Programmed cell death progressively models the development of anther sporophytic tissues from the tapetum and is triggered
in pollen grains during maturation
Anne-Lise Varnier a ,Florence Mazeyrat-Gourbeyre a ,Rajbir S.Sangwan b ,
Christophe Cle
′ment a,*a
Laboratory of Plant Stress Defence and Reproduction,URVVC EA 2069,University of Reims,P.O.Box 1039,51687Reims Cedex 2,France
b
Laboratory of Androgenesis and Biotechnology,University of Amiens,80039Amiens Cedex,France
Received 31May 2005;received in revised form 12July 2005;accepted 14July 2005
Available online 7October 2005
Abstract
To characterize the spatial and temporal occurrence of programmed cell death (PCD)in Lilium anther tissues,we used both micro-scopical and molecular markers of apoptosis for developmental stages from meiosis to pollen release.The ?rst hallmarks of PCD include cell condensation and shrinkage of the cytoplasm,separation of chromatin into delineated masses,and DNA fragmentation in the tape-tum as early as the premeiosis stage.PCD then extended to other anther sporophytic tissues,leading to anther dehiscence.Although the PCD clearly a?ected the endothecium and the epidermis,these two cell layers remained alive until anther dehiscence.In pollen,no sign of PCD was found until pollen mitosis I,after what apoptotic features developed progressively in the vegetative cell.In addition,DNA ladders were detected in all sporophytic tissues and cell types throughout pollen development,whereas in the male gametophyte DNA ladders were only detected during pollen maturation.Our data suggest that PCD is a progressive and active process a?ecting all the anther tissues,?rst being triggered in the tapetum.ó2005Published by Elsevier Inc.
Keywords:Anthers;Lilium ;Mitochondria;Nucleus;Programmed cell death;Tapetum
1.Introduction
Programmed cell death (PCD)is a genetically regulated cell death allowing the elimination of speci?c cells,tissues or whole organs.Most of stereotypic signs of PCD,?rst reported in animal cells,were progressively described in plant cells during the last decade.In plants PCD is correlated with developmental processes,including leaf senescence,the formation of sexual organs,the disappearance of aleurone cells,the removing of root cap cells or the di?erentiation of specialized cell types such as tracheary elements (Jones,2000;Kuriyama and Fukuda,2002;Pennell and Lamb,1997).Environmental variations resulting in biotic or abiotic
stresses such as climatic change (Chen et al.,1999),toxic
compounds (Yao et al.,2002a ),oxidative stress (Pedroso et al.,2000)or pathogen attack (Hoeberichts et al.,2003)also generate physiological reactions involving PCD.
In angiosperms,PCD is widely involved in the achieve-ment of reproduction (Pennell and Lamb,1997;Wu and Cheung,2000).In the male organs,PCD occurs before pol-lination and is pronounced in the anther.During normal development,it has been shown that PCD takes place in some anther sporophytic tissues,resulting in tapetum dis-appearance and development of the dehiscence zone (Cle
′ment et al.,1998;Papini et al.,1999;Schreiber et al.,2004;Thomas et al.,2003;Wang et al.,1999;Wu and Cheung,2000).Furthermore,it was shown that the epider-mis and the endothecium undergo PCD like process in Solanum melongena and Zea mays during pollen
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Journal of Structural Biology 152(2005)
118–128
Journal of
Structural Biology
1047-8477/$-see front matter ó2005Published by Elsevier Inc.doi:10.1016/j.jsb.2005.07.011
*
Corresponding author.Fax:+33326913339.
E-mail address:christophe.clement@univ-reims.fr (C.Cle
′ment).
maturation(Schreiber et al.,2004;Xu and Chye,1999).In some species,male gametophytic cells may also be tran-siently a?ected by PCD.In Hordeum vulgare,the nucleus of uninucleate microspores exhibits positive reaction to the TUNEL(Terminal deoxynucleotidyl transferase-medi-ated dUTP Nick End-Labeling)staining,indicating degra-dation of their DNA during the process of vacuolization (Wang et al.,1999).During the same period,endonuclease genes are expressed and translated in the microspores (Marchetti et al.,2001;Zaina et al.,2003).Nothing has been reported so far on the other phases of pollen develop-ment though the male gametophyte is a temporary struc-ture.Moreover,the available data are incomplete concerning all the anther cell types,either from the sporo-phytic tissues(middle layers,endothecium or epidermis)or from the gametophytic tissue(microspores and pollen grains).Although,the developmental events leading to an-ther formation,pollen release,cell di?erentiation,and dehiscence events occur in a precise order and are well de-?ned(Sanders et al.,2005;Scott et al.,2004),the cellular processes that regulate anther cell di?erentiation,establish anther tissue patterns,and cause the anther to switch from a histospeci?cation program to cell degeneration and dehiscence program remain widely unknown.Interestingly, failure of PCD in the anther leads to abnormal pollen development and sterility(Sanders et al.,2000;Wu and Cheung,2000),thus indicating a critical role of PCD in normal anther/pollen development.
Recently,new evidence for the occurrence of PCD in plant tissues has been provided by observations of chroma-tin condensation,nuclear fragmentation,increased expo-sure of phosphatidylserine on the outer surface of the plasma membrane,and internucleosomal DNA cleavage (Gunawardena et al.,2004).The involvement of mitochon-dria at precocious stages of PCD has also been demonstrat-ed in plants(Hanson and Bentolila,2004).It is characterized by H2O2release from permeability transition pores that are similar to those present in animal mitochon-dria preceding the apoptotic process(Yao et al.,2002b).
Lilium is one of the most routinely used model plant for the study of anther physiology.In particular,using this model genus the understanding of the relationships be-tween the anther diploid sporophytic tissues and the hap-loid gametophytic cells via the locular?uid have been signi?cantly enhanced in the last decade(Aouali et al., 2001).The identi?cation of active proteinases in the anther of Lilium temporally correlated with apoptotic events pre-ceding anther and pollen maturation indicates that PCD may a?ect both the sporophytic and gametophytic tissue during development(DeGuzman and Riggs,2000).Recent-ly,we have optimized a protocol allowing the accurate sep-aration of the sporophytic and the gametophytic parts of the anther(Aouali et al.,2001).This relative accessibility and predictability of Lilium anther and pollen develop-ment,thus provides an attractive model system for the study of developmental PCD in planta.Therefore,we undertook a more detailed analysis of the PCD process in anther by light and electron microscopy in conjunction with analysis of DNA degradation to better understand the occurrence of PCD in the whole anther.Our data sug-gest that the PCD may play a key role in normal anther and or pollen development leading to e?cient pollen re-lease and fertile pollen production.
2.Materials and methods
2.1.Plant material
Bulbs of Lilium hybrida cv.Citronella were grown in the laboratory greenhouse at25°C,under a16hours photope-riod provided by400W mà2‘‘SAUDICLAUDE’’lamps, and a relative humidity of50%.To determine the exact stage of pollen development,one fresh anther per?ower bud was squashed in acetic carmine(5%(w/v)in45%acetic acid boiled for1h and?ltered)and observed under the microscope.The di?erent stages of pollen ontogenesis were used as chronological marker of anther development(Cle′-ment et al.,1994).
2.2.Separation of anther compartments
The separation of anther tissues was carried out at4°C according to Aouali et al.(2001).Anthers were collected from the?ower bud and cut transversely into1mm slices, placed in hemolysis tubes containing1mL of0.4M manni-tol and lightly vortexed for1min.The samples were?ltered on metal grids(400l m mesh),which retained the anther wall fraction,allowing the pollen fraction to be harvested in a new hemolysis tube.The anther wall fraction was rinsed with1mL of0.4M mannitol,gently vortexed once more and?ltered in order to remove remaining pollen grains.This fraction was ground with liquid nitrogen into a?ne powder which was stored atà80°C until use.The pollen fraction was centrifuged at100g for5min.The resulting pellet,corresponding to the pollen fraction,was homogenized in liquid nitrogen and stored atà80°C. 2.3.Light microscopy
Anthers were?xed for24h at room temperature in2% glutaraldehyde in0.1M phosphate bu?er(pH7.2)with1% (w/v)sucrose and1&(v/v)Tween20.Then the samples were treated separately either for topographic cytology or for ?uorescence destined to examine the aspect of the nucleus.
For general cell structure,each sample was rinsed three times for5min in0.1M phosphate bu?er(pH7.24)with 1%(w/v)sucrose.The samples were post-?xed for4h in 1%(w/v)osmium tetroxide in0.1M phosphate bu?er (pH7.24)with1%(w/v)sucrose,dehydrated in alcohol ser-ies,and embedded in araldite.Semithin sections(1l m) were collected on glass sides and the periodic acid Schi?(PAS)polysaccharide speci?c reaction was carried out.Sec-tions were?rst plunged in1%(w/v)periodic acid for4h, then in Schi??s reagent without rinsing for16h,and?nally
A.-L.Varnier et al./Journal of Structural Biology152(2005)118–128119
in 5%(w/v)sodium metabisul?te for 20min.Sections were then rinsed in distilled water,air-dried,and mounted in Eukitt.
For ?uorescence microscopy,the samples were rinsed three times for 5min in distilled water before being dehydrat-ed in an alcohol series and embedded in historesin.Semi-thin (1l m)sections were cut with a Leica JungRM 2055micro-tome.Sections were stained with 5l M Hoechst 33342for 15min in the dark,washed with a phosphate bu?er saline Na 2HPO 4/NaH 2PO 40.1M,NaCl 9&,and examined under an Olympus BH-2?uorescence microscope.2.4.Electron microscopy
For transmission electron microscopy,anthers were ?xed for 24h at room temperature in a solution of 2%(w/v)glutaraldehyde in 0.1M phosphate bu?er (pH 7.24)with 1%(w/v)sucrose and 1&(v/v)Tween 20.Each sam-ple was rinsed three times for 5min in the 0.1M phosphate bu?er (pH 7.24)with 1%(w/v)sucrose.The samples were post-?xed for 4h in 1%(w/v)osmium tetroxide in 0.1M phosphate bu?er (pH 7.24)with 1%(w/v)sucrose,dehy-drated in an alcohol series,and embedded in araldite.Ul-tra-thin sections were mounted onto 150-mesh copper grids with a diamond knife on a Reichert OM U2ultratom-e,stained with uranyl acetate/lead citrate,and examined on a JEOL JEM 100S electron microscope at 80kV.2.5.DNA extraction and analysis
Anther wall and pollen fractions were grounded in liquid nitrogen.The powder (200mg)was suspended in b -mercaptoethanol (5l L)and Sarkosyl 20%(100l L).Next,300l L of extraction bu?er (EDTA 25mM,NaCl 250mM,SDS 0.5%(w/v),and Tris 200mM,pH 8)was added and the whole mixture was incubated at 65°C for 10min.Polyvinylpolypyrrolidone (6%,24mg)and ammo-nium acetate 7.5M (200l L)were added.Samples were
mixed and incubated for 15min in ice before centrifugation at 10000g during 10min.The supernatant was mixed with an equal volume of chloroform/isoamyl alcohol (24:1,v/v)and centrifuged for 10min at 10000g .DNA was precipitat-ed by adding an equal volume of isopropanol to the super-natant.Samples were centrifuged for 10min at 10000g .The pellet was washed with 70%ethanol and resuspended in distilled water (30l L).Contaminating RNAs were re-moved by incubation at 37°C for 30min in the presence of RNAse (1mg mL à1).After addition of ammonium ace-tate 4M (35l L)and 95%ethanol (175l L),samples were mixed and immediately centrifuged 20min at 10000g .The pellet was washed with 80%ethanol and centrifuged 10min at 10000g .The pellet was next air-dried and resus-pended in distilled water.DNA samples (500ng)were run on a 1%(w/v)agarose gel at 110V for 45min.The DNA was stained with SYBR Green I (1/10000)and revealed un-der UV.As a control,500mg of leaves were treated according to the same protocol.3.Results
3.1.Cell structure at di?erent developmental stages of the anther
To date,each anther is composed of four locules all in contact with the connective tissue (Fig.1A).The locule supports the gametophytic pollen grains and is surround-ed by the sporophytic anther wall layers composed of four cell types (Fig.1B)whose function were previously de?ned (Cle
′ment et al.,1994):(i)the most internal one is the tape-tum lining the anther cavity and providing nutrients for the developing microspores;(ii)three middle layers sur-round the tapetum and are devoted to starch accumula-tion and mobilization;(iii)the endothecium is a single cell layer destined to store reserves during the ?rst half of pollen development and next to develop cell wall thick-enings involved in the anther dehiscence process;and (iv)
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1.Anther anatomy during pollen development.(A)Anther hand section at early stage showing four locules (L)linked by the connective tissue including a vascular bundle (asterisk)and separated by the stomium (Stm).Bar scale =300l m.(B)Microscopical section of the anther locule stained reaction.The gametophytic cells (Ga)were surrounded by the sporophytic tissues (Sp)and the dehiscence zone (arrowhead).The anther wall formed by the epidermis (Ep),the endothecium (End),the middle layers (ML),and the tapetum (T).Bar scale =200l m.(C)Detail of the connective tissue showing the vascular bundle including xylem (Xy)and phloem (Ph)close to transfer cells (TC)boarding the locule.Cell layers around the locule included amount of starch (arrowhead).Bar scale =200l m.
120 A.-L.Varnier et al./Journal of Structural Biology 152(2005)118–128
the epidermis including stomatas acting as a protective cell layer and participating in anther autotrophy.The connec-tive tissue includes two types of tissue(Fig.1C):(i)the vascular bundle supplying the whole anther with both types of sieve,and(ii)the transfer cells contributing to the distribution of nutrients to the locules.Moreover,be-tween adjacent locules,the cells of the stomium are the site of dehiscence for further pollen release and dispersion.
To check the timing of PCD in the various anther frac-tions,we examined the cytological aspects of organelles in all the anther cell types,focusing particularly on the struc-ture of the nucleus and mitochondria.To provide a general overview of anther cell behaviour,PCD occurrence was followed throughout pollen development and is summa-rized in Table1.
3.2.Fate of anther sporophytic cell layers
Each anther cell type exhibited a speci?c developmental pathway(Fig.2).In the anther wall both tapetum and mid-dle layers were ephemerous.The tapetum completed endo-mitosis at early meiosis(Fig.2A)and then transformed into a polarized secretory cell soon after meiosis(Figs.2B and C).Afterwards the tapetum accumulated large amounts of lipid globules(Figs.2C and D)and the cytoplasm degener-ated,leading to cell death and lipid release in the loculus at the time of mitosis.At meiosis,the middle layers contained starch grains that are progressively mobilized during the process of microspore vacuolization(Figs.2A–C).After pollen mitosis I,the cytoplasm was degraded and cells shrank during pollen maturation(Figs.2E and F).The endothecial cells did not degenerate but during pollen mat-uration their nuclei invaginated and wall thickenings devel-oped(Figs.2E and F).Epidermal cells and connective tissue were similar to the endothecium with regard to the nucleus.
The stomium cells between adjacent anther locules looked like anther wall layers until the end of meiosis (Fig.3A).Afterwards,drastic modi?cations occurred beginning during microspore vacuolization.These were characterized by the loss of cell adhesion and cytoplasm degeneration(Fig.3B).Later on,the epidermal cells close to the stomium increased in size and accumulated pigment in their vacuole(Fig.3C).Altogether this contributed to the disruption of the tissues between adjacent locules (Fig.3D)and subsequently led to anther dehiscence,allow-ing the release of pollen grains(Figs.3E and F).
3.3.DNA is cleaved in both sporophytic and gametophytic tissues during pollen development
The detection of DNA laddering was carried out sepa-rately in sporophytic and gametophytic compartments of the anther.In the sporophytic compartment,typical apop-totic DNA fragmentation was identi?ed as early as the microspore mother cell stage up to the end of pollen development(Fig.4A).This indicates that PCD is active in the sporophytic tissues of the anther from meiosis to mature pollen stage.Conversely,in the gametophytic frac-tion,no DNA laddering was observed either in the micro-spore mother cells,tetrads or microspores during vacuolization(Fig.4B).However,DNA ladders were clearly detected in the pollen grains after pollen mitosis I,indicating that pollen maturation involves apoptotic processes.
3.4.The?rst hallmarks of apoptosis appeared in the tapetum at the microspore mother cell stage
From the microspore mother cell until the tetrad stage, the tapetum nuclei were invaginated and the chromatin showed the?rst signs of condensation(Fig.5A).In the cytoplasm,some of the mitochondria showed abnormal features characterized by the appearance of internal vacu-oles(Fig.5B).During microspore vacuolization,the con-densation of the chromatin was progressively enhanced (Fig.5C)and invaginations of the nuclei increased (Fig.5D)due to the development of cytoplasmic enclosures in the nucleoplasm.Close to microspore vacuolization,the chromatin was strongly condensed(Fig.5E)and the nuclei were highly fragmented(Fig.5F).In the mitochondria,the
Table1
Appearance of PCD hallmarks in anther
tissues
MMC,microspore mother cell;Tet,tetrad;YM,young microspore;VM,vacuolated microspore;Mi,mitosis;YPG,young pollen grain;APG,amylaceous pollen grain;MPG,mature pollen grain.
A.-L.Varnier et al./Journal of Structural Biology152(2005)118–128121
vacuolization process had developed though some of them still exhibited typical functional cristae (Fig.5G).Later on the tapetum was disintegrated and its remnants were dis-persed in the loculus.These results indicate that PCD af-fects the tapetum as early as the premeiotic stage and extends until ?nal cell degradation and lysis.
3.5.The middle layers were a?ected by PCD during microspore vacuolization
The ?rst signs of PCD could be observed in the middle layers during microspore vacuolization.Until the mid vacu-olated microspore stage,the nucleus appeared spherical and
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the chromatin was relatively homogeneous though some heterochromatin could be observed (Fig.6A).Mitochon-dria did not seem to be a?ected at this stage.During microspore vacuolization,the heterogeneity of the chroma-tin was emphasized (Fig.6B)and the nucleus showed invag-inations (Figs.6C and D).In the meantime,mitochondria were a?ected by abnormal vacuolization (Fig.6E).The cytoplasm progressively degenerated after microspore mito-sis and the middle layers had entirely disappeared at the late binucleate,starch-?lled pollen grain stage.
3.6.Apoptotic features extended simultaneously in the
endothecium,the epidermis and the connective tissue at the late vacuolated microspore stage
Just before microspore mitosis,the signs of PCD simul-taneously reached the endothecium,the epidermis and the connective tissue (Table 1).In these cell types,the nucleus exhibited the ?rst invaginations (Figs.6C and D)and some mitochondria were a?ected simultaneously by the develop-ment of internal vacuoles and dark vesicles (Fig.6E).These
features were maintained until pollen maturation and an-ther dehiscence (Fig.6F),but neither endothecial nor epi-dermal nor connective cells degenerated.
3.7.The dehiscence zone showed PCD signs synchronously with the middle layers
From the microspore mother cell stage until the ?rst steps of microspore vacuolization,the stomium cells in-volved in anther dehiscence did not show signs of PCD (Table 1).During microspore vacuolization,the shape of nuclei became irregular (Fig.7A)and heterogeneous chro-matin appeared in cells between adjacent anther locules (Fig.7B).This was preceded by the progressive appearance of vesicles in some mitochondria.At the vacuolated micro-spore stage,all the nuclei of this area were characterized by heterogeneous chromatin (Fig.7C).At microspore mitosis,nuclei further invaginated and the heterogeneity of chro-matin increased,whereas most of mitochondria had abnor-mal features (Fig.7D).During pollen maturation the cells of the dehiscence zone degenerated,allowing connections between two adjacent locules and release of pollen at ?ower opening.The results obtained here indicate that PCD oc-curs simultaneously in the middle layers and in the cells in-volved in anther dehiscence.
3.8.The vegetative cell of the pollen grain shows apoptotic features during maturation
In the microspore mother cell,the diploid nucleus appeared spherical and mitochondria had normal patterns (Table 1).After pollen mitosis I,the two nuclei in the young pollen grain had di?erent features (Fig.8A).The vegetative nucleus had a spherical shape including homogeneous chro-matin with low density,whereas the chromatin of the gener-ative nucleus was more dense.During pollen maturation,the chromatin of the generative nucleus remained condensed.Conversely,the nucleus of the vegetative cell showed increas-ing invaginations (Fig.8B).In the mature pollen grain,the nucleus was strongly invaginated leading to the appearance of some nuclear bodies that were released from the nucleus (Fig.8C).No mitochondria were present in the generative
WM
L AW Tet YM VM Mi YPG MPG
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Fig.4.DNA laddering in the anther tissues during pollen development.(A)Sporophytic fraction.(B)Lower part of the gel for the gametophytic fraction.(WM)Weight markers,(L)leaf,(AW)mixed anther wall,(Tet)tetrad stage,(YM)young microspore stage,(VM)vacuolated microspore stage,(Mi)mitosis,(YPG)young pollen grain stage,(MPG)mature pollen grain stage,and (bp)base pair.
Fig.2.Development of the locule envelopes during pollen ontogenesis.Light microscopy of anther sections stained with PAS reaction.(A)Microspore mother cell stage.Microspore mother cells (asterisk)in the locular ?uid (LF).The locule was edged by the tapetum (T)undergoing endomitosis (arrowhead),the middle layers (ML),the endothecium (End),and the epidermis (Ep).Bar scale =100l m.(B)Disconnection of the cell wall (arrowhead)from the binucleate tapetum (asterisk)at the tetrad (Tet)stage.(St)stomata.Bar scale =100l m.(C)Appearance of the ?rst lipid globules in the tapetum (arrowhead)during microspore (asterisk)vacuolization.Bar scale =100l m.(D)Increase of lipid accumulation in the tapetum (white asterisk)at the vacuolated microspore (VM)stage.Bar scale =100l m.(E)Nucleus invaginations in the middle layer and endothecium cells (arrowheads)at the young pollen grain (YPG)stage.Bar scale,100l m.(F)Flattened epidermal cells at the mature pollen grain (MPG)stage and ?nal thickening of endothecium cell wall (arrowhead).Bar scale =100l m.
Fig.3.Development of the dehiscence zone observed from anther sections stained with the PAS reaction.(A)Zone between two adjacent anther locules at the microspore mother cell (MMC)stage showing the tapetum (T)of the two locules,the epidermis (Ep),the endothecium (End),the middle layers (ML),and the future location of anther opening (arrowhead).Bar scale =200l m.(B)Appearance of intercellular spaces (arrowheads)from the young microspore (YM)stage.Bar scale =200l m.(C)Loss of cell adhesion (arrowheads)and accumulation of secondary metabolites (asterisks)in the distal epidermal cells.Bar scale,200l m.(D)Disconnection of epidermal cells and cell wall breakage (arrowheads)just before microspore mitosis.Bar scale =200l m.(E)Communication (asterisk)between two adjacent locules (L1and L2)at mitosis (Mi)though the epidermis (Ep)of both locules were still connected (arrowhead).Bar scale =100l m.(F)Complete anther opening (asterisk)at the young pollen grain stage.Bar scale =100l m.
b
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123
cell,but in the vegetative cell,mitochondria exhibited either normal features or were enlarged,lost their density and were characterized by internal vacuoles(Fig.8D).
4.Discussion
We characterized the spatial and temporal occurrence of PCD in Lilium anther tissues during pollen development using both light microscopy,TEM(mitochondria and nucleus structure,cytoplasm retraction)and molecular/ DNA markers of apoptosis.PCD in Lilium anther tissues implies that there are cells within anthers that possess par-ticular metabolic states which enable them to respond to certain physiological signals or stimuli and to enter the pro-cess of cell death—a process apparently required for nor-mal pollen development,dehiscence,and release of pollen grains at?ower opening(Sanders et al.,2000;Wu and Cheung,2000).The key questions we asked were:which cell types are most a?ected by PCD?Where are these locat-ed within anther?And is there any sequential,spatial and temporal onset of PCD during anther development?Our major?ndings are summarized in Table1and a schematic representation of the PCD events that occur within the an-ther sporophytic and gametophytic tissues is shown in Fig.9.4.1.PCD models the development of the anther sporophytic tissues
Our results show that the anther of Lilium undergoes PCD throughout anther/pollen development,from male meiosis until pollen grain maturation.Previously,PCD in the anther was reported to occur primarily during tapetum disappearance(Cle′ment et al.,1998;Papini et al.,1999; Wang et al.,1999;Wu and Cheung,2000)and during an-ther dehiscence(Schreiber et al.,2004;Wu and Cheung, 2000),however,we have shown here that PCD a?ects all the anther tissues including both sporophytic diploid and gametophytic haploid tissues,which is also consistent with the presence of proteinase(a PCD marker)in this organ (DeGuzman and Riggs,2000).Moreover,following each step of pollen development,we have further determined the spatial and temporal location of PCD in the whole anther.
In the anther cell layers surrounding the pollen locule, PCD hallmarks?rst appeared in the tapetum as early as pre-meiosis and extended exclusively in this cell type until the middle of microspore vacuolization.During these stag-es,cytological and molecular data indicate that the DNA ladders detected in the sporophytic compartment may be attributed solely to the binucleate tapetum.The cytology Mi
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Fig.5.Cytological features of tapetum development.(A)TEM section showing invaginations(arrowheads)of the nucleus(Nu)at meiosis.Bar scale=10l m.(B)Higher magni?cation showing alterations of some mitochondria re?ected by abnormal vesicles(arrowhead).Bar scale=3l m.(C) Hoechst33342stained anther section showing chromatin condensation(arrowhead)in the nucleus of the tapetum(T)but not in the middle layers(ML)nor in the endothecium(End)at the young microspore(YM)stage.Bar scale=150l m.(D)TEM section of the tapetum showing increased invagination (arrowhead)of the nucleus(Nu).Bar scale=15l m.(E)Enhancement of chromatin condensation(arrowhead)in the tapetum(T)nucleus at the vacuolated microspore(VM)stage,revealed by Hoechst33342staining.Bar scale=300l m.(F)Fragments of the nucleus(white asterisks)during late microspore vacuolization(VM).Bar scale=12l m.(G)Altered mitochondria(Mi)with widely developed vesicle(arrowhead)in the stroma.Bar scale=3l m.
124 A.-L.Varnier et al./Journal of Structural Biology152(2005)118–128
of tapetal cell degradation has been extensively described in
many species (Buckner et al.,1998;Cle
′ment et al.,1998;Lesniewska et al.,2000;Papini et al.,1999).Our results provide further evidence that tapetum degradation corre-sponds to PCD,in accordance with the results presented earlier (Balk and Leaver,2001).Previously,it has been reported that the anther undergoes PCD,but not before microspore release from the tetrads (Wu and Cheung,2000).However,the presence of DNA ladders,the alter-ation of some mitochondria and the fragmentation of the nucleus into nuclear ‘‘apoptotic body-like’’structures pre-sented here,clearly suggest that the tapetum is a?ected by PCD in Lilium as early as the microspore mother cell stage.
In the sporophytic tissue,the PCD not only a?ects the tapetum and the dehiscence zone,but during microspore vacuolization,the PCD extends radially from the tapetum and progressively reaches the most peripheral layers.The propagation/extension of PCD begins at early stages of microspore vacuolization and ?rst reaches the middle lay-ers and the dehiscence zone,which are both in close contact with the https://www.doczj.com/doc/9e11170692.html,ter on at the late vacuolated micro-spore stage,the endothecium,the epidermis and the con-nective tissue show the ?rst apoptotic features,in
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S T O M I U M VM VM VM Mitosis
Fig.7.Cytological features of the dehiscence zone.(A)At the vacuolated microspore (VM)stage,the ?rst signs of nucleus (Nu)invaginations (arrowheads)can be detected using TEM.Bar scale =20l m.(B)Hoechst staining of the stomium showing the increased condensation of chromatin at the vacuolated microspore (VM)stage.Bar scale =200l m.(C)Higher magni?cation of nucleus invaginations.Bar scale =100l m.(D)Advanced invaginations of the nucleus (Nu)at pollen mitosis,as well as normal (Mi)and abnormal (asterisk)mitochondria.Bar scale =20l m.
A B
C
D F
Mi
*
Nu
Nu
E X T E R N A L E N V E L O P E S
YM VM YPG
YPG
YPG
E Mi
*
VM
Fig.6.Developmental features of middle layers,endothecium,and epidermis.(A)TEM section of a middle layer cell showing the spherical shape and moderate condensation of the chromatin in the nucleus at the young microspore (YM)stage.Bar scale =10l m.(B)Appearance of nucleus invagination (arrowhead)in the middle layers at the vacuolated microspore (VM)stage revealed by the Hoechst staining.Bar scale =250l m.(C)TEM section of endothecium nucleus exhibiting moderate invagination (arrowhead)of the endothecium nucleus (Nu)at the young pollen grain (YPG)stage.Bar scale =20l m.(D)Similar features (arrowhead)in the nucleus (Nu)of the epidermis at the same stage.Bar scale =20l m.(E)TEM view at high magni?cation illustrating the abnormal development of some mitochondria in the endothecium at the vacuolated microspore (VM)stage.Bar scale =1l m.(F)Electron microscope view of the 2mitochondrial populations in the endothecium,normal (Mi)and vesiculated (asterisk)at the young pollen grain (YPG)stage.Bar scale =8l m.
A.-L.Varnier et al./Journal of Structural Biology 152(2005)118–128125
agreement with recent ?ndings (Schreiber et al.,2004).This strongly suggests that a wave of PCD extends from the tapetum to the outer anther cell types and progressively af-fects all the anther diploid cell types,as proposed in Fig.9.The involvement of PCD in the dehiscence zone con-?rms previous data (Kuriyama and Fukuda,2002;Sanders et al.,2000,2005).However,we could demonstrate here for the ?rst time the concomitant occurrence of apoptosis in the middle layers of anther.Besides,only few information is available concerning the development of apoptosis in the
epidermis,the endothecium and the connective tissue (Schreiber et al.,2004;Xu and Chye,1999).Our data now suggest that PCD,which is ?rst triggered in the tape-tum,progressively extends to the external anther cell layers a?ecting concomitantly the middle layers as well as the dehiscence zone and later on the endothecium plus the epi-dermis (Fig.9).
It is of interest to note that male sterile mutants and those defective in anther dehiscence have defects in the PCD process (Sanders et al.,2005;Yang et al.,2003),indi-cating that PCD is required for normal pollen develop-ment,anther dehiscence and release of pollen grains at ?ower opening (Sanders et al.,2000;Wu and Cheung,2000).
4.2.PCD a?ects the vegetative cell in the pollen grain The various cytological and molecular tools used in this study to characterize PCD allow us to conclude that PCD a?ects the vegetative cell of the pollen grain during maturation but not the generative cell.Previous works has demonstrated that apoptotic events can a?ect the microspore of Hordeum vulgare during vacuolization (Marchetti et al.,2001;Wang et al.,1999;Zaina et al.,2003).This may correspond to chromatin remodeling,fol-lowing the transition from the diploid to the haploid state,which is a key step for the reprogramming of tran-scription associated with development and cell di?erentia-tion (Farrona et al.,2004).However no data are available regarding the pollen grain.
Moreover,studies concerning PCD in plant cells usually report that no apoptotic bodies are formed (Danon et al.,2000).However,the formation of nuclear fragments was described in tobacco protoplasts or BY2cells treated with PCD inducers,respectively,menadione and isopentenylad-enosine (Mlejnek and Prochazka,2002;Sun et al.,1999),which is in accordance with our data.
The triggering of PCD in the vegetative cell is consistent with the fate of this cell,which disappears after pollen tube germination,i.e.,a few hours after maturation and anther
A
D
GN VN C
VN
B
VN
Mi
YPG YPG MPG
MPG
P O L L E N
Fig.8.Development of the microspore/pollen grain.(A)Hoechst stained section of a young pollen grain (YPG)showing homogeneous chromatin in the generative nucleus (GN)and heterogeneous chromatin in the vegetative one (VN).Bar scale =100l m.(B)Moderate invaginations (arrowheads)of the vegetative nucleus (VN)in the young pollen grain (YPG)revealed by TEM.Bar scale =15l m.(C)Vegetative nucleus (VN)in the mature pollen grain (MPG)showing nuclear bodies (arrowheads).Bar scale =100l m.(D)Corresponding TEM feature con?rming the presence of normal (Mi)and abnormal (arrowhead)mitochondria in the vegetative cell of the mature pollen grain.Bar scale =10l
m.
Fig.9.Possible progression of PCD in the anther of Lilium during pollen development.Initial stage,PCD occurs in the tapetum.Phase 1,progression of PCD to the middle layers and thus to the stomium.Phase 2,occurrence of PCD to the endothecium and epidermis (Ep-End).Phase 3,hypothetical transfer of PCD signal into the loculus to the pollen grains.
126 A.-L.Varnier et al./Journal of Structural Biology 152(2005)118–128
dehiscence.Indeed,the progressive degradation of the veg-etative cell organelles,especially mitochondria and plas-tids,has been shown to occur at various stages of pollen development(Mogensen,1996).In the case of barley with maternal cytoplasmic inheritance,the degradation of plastids begins in the microspores during vacuolization (Carreda et al.,2000),suggesting that an apoptotic like process takes place in these gametophytic cells.Both the vegetative and the generative cells originate from the microspore after mitosis.The question remains whether PCD a?ects the vegetative cell or the generative cell or both.Sato et al.(2004)have shown that the mitochondrial DNA progressively decreased during pollen development and disappeared in mature pollen.Moreover,recent data also show that the ZmMADS2transcription factor whose expression is indispensable for anther dehiscence is re-quired for pollen maturation and correlated with apoptosis (Schreiber et al.,2004).It seems further that this MADS box gene is also expressed in the pollen tube during elonga-tion(Heuer et al.,2000).This may indicate that the PCD is still active in the pollen tube,which is consistent with the fate of the pollen tube elements that are not involved in the fertilization process.Based on the appearance of DNA laddering in the pollen fraction and the cytological data,we conclude that PCD is progressively triggered after pollen mitosis I and a?ects only the vegetative cell of the pollen grain.
4.3.Anther as a model system for studying plant PCD triggering and propagation/extension
The radial extension of PCD in the anther cell layers from the tapetum to the peripheral layers suggests that a PCD hormonal signal is conveyed from the internal to-wards the peripheral cell layers(Kuriyama and Fukuda, 2002).Both jasmonic acid and ethylene may be good candidates for the spread of PCD in the anther.For example,the use of Arabidopsis thaliana mutants revealed a role for jasmonic acid signaling in controlling the time of anther dehiscence(Ishiguro et al.,2001;Sanders et al., 2000).Arabidopsis mutants that are defective in either jasmonic acid biosynthesis(e.g.,dde1,dde2,and da1) or perception(e.g.,coi1)are male sterile and have an-thers that dehisce too late for successful pollination to occur(Ishiguro et al.,2001;Sanders et al.,2005).Other phytohormones,such as ethylene,auxin and GA,have also been shown to play a role in anther dehiscence e.g.,it has been shown in Nicotiana tabaccum that ethyl-ene is directly involved in the?nal process of anther dehiscence and could act in synergy or in concurrence with jasmonic acid(Rieu et al.,2003).
The question remains,however,about the nature of the signal triggering the PCD in the vegetative cell of the pollen grain during maturation.In Lilium longi?orum,serine pro-teases that are known to be implicated in PCD are secreted in the loculus by the tapetum during microspore vacuoliza-tion(Taylor et al.,1997).Similar proteinases were found in the anther of Lilium(DeGuzman and Riggs,2000)and Solanum melongena(Xu and Chye,1999)at developmental stages correlated with apoptotic events.Proteinases may thus be good candidates for spreading of PCD in the an-ther in response to hormonal signalling.
In conclusion,anther is an interesting system for the study of plant PCD since the phenomenon is indispensable for the achievement and release of fertile pollen grain (Sanders et al.,2000;Wu and Cheung,2000).The process is?rst triggered in the tapetum which appears as the key cell layer from which apoptotic events are initiated in the anther.From the tapetal cells,the wave of PCD progresses radially in opposite directions,to both the outer anther sporophytic cell layers and the inner gametophytic devel-oping pollen grains via the locular?uid.The exact mecha-nisms of PCD signal transmission and the molecular nature of the apoptotic machinery in anther,however,remain to be elucidated.
Acknowledgment
The authors thank Dr.Antoine Danon for critically reading the manuscript.
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