coleoptile senescence in rice (oryza sativa l.)
TRANSCRIPT
Annals of Botany 86: 405±414, 2000doi:10.1006/anbo.2000.1199, available online at http://www.idealibrary.com on
Coleoptile Senescence in Rice (Oryza sativa L.)
MAKI KAWAI*{ and HIROFUMI UCHIMIYA{{
{Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032,
Japan and {Advanced Science Research Center, Japan Atomic Energy Research Institute, Takasaki 370-1292, JapanReceived: 6 January 2000 Returned for revision: 1 April 2000 Accepted: 29 April 2000
and acts lik
0305-7364/0
* For corrimcbns.iam.u
We investigated the cellular events associated with cell death in the coleoptile of rice plants (Oryza sativa L.). Seedsgerminated under submergence produced coleoptiles that were more elongated than those grown under aerobicconditions. Transfer of seedlings to aerobic conditions was associated with coleoptile opening (i.e. splitting) due todeath of speci®c cells in the side of the organ. Another type of cell death occurred in the formation of lysigenousaerenchyma. Senescence of the coleoptile was also noted, during which discolouration of the chlorophyll and tissuebrowning were apparent. DNA fragmentation was observed by deoxynucleotidyltransferase-mediated dUTP nick endlabelling (TUNEL) assay, and further con®rmed by the appearance of oligonucleosomal DNA ladders in senescentcoleoptile cells. Two nucleases (Nuc-a and Nuc-b) were detected by in-gel-assay from proteins isolated fromcoleoptiles. Nuc-a, commonly observed in three cell death phases required either Ca2� or Mg2�, whereas Nuc-bwhich appeared during senescence required both Ca2� and Mg2�. Both nucleases were strongly inhibited by Zn2�.
# 2000 Annals of Botany Company
Key words: Aerenchyma, rice, cell death, coleoptile, fragmentation, nuclease, Oryza sativa, senescence, split,submergence, TUNEL.
ase activity during senescence of the coleoptile is reported.
INTRODUCTION
Inundation that gives rise to soil ¯ooding, or completesubmergence, is the most common environmental cause ofoxygen shortage for vascular plants. Rice is well known forits adaptation to ¯ooded conditions (Avadhani et al., 1978)and is one of the plant species whose coleoptile can elongatein the complete absence of O2. The coleoptile serves toprotect true leaves against soil pressures and other physicalconstraints and also provides nutrients for the developingtissues (FroÈ hlich and Kutschera, 1995). According to Esau(1965), the foliar interpretation of the coleoptile is notuniversal. Some regard it as an outgrowth of the scutellum,the scutellar sheath, rather than a product of the apicalmeristem. Another hypothesis is that the coleoptile andmesocotyl are new acquisitions without homologues inother embryos.
In rice plants, aerenchyma develops in the coleoptile,root, mid-rib of the leaf blade, and leaf sheath throughlysigeny (Hoshikawa, 1989; Matsukura et al., 2000). Wehave recently reported that aerenchyma formation in riceroots is characterized by position-speci®c cell death (Kawaiet al., 1998; Samarajeewa et al., 1999). There is ampleevidence indicating that aerenchyma acts as a di�usion pathfor the transport of oxygen from aerial plant parts to rootsor rhizomes in a waterlogged O2-de®cient environment(Armstrong, 1971; Kawase and Whitmoyer, 1980). In the®rst stage of germination, the coleoptile elongates rapidly,
e a `snorkel' to reach the air.
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Inada et al. (1998a, b) reported that whitish-yellowcoleoptiles emerged from imbibed rice seeds after sowing,grew rapidly to their full size and became pale green incolour within 3 d. Upon cessation of coleoptile growth, the®rst leaf increased in size, splitting the cylindrical coleoptilelongitudinally. As the ®rst, second and third leaves grew,the coleoptile turned brown and ®nally died completely.Thus, the coleoptile is the ®rst organ in the rice plant tosenescence after germination.
When a leaf reaches a certain age, or when the repro-ductive phase of the plant reaches a certain stage, sene-scence is initiated in such organs even if the plant is growingunder favourable conditions (Buchanan-Wollaston, 1997;OrzaÈ ez and Granell, 1997). McManus et al. (1998) reportedconsiderable changes in the size of the nuclei and chromatinwithin the nuclei of the senescent leaves. Furthermore, Yenand Yang (1998) also con®rmed the presence of DNAfragmentation in senescent leaves.
The objectives of this study were to characterize thepattern of cell death events in elongated coleoptiles of riceplants, which had been kept under submerged conditionsbefore being transferred to aerobic conditions. In this study,we show that splitting of the coleoptile is spatially pre-determined. Furthermore, the occurrence of unique nucle-
MATERIALS AND METHODS
Plant material
Rice (Oryza sativa L. `Yamahoushi') caryopses were placedon moist paper towels in glass tubes. These were kept under
continuous light at 238C for 4 d. For submersion studies,# 2000 Annals of Botany Company
shown is a negative-image.
le
rice caryopses were completely submerged (8 cm below thewater surface) in autoclaved water within glass bottles,which had been de-gassed under vacuum for 30 min toremove dissolved oxygen, and sealed. To facilitate theinitiation of cell death, seedlings that had been submergedfor 7 d were exposed to air. All plants were kept at 238C. Tomeasure O2 concentration in the water, a dissolved O2 meter(OM-14, HORIBA, Tokyo, Japan) was used according tothe manufacturer's instructions. For observation of senes-
406 Kawai and UchimiyaÐCo
cent coleoptiles, 14-d-old seedlings were used.
Examination of coleoptiles
The 5 mm long median hand-sections of coleoptiles were®xed overnight in 4% paraformaldehyde in 20 mM caco-dylate bu�er (pH 7.0) at 48C. They were then dehydrated inan ethanol series to 100% ethanol and embedded inTechnovit 8100 (Kulzer, Wehrheim, Germany) containing5% butoxyethanol. For microscopic examination, cross-and longitudinal-sections (10 mm thick) were cut with amicotome (Yamato Kohki, Japan), and stained with 0.1%toluidine blue. Cell length was determined using magni®ed
photo-images.Coomassie brilliant blue R-250 (CBB).
Evans blue staining
Fresh sections cut with a razor blade were stained with2.5% Evans blue (Nakarai Chemicals, Kyoto, Japan) asdescribed previously (Kawai et al., 1998). After 1 min, eachsection was washed with water and examined under a lightmicroscope (FX-35WA; Nikon, Tokyo, Japan) and photo-graphed. For examination of the abscission site, wholeseedlings were treated with 2.5% Evans blue and washedwith water before being examined with a stereomicroscope
(MZ APO, Leica, Wetzlar, Germany).TUNEL assay
An in situ cell death detection kit (Boehringer, Tokyo,Japan) was used for the terminal deoxynucleotidyl trans-ferase (TdT)-mediated dUTP nick end in situ labelling(TUNEL; Gavrieli et al., 1992) assay to visualize 30-OHgroups in the DNA of nuclei. Sections of tissues obtainedfrom samples embedded in Technovit were cut to a thick-ness of 10 mm, dried on cover glasses, and incubated with0.1% sodium citrate containing 0.1% Triton X-100 for15 min. After washing with PBS, tissues were subjected tothe TUNEL reaction according to the manufacturer'sinstructions: tissues were stained with DAPI (40,60-diami-dino-2-phenylidole) and mounted with 50% glycerolcontaining 1 mg mlÿ1 n-propyl gallate, followed by exam-ination under a ¯uorescence photomicroscope (LEITZ DM
RD, Leica, Wetzlar, Germany).DNA extraction and fragmentation analysis
Coleoptiles were ground in liquid N2 with a mortar andpestle to a ®ne powder. The extraction bu�er (5 mM Tris-HCl, pH 8.0, 20 mM EDTA and 0.5% Triton X-100) was
then added, followed by an equal volume of phenol-chloroform (1 :1, v/v), and the samples were centrifuged at8000 g for 15 min. Total nucleic acid was precipitated bythe addition of an equal volume of isopropanol. Ten mg ofDNA treated with RNase (0.1 mg mlÿ1) was electrophor-esed on a 1.5% agarose gel, stained with ethidium bromide,and photographed on a UV light box. The photograph
optile Senescence in Rice
Nuclease in-gel activity assay
Crude extracts were isolated from plant tissues byhomogenization with 50 mM HEPES (pH 8.0) containing330 mM sorbitol, 20 mM EDTA and 1 mM phenylmethyl-sulfonyl (PMSF). Homogenates were sonicated (UR-20Psonicator; TOMY SEICO, Japan), and centrifuged at5000 g for 15 min at 48C. The supernatant (10 mg protein)was applied to SDS-PAGE. The 12% acrylamide gelcontained 0.2 mg mlÿ1 denatured salmon sperm DNA(Sigma, St. Louis, MO, USA) as a substrate for nucleases.Following electrophoresis, gels were incubated in a reactionsolution containing 20 mM Tris-HCl (pH 7.5) and 5 mM 2-mercaptoethanol for 1 h at 508C, and in 20 mM Tris-HCl(pH 7.5) overnight at 48C. Gels were then incubated in50 mM Tris-HCl (pH 7.5), 3 mM CaCl2, 3 mM MgCl2 and1 mM 2-mercaptoethanol for 10±24 h at 378C. To detectnuclease activity, the gel was stained with ethidiumbromide, and examined under UV light. For the analysisof ion requirements of nucleases, 10 mM EDTA (pH 8.0) or3 mM ZnSO4 were added in the reaction solutions. Fordetection of the total protein, the gel was stained with
RESULTS
Comparison of coleoptile parenchyma cells of rice seedlingsgerminated under submergence or aerobic conditions
To investigate the growth of coleoptiles, seedlingsgerminated under aerobic or submerged conditions werecompared. Coleoptile growth of rice seedlings submergedfor 4 d was greater than that of seedlings grown in aeratedconditions for the same period (Fig. 1A). There was nogreening of the coleoptile under submerged conditions androot growth was suppressed. To determine the cellular basisof di�erences in coleoptile elongation under aerobic andanaerobic conditions, we compared the cell length ofparenchyma tissue using longitudinal-sections obtainedfrom the middle part of each coleoptile (Fig. 1B, C). Thecell length of coleoptiles obtained from seedlings germinatedunder submergence was several times longer than that ofcoleoptiles of seedlings under aerobic conditions (Fig. 1D),demonstrating that the cellular basis of coleoptile elongationunder anaerobic conditions is mostly due to cell elongation.
Examination of longitudinal-sections of coleoptiles ofrice seedlings grown in aerobic conditions showed aerench-ymatous cavities (Fig. 1B), but such cavities were notobserved in coleoptiles of seedlings grown under submerg-ence (Fig. 1C). In submerged conditions, the concentrationof dissolved O was 3.00 mg lÿ1 on the ®rst day, but
2decreased to 0.38 mg lÿ1 by 48 h after germination,
grown under submerged conditions (Fig. 2C).
senescence of coleoptiles.
FIG. 1. A, Rice coleoptiles germinated under aerobic or submergedconditions for 4 d. Arrows indicate coleoptiles. B and C, Longitudinalsections of coleoptiles obtained from seedlings germinated underaerobic (B) or submerged conditions (C). ae, Aerenchyma;bar � 100 mm. D, Cell length of parenchyma cells of coleoptiles.
Kawai and UchimiyaÐCole
con®rming that submergence was associated with low O2
(hypoxia). Water in equilibrium with air has a dissolved O2
concentration of 8.39 mg lÿ1 at 238C.To examine dead cells, hand-sections obtained from the
Data are mean+ s.e. (n � 60).
mid-region of coleoptiles were stained with Evans blue
(Fig. 2). Semi-permeable plasma membranes of living plantcells exclude Evans blue, whereas those with damagedmembranes incorporate the dye (Ga� and Okong'O-Ogola,1971; Kanai and Edward, 1973). Two large vascularbundles can be seen in coleoptiles regardless of aerobic(Fig. 2A, B, D) or submerged conditions (Fig. 2C).Aerenchyma was seen adjacent to and on both sides ofthe vascular bundles of coleoptiles only under aerobic con-ditions (Fig. 2A, B, D). Cells stained with Evans blue wereobserved around the cavity and collapsing region, wherethese cells are destined to form aerenchyma (Fig. 2B, D).Furthermore, we also observed a strong Evans blue stainingin the peripheral cell layer of the coleoptile where splittingwas apparent (Fig. 2E). In contrast, we could not detectcavities nor cells stained with Evans blue in coleoptiles
optile Senescence in Rice 407
Aerobic conditions induce rapid maturation and cell death incoleoptiles
As demonstrated in Fig. 1, coleoptiles of rice plantsgerminated under submerged conditions showed substantialelongation. Using such coleoptiles, we were able to identifycell death events that were associated with maturation andsenescence as shown in Fig. 3A. When the elongatedcoleoptiles grown under submergence for 7 d were trans-ferred to aerated conditions, greening of coleoptiles wasseen after 72 h, and the ®rst leaf emerged completely bysplitting of the coleoptile 96 h after transfer. Chlorophylldegradation, a typical symptom of senescence, was alsoseen at 96 h. For further examination of the splitting zonein the coleoptile, seedlings were treated with Evans blue atdi�erent periods (Fig. 3B±E). After 6 h of exposure toaerobic conditions, a small amount of blue colouration wasnoted along the longitudinal axis in the adaxial region(Fig. 3C, E), where death resulted in splitting of thecoleoptile after 24 h (Fig. 3D, F). After 6 h, the ®rst leafhad not elongated inside the coleoptile (data not shown).
To determine the origin of cell death, further examina-tion of the splitting zone was carried out. Seedlings grownunder submergence for 7 d (Fig. 4A, C) were exposed to air.A few compact cellsÐthat subsequently diedÐwereobserved before splitting occurred (Fig. 4C). The splitcommenced from the adaxial surface outwards after 6 h(Fig. 4D). Coleoptile opening occurred most frequently inthe centre of the long axis (Fig. 4E). Thus, the split zone ispredetermined and is exactly in the centre of two vascularbundles. Thus, we found that the opening of the coleoptileoccurs in speci®c cells. Coleoptile splitting was completedby 24 h (Fig. 3D). Furthermore, Evans blue staining andinitiation of cell collapse associated with aerenchymaformation were observed after 48 h (data not shown).Aerenchymatous cavities developed at 96 h (Fig. 4B).Porosity expansion continued throughout maturation and
DNA fragmentation is associated with senescence
In animals, apoptosis which is a type of programmed cell
death (PCD), involves chromatin condensation, cellular
shown).
strongly inhibited by Zn2� as well as by EDTA.
FIG. 2. Visible cell death area in coleoptiles obtained from seedlings grown under aerobic conditions for 4 d (A, B, D and E) and submergedconditions for 7 d (C). Hand cross-sections obtained from the middle part of the coleoptiles (A) were treated with Evans blue to stain dead cells(B±E). D, A magni®ed image of (B). ae, Aerenchyma. E, Split zone stained with Evans blue. Arrows in A±D indicate vascular bundles.
10
408 Kawai and UchimiyaÐColeoptile Senescence in Rice
shrinkage, membrane blebbing, the formation of apoptoticbodies and digestion by macrophages (Kerr et al., 1972).Fragmentation of nuclear DNA is the hallmark of theapoptotic process (Wyllie, 1980). To determine whetherDNA fragmentation occurs in the coleoptile, we performedTUNEL (Gavrieli et al., 1992) on rice coleoptile cells.Accumulation of DNA 30-OH groups occurred in senescentcoleoptiles (Fig. 5B). Dead cells associated with coleoptilesplitting and aerenchymatous cavities have no TUNEL-positive cells (Fig. 5A). TUNEL-positive signals weredetected in the coleoptile 120 h after transfer from sub-mergence to aerobic conditions (data not shown). Thistemporal occurrence of DNA fragmentation seems tocorrelate with the progression of senescence. There was noevidence of a TUNEL signal in the split and aerenchyma-tous zones at any stage.
To observe the DNA ladders during senescence, totalDNA was extracted from coleoptiles at 0, 48, 96, 120 and144 h after exposure to air, and 10 mg of DNA wasseparated by electrophoresis on a 1.5% agarose gel. Theintensity of the DNA ladder was faint at 96 h, but presentat 120 h, and shifted to smaller bands at 144 h (Fig. 6). Thebands of large molecular DNA at 0 or 48 h were weak evenwhen DNA was equally or over loaded, because ethidiumbromide binding to intact DNA is low. The sizes of thebands (indicated by arrows) were calculated to be around200 and 400 bp, respectively, suggesting that they representoligonucleosomal fragmentation. The smallest molecules(indicated by an outlined arrow) increased in intensityduring coleoptile aging but are not degraded RNA, becausea nucleic acid of this size was not present at 0 and 48 h. Inthe completely dried, brown coleoptile, DNA recovery wasreduced and the laddering became smeared (data notshown). On the other hand, even in the coleoptiles kept
Bar �
under submergence for 3 weeks, DNA was intact and no
evidence of DNA fragmentation occurred (data not
0 mm.
Nuclease activity associated with cell death processes
There is ample evidence indicating that the breakdown ofDNA during PCD is due to cleavage by nucleases (Wyllieet al., 1984; Shiokawa et al., 1994; Mittler and Lam, 1995a).To detect nuclease activity during coleoptile development,protein extracts obtained from coleoptiles transferred toaerobic conditions were subjected to nuclease in-gel-assay.CBB stained gel showed degradation of several proteinsduring the progression of senescence (Fig. 7A). Nucleaseassay demonstrated that the development of the coleoptilewas associated with induction of activity of at least twonucleases (Fig. 7B). One nuclease (Nuc-a, 38 kDa) wasinduced at 24 h by exposure of seedlings grown under sub-mergence to aerobic conditions. A second nuclease (Nuc-b,23 kDa) was detected in extracts of senescent coleoptiles,and at 120 h after exposure to aerobic conditions. When thegel was incubated at low pH (pH 5.5), we could not detectany nuclease activity (data not shown). To analyse the ion-dependency of these nucleases, gels were incubated with areaction solution containing several combinations of Mg2�,Ca2�, Zn2� and EDTA (Fig. 8). Regardless of the presenceof Ca2� or Mg2�, the activity of Nuc-a was detectable. Onthe other hand, stimulation of Nuc-b activity required bothMg2� and Ca2�. The activities of both enzymes were
DISCUSSION
A unique feature of growth and development in coleoptilesof graminaceous monocots is the rapid transition from a
living state to senescence. By using seedlings that had
FIG. 3. A, Rice seedlings that had been kept under submergence for 7 d (0 h) were exposed to air for 24, 48, 72, 96 and 144 h. Arrow indicates thesenescent coleoptile. Bar � 1 cm. Coleoptiles exposed to air for 0 h (B), 6 h (C and E) and 24 h (D) were treated with Evans blue to detect deadcells. E is a magni®ed image of C. Arrows in C and E indicate sites stained with Evans blue. Bars � 0.2 cm (B±E). F, Schematic image of split
zone (blue colour) in coleoptile of seedling after transfer from submergence to aerobic conditions.
Kawai and UchimiyaÐColeoptile Senescence in Rice 409
germinated under submergence, and that were subsequentlytransferred to aerobic conditions, we had the opportunityto analyse cell death events in elongated coleoptiles.
There was no evidence of cell death events leading to theopening of the coleoptile, aerenchyma formation andsenescence of the coleoptile of rice seedlings while growingunder submergence. Thus, submergence/hypoxia seems toblock maturation and subsequent cell death in coleoptiles.Once submerged coleoptiles were exposed to air, at leastthree death events, splitting, aerenchyma formation and
senescence occurred.The speci®c programme involved in the induction of celldeath in aerated coleoptiles cannot be identi®ed unambig-uously based on the present evidence. It is interesting tospeculate that the initiation of cell death may be due to theresponse to reactive oxygen species. It is reported that H2O2
produced as an oxidative burst, induces cell death in higherplants (Levine et al., 1994; Tenhanken et al., 1995). It wouldbe interesting to investigate further the interaction of thesesignals in conjunction with senescence of coleoptiles.
Light microscopic examination of cross-sections through
the splitting region of coleoptiles showed that this zone is
FIG. 4. Cross-sections of coleoptiles obtained from seedlings transferred from submergence to aerobic conditions. 0 h (A) and 96 h (B) aftertransfer to aerobic conditions. Arrows in B indicate aerenchyma. Magni®ed images of split zones at 0 (C) and 6 h (D) after exposure to air arepresented. A portion of a coleoptile stained with Evans blue was ®xed as described in the Materials and Methods. Arrows in C and D show the sitewhere splitting takes place. Bar � 50 mm. E, The abscission zone was determined in coleoptiles 6 h after transfer from submergence to aerobicconditions. The site of Evans blue staining was plotted and results are expressed as a percentage of the number of seedlings examined. The position
of the ®rst leaf inside the coleoptile is shown in E. n � 60.
410 Kawai and UchimiyaÐColeoptile Senescence in Rice
characterized by the presence of compact cells. We foundhere that splitting of the coleoptile occurs spontaneously inspecialized parenchyma tissues.
Apoptosis, which is a speci®c type of PCD in animalcells, involves activation of endonucleases and the appear-ance of oligonucleosome-sized DNA fragments as shownby agarose gel electrophoresis (Wyllie et al., 1984). How-ever, this is not always the case, and sometimes PCD inanimal cells does not involve fragmentation of DNA intooligonucleosome-sized pieces (Obserhammer et al., 1993;Schwartz et al., 1993). Some PCD in plants can also involvethe generation of oligonucleosome-sized DNA fragments(Wang et al., 1996a, b; Koukalova et al., 1997; OrzaÈ ez andGranell, 1997; Young et al., 1997), although some otherforms of cell death that are also thought to occur by PCDdo not involve the processing of DNA into such oligo-
nucleosomal fragments (Mittler and Lam, 1995a, b). Thus,there are several types of cell death mechanisms in PCD,including apoptosis.
OrzaÈ ez and Granell (1997) reported that cells of thesenescent carpel of pea showed both DNA laddering andcondensed nuclei. More recently, using the TUNEL assay,Yen and Yang (1998) reported the detection of DNAfragmentation in naturally senescent leaves of some plantspecies. In rice coleoptiles, Inada et al. (1998a, b) demon-strated degradation of chloroplast DNA and nuclear dis-organization using microscopic techniques. In this study,we found TUNEL-positive signals in nuclei of peripheralcells and oligonucleosomal DNA ladders in senescentcoleoptiles. The size of the bands was calculated to bearound 200 and 400 bp, and smaller molecules increased inintensity with time. These results con®rm the link betweenDNA fragmentation and senescence. Our results have
demonstrated that such a hallmark of PCD may be below
FIG. 5. Detection of fragmented nuclear DNA by TUNEL assay.Cross-sections of young coleoptile obtained from 4-d-old seedlingsgrown under aerated conditions (A), and senescent coleoptile of 14-d-old seedlings grown under aerobic conditions (B). Each cross-sectionwas treated with DAPI (left) and analysed by TUNEL assay (right).Green-coloured signals indicate cells positive for the TUNEL assayshowing accumulated DNA 30-OH groups. Arrow in A indicates split
zone. Bar � 30 mm. ae, Aerenchyma.
0 48 96 120 144 h
kbp
23.19.6
1.5
0.9
0.3
0.1
FIG. 6. Detection of DNA laddering in coleoptiles. DNA wasextracted at 0, 48, 96, 120 and 144 h after exposure to air andseparated by electrophoresis on a 1.5% agarose gel. Ten mg of DNAtreated with RNase was loaded into each lane. Molecular sizes in bases(kbp) are indicated. An open arrowhead indicates a position of well.
An closed arrowhead indicates large genomic DNA.
kDaA
B kDa
0 24 48 72 120 168 SC
Nuc-a
Nuc-b
0 24 48 72 120 168 SC
83
62
47
32
25
16
83
62
47
32
25
16
FIG. 7. Induction of nuclease activity in coleoptiles obtained fromseedlings that had been transferred from submergence (7d) to aerobicconditions. Proteins were extracted at 0, 24, 48, 72, 120 and 168 h afterexposure to air. Senescent coleoptile (SC) obtained from seedlingsgerminated under aerobic conditions for 14 d. Gels stained with CBB(A) and assayed for nuclease activity (B) are presented. Molecular massmarkers are shown on the left in kilodaltons (kDa). After electro-phoresis, the gel was incubated with a reaction solution containing3 mM CaCl2 and 3 mM MgCl2. Nuc-a, Nuclease-a; Nuc-b, nuclease-b.
Kawai and UchimiyaÐColeoptile Senescence in Rice 411
the detection level in cells forming gas-spaces or the splitzone. There was no evidence of a TUNEL signal in thevicinity of aerenchyma formation or coleoptile splittingwhich included collapsing and Evans blue-stained cells. Inthe early phase of aerenchyma formation or coleoptileopening, nuclei seemed to remain intact. Even in collapsingcells, we observed that some cells contained nuclei. Themechanisms of cell death in these cell types may be di�erentfrom those occurring with senescence. Thus, further studiesof the mechanisms of DNA degradation are needed in oursystem.
We also examined endonuclease activity by in-gel-nuclease assay. We detected two activities, namely Nuc-a(38 kDa) and Nuc-b (23 kDa), which could be inducedduring coleoptile development. Nuc-a required either Mg2�
or Ca2�, was inhibited in coleoptiles grown under submerg-ence, but was induced 24 h after exposure to air. Nuc-brequired both Mg2� and Ca2� and was detected during theprocess of senescence. Because Nuc-a was induced in theearly stages of coleoptile development when DNA frag-mentation was not seen, it is possible that Nuc-a may be
involved in the general recycling of DNA. The ®nding that
Mg, Ca1 2 SC
Ca1 2 SC
Mg1 2 SC
EDTA1 2 SC
Ca, Mg, Zn1 2 SC
Nuc-a
Nuc-b
FIG. 8. Comparison of ion-dependency of nucleases. Protein extracts obtained from coleoptiles of seedlings at 48 h (lane 1), 168 h (lane 2)following transfer from submergence to aerobic conditions and senescent coleoptile (SC) were analysed for nuclease activities as described in theMaterials and Methods, except that gels were incubated with a reaction solution containing various cations as indicated. Nuc-a, Nuclease a;
Nuc-b, nuclease b.
FIG. 9. A schematic diagram illustrating cell death events in coleoptiles. A, Schematic diagram of coleoptile development under aerobic andsubmerged conditions. When seedlings grown under submergence were transferred to aerobic conditions, a rapid progression of cell deathassociated with splitting, aerenchyma formation and senescence can be seen. The location of cells stained with Evans blue (EB) and TUNELpositive are indicated. B, Temporal ¯ow chart indicating di�erential cellular and molecular events in coleoptiles obtained from seedlings
en
412 Kawai and UchimiyaÐColeoptile Senescence in Rice
Nuc-b activity corresponded to the senescence stage mayhave a functional signi®cance in nuclear fragmentationdetected by TUNEL assay.
Thelen and Northcote (1988) demonstrated that activityof a 43 kDa nuclease is closely associated with cell deathduring tracheary element formation in Zinnia cells.Recently, Aoyagi et al. (1998) isolated cDNAs encoding avacuolar 43 kDa endonuclease of Zinnia (ZEN), and a35 kDa nuclease of barley (BEN) which were reported to be
transferred from submerg
secreted from the aleurone layer into the endosperm during
germination. Both enzymes are S1-type DNases, and haveendonucleolytic activity in the presence of Zn2�. The cationrequirements of BEN and ZEN are clearly distinguishablefrom the biochemical properties of Nuc-a and Nuc-b,whose activities were strongly inhibited by Zn2�. In thisregard, Mittler and Lam (1995a, 1997) reported that thehypersensitive response (HR) of plants to avirulentpathogens is accompanied by an increase in the activity ofa 36 kDa nuclease. HR-associated nuclease activities were
ce to aerobic conditions.
stimulated by Ca2�, but not by Mg2�, and inhibited by
le
Zn2�. The speci®c DNase engaged in DNA fragmentationhas not yet been identi®ed in plants.
Interestingly, in animals, DNase g (Shiokawa et al., 1994)and NUC18 (Gaido and Cidlowski, 1991), found in nucleiof rat thymocytes that were induced to undergo apoptosis,requires both Ca2� and Mg2�, and was inhibited by Zn2�.It is possible that Nuc-b identi®ed in the present study mayrepresent the plant counterpart of the animal nucleasesassociated with apoptosis. Since a direct interactionbetween Nuc-b and DNA fragmentation has not beendemonstrated, molecular cloning of the gene encoding thisnuclease is necessary in future studies.
As summarized in Fig. 9, developing coleoptiles grownaerobically showed three types of cell death processes:splitting, aerenchyma formation and senescence. Submerg-ence, i.e. anaerobic conditions, inhibited such cell deathevents. Following exposure of submerged seedlings to air,cell death events are initiated. As soon as 6 h after transferto aerobic conditions, cell death occurred in a restricted cellposition in the parenchyma, and resulted in coleoptileopening. Aerenchyma formation was visible after 48 h asEvans blue-stained dead cells. A TUNEL assay showed thatsenescence included fragmented DNAs, although coleoptilesplitting and aerenchyma formation would not include suchevents. Further analysis showed induction of Nuc-anuclease after 24 h. At 120 h, induction of Nuc-b nucleaseand DNA fragmentation were observed, and thesecoincided with the progression of coleoptile senescence.
We have demonstrated that the coleoptile is a uniqueorgan associated with several cell death events. Furtherwork to de®ne the cellular mechanisms of such events is
Kawai and UchimiyaÐCo
now required.
ACKNOWLEDGEMENTS
We thank Dr M. C. Drew (Texas A & M University) forcritical reading of the manuscript. This research wassupported by Grants-in-Aid for Scienti®c Research fromthe Ministry of Education, Culture and Science, Japan; byResearch for the Future from the Japan Society for
Promotion of Science (grant no. JSPS-RFTF96L00604).LITERATURE CITED
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