the healthy ovules shown in B and C, respectively. A, In an ovule
at stage 2-IV, the megaspore mother cell (mmc) occupies the central region of the nucellus. B, One day later, the megaspore
mother cell went through meiosis, yielding one large haploid megaspore (m). C, This megaspore underwent three rounds of
mitotic division and the products differentiated into a gametophyte. Within the gametophyte, the egg cell (ec), two synergid cells
(sc), two central cells (cc), and three antipodal cells (ac) were present. D, When ovules were stressed at stage 2-IV, they
completed meiosis, although the outer integument (oi) and inner integument (ii) grew more slowly than healthy ovules. E, The
megaspore from this stressed plant went through only one mitotic division, yielding a gametophyte (g) with just two nuclei.
during PCD. The anatomy of the remaining gametophytes was indistinguishable from wild type.
Senescence of Stressed Zygotes and Embryos
DISCUSSION
Ovule Abortion in Arabidopsis
Sun et al.
Figure 7. Maternal cells in stressed ovules (ov) underwent extensive DNA nicking or fragmentation. In B, D, and F to H, nicked
DNA was fluorescein labeled (yellow), while cell walls autofluoresced (red). In A, C, and E, DAPI-stained nuclei were observed
(blue). A and B, In healthy pistils, DNA was intact or unbroken. C and D, In stressed pistils, the DNA in many integument nuclei
and the chalaza nuclei was nicked. By contrast, nuclei in the carpel walls (cw), stigma (st), and style were not modified in this
manner. E and F, While maternal cells from stressed ovules exhibited DNA fragmentation, the nuclei in the gametophyte (arrows)
were undamaged. G and H, Embryo and endosperm nuclei from stressed plants did not exhibit any DNA fragmentation or
nicking. tt, Transmitting tract.
(2) many cells in the chalaza and maternal integuments exhibited DNA fragmentation (Fig. 7). PCD of
these maternal cells frequently lagged behind the
breakdown of gametophytic cells. In a number of crop
plants, including bean, rapeseed, maize, and soybean,
stress induces mature gametophytes to undergo PCD
(Moss and Downey, 1971; Sage and Webster, 1990;
Kokubun et al., 2001; Young et al., 2004). Thus, adverse
environmental conditions can cause plants to suspend
normal development of gametophytes, embryos, and
pollen grains, but the phenotype of abortion depends
upon the developmental stage at which stress was
experienced.
Callose Synthesis
CONCLUSIONS
Plants use a variety of mechanisms to regulate reproduction and maximize plant fitness. Lloyd (1980)
proposed that plants adjust the expenditure of maternal resources at a series of developmental stages,
regulating the number of flowers, gametophytes, and
embryos that advance further in development. Arabidopsis ovule abortion and embryo senescence were
regulated phenomena, which agrees with the conclu2365
Sun et al.
sions drawn by Lloyd in his maternal resources hypothesis. This strategy conserves plant resources since
terminating reproduction in an early developmental
stage expends relatively few resources. The resources
conserved by reducing plant fertility, thereby, can be
shunted into processes that permit plants to acclimate
to stressful environmental conditions. Even when
severely stressed, however, Arabidopsis allocates
sufficient resources to produce a few seeds in each
fruit, which ensures the genetic line is continued. We
hypothesize that by readjusting reproduction and resource allocation during stress, Arabidopsis plants
are able to tolerate harsh environmental conditions
and, if moderate environmental conditions return,
increase reproductive output. This hypothesis will
be tested by precisely monitoring resource allocation
to ovules and embryos during and after stress.
Light Microscopy
For paraffin sections, flowers were fixed overnight in 10% formalin, 5%
acetic acid, and 50% ethanol. Samples were dehydrated in a graded ethanol
series then embedded in Paraplast1. Samples were sectioned at 5-mm
intervals, then stained with Safranin O (Polysciences, Warrington, PA) and
fast green (Johansen, 1940). For plastic sections, pistils were fixed overnight in
3% (v/v) glutaraldehyde in 50 mM sodium cacodylate buffer, pH 7. Samples
were postfixed with 2% osmium tetroxide, dehydrated, and infiltrated
according to Spurr (1969). Polymerized resin was sectioned at 0.5- to 2-mm
intervals and stained with thionine and acridine orange according to Paul
(1980). To observe callose deposition, ovules were cleared in BB41/2 (Herr,
1971) supplemented with 0.001% aniline blue, and observed by fluorescence
microscopy using 4#, 6-diamino-phenylindole (DAPI) excitation and emission
filters. A TUNEL assay detected DNA fragmentation. Using sectioned pistils,
the DeadEnd Fluorometric TUNEL kit (Promega, Madison, WI) labeled
nicked-DNA with fluorescein. A fluorescent DNA intercalating agent, DAPI
(Sigma, St. Louis), stained nuclei. An Axioscope microscope (Zeiss, Thornwood, NY) equipped with a Kodak MDS290 camera (Rochester, NY) captured
microscopic images. Digital images were adjusted for publication using
Adobe Photoshop 5 (San Jose, CA).
ACKNOWLEDGMENTS
We thank Greg Erdos and Karen Kelley of the ICBR Electron Microscopy
Facility (University of Florida) for technical assistance, Tim Martin (Univer-
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sity of Florida) for assistance with pressure chambers, and Margaret Joyner
(University of Florida) and two anonymous reviewers for helpful comments.
Received March 26, 2004; returned for revision June 29, 2004; accepted July 1,
2004.
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