Roles of extensins in cotyledon primordium formation and shoot apical meristem activity in ...

14 Pages·2008·1.21 MB·English

JournalofExperimentalBotany,Vol.59,No.14,pp.4045–4058,2008
doi:10.1093/jxb/ern245
Thispaperisavailableonlinefreeofallaccesscharges(seehttp://jxb.oxfordjournals.org/open_access.htmlforfurtherdetails)
RESEARCH PAPER
Roles of extensins in cotyledon primordium formation and
shoot apical meristem activity in Nicotiana tabacum
XueLian Zhang,YuJunRen andJie Zhao*
Key Laboratory ofthe Ministry ofEducation for Plant Developmental Biology, College ofLife Sciences, Wuhan
University, Wuhan 430072, China
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Received2July2008;Revised27August2008;Accepted8September2008 n
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Abstract Introduction h
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Epexptetindseinbsacakrebocnelelwthaaltlibsaesxictrgelmyceolyprrioctheiinnshwyidthroaxypporloy-- Emxatneynspinlasntarceelhlywdarlolxsyapsraolminaej-orircphrogtleyicnocporomtepionnsenfotu(nCdheinn s://ac
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line. In this paper, the function of extensins in embryo and Varner, 1985). They are composed of a polypeptide de
m
development was studied in Nicotiana tabacum. By backbone which is extremely rich in hydroxyproline ic
using Western blot and immunohistochemistry, the (Hyp) and also contains much serine, lysine, tyrosine, and .ou
p
extensin JIM20 epitopes were found to express in valine residues, and oligosaccharides of arabinose and .c
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different developmental stages of embryos, and spe- galactose (Wilson et al., 1986). This structure is the result m
cifically in the top of the embryo proper (EP) and the of considerable post-translational processing of the poly- /jxb
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suspensor of the late globular embryos. In order to peptide gene product. Extensin family members exhibit rtic
clarifythefunctionsofextensins,apotenthydroxypro- a high degree of post-translational modification (PTM) as le
-a
line synthesis inhibitor, 3,4-dehydro-L-proline (3,4- more than 50% of the amino acids were glycosylated bs
DHP), was used in ovule and embryo culture. The (Kieliszewski and Lamport, 1994). Among the proteins tra
c
results showed that the addition of 3,4-DHP caused which are present in the plant cell wall, the extensins are t/5
9
abnormal embryos with single, asymmetry and super- implicated in nearly all aspects of plant development. It /1
4
numerarycotyledonprimordia,andcontinuousculture was found that the expression of extensins and their /4
0
led to cotyledon defects in the germinated seedlings. mRNAs occurred in response to a varieties of biotic and 45
Histological sections showed that the shoot apical abiotic stresses, such as resistance to pathogens (Deepak /55
2
meristem (SAM) of the abnormal seedlings was dis- et al., 2007), wounding (Ahn et al., 1996), ozone 00
7
similar from the controls, especially in the seedlings (Schneiderbauerf et al., 1995), and superoxide and b
y
with cup-shaped cotyledons. Furthermore, the vascu- hydrogen peroxide (Wisniewski et al., 1999). There is g
u
lature of the abnormal cotyledons was in an out-of- also some evidence showing that extensins participate in es
order format and contained at least two main veins. many events during plant development, such as pollen t on
Finally,boththe hydroxyprolineassay andfluorescent recognition and fertilization (Wu et al., 2001), cell 12
immunolocalization confirmed that 3,4-DHP treatment division and differentiation, cessation of cell elongation Ap
reduced the level of extensins in the cultured ovules (Ito et al., 1998), abscission and senescence (Merkour- ril 2
0
and embryos. These results indicate that extensins opoulos and Shirsat, 2003), and flower organ formation 1
9
may play important roles in the cotyledon primordium (Neale et al., 1990).
formation,SAMactivity,andvasculaturedifferentiation In order to provide direct evidence for the functions of
duringembryodevelopment. extensins, researchers adopted different methods to alter
the level of extensins in plant cell walls. A transgenic
Key words: Cotyledon, embryo development, extensin, approach was one of the methods used by some
Nicotianatabacum,shootapicalmeristem,3,4-DHP. researchers. Memelink et al. (1993) studied the extensin
gene pCNT1 in Nicotiana tabacum L. with sense and
*Towhomcorrespondenceshouldbeaddressed:E-mail:[email protected]
Abbreviations: 3,4-DHP, 3,4-dehydro-L-proline; DAP, days after pollination; DP, 3,4-DL-dehydroproline; EP, embryo proper; HRGPs, hydroxyproline-rich
glycoproteins;Mb,monoclonalantibody;SAM,shootapicalmeristem;TEM,transmissionelectronmicroscopy.
ª2008TheAuthor(s).
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense(http://creativecommons.org/licenses/by-nc/2.0/uk/)which
permitsunrestrictednon-commercialuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
4046 Zhangetal.
antisense constructs under the control of the CaMV35S in pollination, but a few HRGPs have been reported to be
promoter. In studying the Atext1 gene in transgenic involved in embryo development. Up to now, only the
Arabidopsis, it was found that increasing the extensin RSH (Root–Shoot–Hypocotyl–Defective) extensin protein
level not only decreased the pathogen invasiveness (Wei has been proved to be essential in regulating the accurate
and Shirsat, 2006), but also affected the inflorescence positioning of the cell plate during embryogenesis of
stem thickness and height (Roberts and Shirsat, 2006). Arabidopsis (Hall and Cannon, 2002). Because embryos
Simultaneously, other methods such as treatment with reside deep in the ovules, it is difficult to observe the
chemical reagents were also adopted. In hydroxyproline- functions of extensins during embryo development in vivo.
rich glycoproteins (HRGPs), most of the Pro residues are Therefore, very few studies have provided direct and
hydroxylated by prolyl hydroxylases and the resulting unequivocal evidence for the functions of extensins during
hydroxyproline (Hyp) residues serve as major sites for embryogenesis of angiosperms. In this paper, an in vitro
oligosaccharide decoration (Kieliszewski and Lamport, embryo micro-culture system was used to solve this
1994). Because of this unique biosynthetic pathway, the problem. By using an immunofluorescence labelling tech- D
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use of specific inhibitors of prolyl hydroxylases could nique,thetemporalandspatialdistributionofextensinswas n
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provide a valuable approach for studying the functions of detected at different embryonic stages. The biological a
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Hyp-rich wall polymers. Such inhibitors should be functions of extensins were also investigated by examining ed
particularly effective for HRGPs because hydroxylase theeffectsof3,4-DHPonembryodevelopment.Theresults fro
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inhibitors can also block the attachment of carbohydrate showed that the inhibition of extensin synthesis increased h
side chains, which are shown to be partial determinants of the abnormal differentiation of embryos and caused defects ttp
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the structure of HRGPs (van Holst and Vamer, 1984; of cotyledon and the shoot apical meristem development. ://a
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StafstromandStaehelin, 1986).AsstronglybasicHRGPs, These indicate that extensins may play important roles in a
d
extensins are extremely rich in Hyp, so the treatment with cotyledon primordial formation and shoot apical meristem em
hydroxylase inhibitors will mostly influence the level of activity. This is the first paper that implicates extensins in ic.o
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extensins in plant cell walls among all types of HRGPs. the developmentoftobaccoembryos. p
.c
The chemical 3,4-DHP (3,4-dehydro-L-proline) was a po- om
tent inhibitor of prolyl hydroxylase and functions at /jx
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micromole concentrations to inactivate prolyl hydroxylase Materials and methods /a
rapidly and irreversibly (Cooper and Vamer, 1983). rtic
Previous studies showed that 3,4-DHP inhibited cell wall Plantmaterials le-a
b
assembly and cell division in Nicotiana tabacum meso- NoficWotuiahnaantUabnaivceurmsityL..C(covn.dSitRio1n)spwlaenrtesaw1e6re/8ghrolwignhti/ndagrrkeecnyhcoleusaet stra
phyll protoplasts (Cooper et al., 1994), and in tomato c
2861 (cid:1)C and the humidity was 65–70%. Flowers were artificially t/5
seedlings, the treatment with 3,4-DHP shortened the root 9
pollinatedduringanthesis. /1
hairs (Bucher et al., 1997). 4
/4
In sexual plant reproduction, there are several complex, 0
Totalproteinextractionofovules 4
5
precise, and key events: pollination, fertilization, and The total proteins of tobacco ovules were extracted as described by /55
embryogenesis. Previous studies have indicated that Qin and Zhao (2006). One gram fresh weight of ovules at different 20
hydroxyproline-rich glycoproteins are potential candidates developmentalstages(1,3,5,6,7,8,9daysafterpollination,DAP) 07
for mediating pollen–pistil interactions in conjunction with were taken from the ovaries and ground to a fine powder in liquid by
partner molecules in female tissues. It has been shown that nitrogen.Theground tissueswereplacedinto2mlextractionbuffer gu
e
thereproductivetissuesarearichsourceofhydroxyproline- (w0a.1sMcenKtr3iPfuOg4e,dpHat71.02).0A0f0terrpimncufboarti2o0namti4n.(cid:1)CThfoers3uphe,rtnhaetamntixwtuares st o
richglycoproteins.Forexample,Pex1(pollenextensin-like) precipitatedwith5volsofcoldacetoneat–20(cid:1)Covernightandthe n 1
2
protein mainly locates in the pollen tube wall and plays precipitate was re-suspended by vortex-mixing in 0.5 ml 50 mM A
raicrholeexitnentusbine-lgikroewptrhot(eRinu)bhinassteainpoestsaibl.l,e1r9o9l5e)i.nCtEheLPpr(oCceysss- T(toRtIaSl-pHroCtle,inpsH)w8a.s0reatnadinetdheanndcestnotrreifdugaetd–.80Fi(cid:1)nCalluyn,titlhueses.upernatant pril 20
1
of pollination and fertilization (Wu et al., 1993). PELPIII 9
SDS-PAGEandimmunoblotassay
(Class III pistil-specific extensin-like proteins) abundantly
and specifically localize in the intercellular matrix (IM) of Thetotalproteinsfromtheovulesatdifferentstageswereanalysedby
SDS-PAGE using a 12.5% acrylamide separating gel and a 5%
tobacco style transmitting tissues,and translocates from IM
acrylamidestackinggelinaMini-ProteanIIelectrophoresiscell(Bio-
to the pollen tube wall after pollination, which indicates
Rad).Equalamountsoftotalproteinswereloadedineachwell.Gels
that the biological function of the glycoprotein is related to wereelectroblotted(88V,3h)ontonitrocellulosetransfermembranes
pollen tube growth (Bosch et al., 2003). It was also found using electro-transfer buffer (20 mM TRIS-base, 150 mM glycine,
thatastyle-specific120kDaprotein,whichislocatedinthe 20% methanol). The nitrocellulose membrane blots were blocked
with5%non-fatdriedmilkinTBSTbuffer(20mMTRIS-base,500
pistil-grownpollentubes,wasrequiredforS-specificpollen
mM NaCl, 0.05% Tween-20, pH 7.5) overnight at 4 (cid:1)C. The
rejection in Nicotiana (Lind et al., 1996; Hancock et al.,
membranes were then incubated with the primary monoclonal
2005). Most glycoproteins have been shown to play roles antibodies(Mb)JIM11,JIM12,JIM19,andJIM20(1:50)respectively
RolesinextensinsincotyledonandshootapicalmeristeminNicotiana 4047
for 2 h at room temperature, and washed with TBST for 3–10 min. conjugate(Sigma)dilutedat1/100with100mMPBSfor1hatRT
The JIM11 and JIM20 antibodies recognize specific arabinosylation inthedarkorat4(cid:1)Covernight.Thesampleswererinsedthreetimes
ofHRGPswhereasJIM12mayrecognizeaproteinepitopeoranon- with 100 mM PBS before microscopic examination (Zhao et al.,
terminal oligosaccharide structure (Smallwood et al., 1994; a kind 2004). Control samples were incubated in 100 mM PBS instead of
giftfromDrJPKnox,CentreforPlantSciences,UniversityofLeeds, theprimaryantibody.Greenimmunofluorescenceofthesampleswas
UK).Theywereincubatedwithalkaline-phosphatase-conjugatedgoat visualizedunderaninvertedmicroscope(OlympusIMT-2).
anti-ratantibody(1:500;Sino-AmericanBiotechnologyCo.)for1h, For subcellular immunogold localization, the late globular stage
washed with TBST three times, and then stained with nitro blue embryos at 7 DAP were fixed in 2% glutaraldehyde, 4% para-
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Sino-American formaldehyde, and 100 mM phosphate buffer (PBS pH 7.2), for
BiotechnologyCo.).Thepre-stainedproteinmolecularweightmarker 2.5 h, then washed in 100 mM PBS, pH 7.2, three times, each for
mixes(Pierce) weremyosin(223 kDa),phosphorylaseB(110kDa), 30min,anddehydratedinaseriesofethanolgradientat4(cid:1)C.The
bovine serum albumin (81.6 kDa), ovalbumin (46.8 kDa), carbonic samples were infiltrated with Lowicryl K4M via three intermediate
anhydrase (31.8 kDa), trypsin inhibitor (24.8 kDa), and lysozyme steps at 2:1, 1:1, and 1:2 mixture of ethanol:Lowicryl K4M (12 h
(16.5kDa). each time). Finally, the mixture was replaced by pure Lowicryl
K4M, kept for 12 h at –20 (cid:1)C, and then changed once with fresh D
Isolationofzygotesandproembryos Lowicryl K4M and kept for 1 d at –20 (cid:1)C. The samples were ow
transferred to capsules with fresh Lowicryl K4M, and cured under n
Tobacco ovules were dissected from ovaries at 3, 5, 6, 7, 8, and 9 lo
two 15 W ultraviolet lamps (360 nm) for at least 24 h at –20 (cid:1)C, a
DAP, placed into an enzyme solution containing 8–13% mannitol, d
p3olmyvMinly2p-y[Nrr-omliodropnheoKlin3o0](PeVthPanKe3su0l)p,h1o%niccelalucliodse(MR-E1S0)a,nd30m.8M% awnedrethceuntcuosnitninguaedScourrvinalglfMorT2-6d00a0tRuTlt.raUmltircartohtionmseecatinodnsc(o6l0lecntmed) ed fro
onto Formvar-coated nickel grids. The grids were floated on PBST m
movaucleersozwyemreewR-a1s0h,edpH3–54.7t,imanedsiwncituhbaatesdolfuotrio3n0cmoinntaaitni2n8g(cid:1)8C–.1T3h%e buffer (60 mM PBS, 0.1% Tween-20, 0.02% NaN3, pH 7.2) http
containing 0.2 M glycine and 1% BSA to block non-specific s
3mamnnMitopl,ol3yvminMylpy2r-r[oNli-dmoonrephKo3li0no(]PVetPhanKe3s0u)l,phaonndicgeanctildy (gMroEuSn)d, binding. The grids were floated in a drop of the primary antibody ://ac
JIM20 for 3 h at 37 (cid:1)C. They were rinsed with PBST three times, a
with a small glass pestle. They were then treated in a second d
and then floated on 1:100 of goat anti-rat IgG antibody conjugated e
enzyme solution supplemented with 8–13% mannitol, 0.25% m
cellulose R-10, and 0.2% macerozyme R-10, pH 5.7, for 10–15 PtoB1S0Tntmhregeotlidmpeasrtaicnldesin(Sdiigsmtilale)dfowra1tehr tahtr3ee7t(cid:1)iCm.eAs,fttherewgraisdhsinwgeirne ic.o
moviunl.eAsftaenrdtheismtbhreyozygsaoctsesuasnidngprmoeimcrbornyeoesdlwesereunisdoelrataend firnovmerttehde air-dried and stained with saturated uranyl acetate for 30 min. The up.c
microscope(OLYMPUSCK30)(QinandZhao,2006). controlsectionsweretreatedsimilarlyexceptthatprimaryantibody om
was substituted with PBS. The samples were examined and /jx
Immunolocalizationtechniques phFotoorgrhaipsthoeldoguincadlersaecHtioTn6s5,0thtreansshmoiostsioapneexleesctroofnsmeeidcrlionsgcsopwe.hich b/artic
For immunoenzyme localization, the isolated ovules were embed- germinated from the cultured ovules were fixed in 4% para- le
ded using a Technovit 7100 Embedding kit (Heraeaus Kulzer, formaldehyde and 100 mM phosphate buffer, pH 7.2, and -ab
Wehrheim, Germany), described by Leroux et al. (2007). Five embeddedinEpon812resin.1lmsemi-sectionswereslicedunder stra
millimetre thick transverse sections were cut with a microtome an ultramicrotome (Sorvall MT-6000), stained with toluidine blue, c
(Sorvall MT-6000 ultramicrotome), and dried on object glasses. examined under an inverted microscope (Olympus IMT-2), and t/5
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Immunoenzyme detection of extensins in the sections used the photographedwithaCoolSNAPCCD(Photometric,USA). /1
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SABC (streptavidin and biotinylated horseradish peroxidase com- /4
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plex) method. The experiments were performed as described by Ovuleculture 45
Yuan et al. (2008) with some modification. The sections were /5
Ovulesattheearlyglobularembryostage(5DAP)weresterilizedin 5
incubated in 3% H O (15 min at room temperature, RT) to block 2
2 2 70% ethanol for 0.5 min and then in 2% NaClO for 4 min. After 0
the endogenous peroxidase activity. After three 5 min washes with 0
rinsing three or four times with sterile water, the ovules were 7
distilled water, the sections were incubated in 10 mM PBS buffer b
cultured in MS medium (pH 5.8) supplemented with 6% sucrose, y
containing 5% BSA (20 min at RT) to block non-specific binding. g
0.25% phytagel, and different concentrations of 3,4-DHP (50, 100 u
Then the sections were incubated with 1:5 dilutions (10 mM PBS, e
and200lM)at25(cid:1)Cinthedark.After10dofculture,someovules s
t1h%reeBtSimAe,spwHit7h.2P)BoSf,eaxntdeninsicnubaantteidbowdyithatbi4oti(cid:1)nC-laobveelrlendighgto,atrinansetid- wotehreersfixweedretotrdaentsefcetrrtehdeetombfrreysohdheavlef-lostpremnegnthtinMsiSdemthedeiuomvulseusp,palned- t on 1
rat IgG antibody for 20 min at 37 (cid:1)C. After that, the sections were 2
mentedwith3%sucroseand0.25%phytagel,pH5.8andculturedat A
rinsed three times with PBS and allowed to react with the SABC p
reagent for 20 min at 37 (cid:1)C. After an extensive washing in PBS 2cu5lt(cid:1)uCredinothveuldesarwkeurnetitlramnastpulraintyte.dThtoessoeieldliinngpsotosriigninoartdeedrftroomtrathcke ril 2
s(tuwpipclee)m, ethnetedsewctiitohns0.w02e%re (svta/vin)edTwweietnh 2th0e(AfoEuCr tikmitesa)t aRnTd.PTBhSe theircontinuedgrowth.Allexperimentswererepeatedatleastthree 019
times,andthestandarddeviationwascalculated.
control sections were treated similarly except that the primary
antibody was substituted with PBS/BSA solution. Sections were
then washed in distilled water and immediately examined under Embryoculture
amicroscope(OlympusIX-70). Ovaries at the late globular embryo stage (7 DAP) were sterilized
For fluorescence inmunolocalization, the isolated embryos were and the embryos were isolated as described above. The isolated
fixedin4%paraformaldehydein50mMPIPESbuffer,pH6.7,2mM embryos were first washed with 8% mannitol and then twice
MgSO , 2 mM EGTA, 8–13% mannitol (PIPES buffer) for 5 h at with KM8p medium (Kao and Michayluk, 1975), and cultivated
4
RT.ThesampleswererinsedthreetimeswiththePIPESbuffer,once in a millicell with a semi-permeable membrane (MILLICELL-CM
with100mMPBS,pH7.4,andthenincubatedintheprimarymono- 3.0 lm PICMO 1250, diameter 12 mm) placed in a 35 mm Petri
antibody JIM20 diluted 1/5 with 100 mM PBS for 3 h at room dish with 1.5 ml KM8p medium (pH 5.8) containing 100–120
temperature.Thesampleswererinsedthreetimeswith100mMPBS, ovules (7.5 DAP) as feeder cells at 25 (cid:1)C in the dark. In order to
and then incubated with the secondary antibody, anti-rat-IgG-FITC affordenoughnutrition,15gl(cid:1)1glucose,50gl(cid:1)1sucrose,15gl(cid:1)1
4048 Zhangetal.
mannitol, 1 mg l(cid:1)1 2, 4-D, 0.5 mg l(cid:1)1 6-BA, 250 mg l(cid:1)1 pH 6.5 for 30 min, rinsed gently and soaked in water for 15 min
lactalbuminhydrolystate,13kindsofaminoacids,and10%coconut twiceinordertoremoveunboundanilineblue.Finally,thesamples
were added to the KM8p medium to form a rich organic chemical were transferred into Hover’s solution (200 g chloral hydrate, 20 g
environment. In order to reduce the biosynthesis of extensins, 2, 5, glycerol, and 50 ml water) until they became transparent. The
10, 50, and 100 lM 3,4-DHP (Sigma) were added to the medium, samples were observed and photographed under an Olympus SZX
respectively.After10dofculture,theembryoswereobservedunder 12stereomicroscopewithaRSphotometricCCD.
an inverted microscope (Olympus IMT-2) and photographed with
aCoolSNAPCCD(Photometric,USA).Thedifferentiatedembryos
were then transferred to fresh half-strength MS medium supple-
mented with 30 g l(cid:1)1 sucrose and 2.5 g l(cid:1)1 phytagel, pH 5.8 and Results
developed into seedlings. The experiments were repeated at least
threetimes,andthestandarddeviationwascalculated. Expression of extensins during ovule development
Western blots were used to detect the expression of
Hydroxyprolineassay extensins in the mixed tobacco ovules at different de-
D
The total Hyp content (lg mg(cid:1)1 dry weight tissue) in the cultured velopmental stages using monoclonal antibodies (Mb) o
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tobacco ovules which were treated with or without 100 lM 3,4- JIM11, JIM12, JIM19, and JIM20 (Fig. 1A). The results n
DHP, as mentioned in the ovule culture, was measured using the showed that the JIM11-labelled extensins were mainly load
method first described by Kivirikko and Liesmaa (1959) and then e
found above 223 kDa, while JIM20 appeared to recognize d
manoddtihfieedstbayndFarryd(d1e9v8i8at)i.oTnhweaesxpgeivriemn.entswererepeatedthreetimes, more abundant and broader extensins from 60 kDa and from
mainly concentrated above 223 kDa. However, the JIM12 h
Cotyledontransparencytechnique and JIM19-recognized extensins were hardly detectable in ttps
the ovules (Fig. 1A). ://a
To investigate the cotyledon vascular bundles, a technique accord- c
a
ing to Bougourd et al. (2000) was used with minor modifications. The expression of JIM20-labelled extensins during d
e
The cotyledons were dehydrated through an ethanol series (15, 50, ovule development were examined further (Fig. 1B). The m
70, 95, and two 100% ethanol steps) for 15 min at each step. The results showed that both of the level and the size of ic.o
fsoarmaptlelseawster3ehle.fTthineyfrwesehre10th0e%n erethhayndoraltaetd4in(cid:1)CanovetehrannigohltsoerrieastRfoTr JIM20-labelled extensins changed dramatically during up.c
o
15mineachstep,andwashedwithdistilledwaterfor15mintwice. ovule development. Extensins ranging from 60 kDa to m
The cotyledons were stained in 0.5% aniline blue and 0.2 M PBS, over 160 kDa were abundant in the unfertilized ovules /jxb
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Fig.1. WesternblotanalysisofextensinsrecognizedbyMbJIM11,JIM12,JIM19,andJIM20andthelocalizationofextensinsintheovuleswithan
egg cell and zygote. (A) Labelling of JIM11, JIM12, JIM19, and JIM20 extensin binding epitopes in the tobacco mixed ovules at 1–9 DAP. (B)
Labelling of JIM20 extensin binding epitopes in the ovules at 1–9 DAP. Molecular mass (kDa) was indicated on the left. (C) A strong extensin
binding signal was detected in the integument and nucellar tissue of ovules with an egg cell. The egg cell and its integument are indicated by an
arrowandanarrowhead,respectively.(D)ExtensinslabelledwithJIM20werefoundintheintegumentandnucellartissueofovuleswithazygote.
Thezygoteanditsintegumentwereindicatedbyanarrowandanarrowhead,respectively.(E)AcontrolwithouttreatmentwithJIM20showedno
signalintheovulewithaneggcell.Bar¼50lm.
RolesinextensinsincotyledonandshootapicalmeristeminNicotiana 4049
(1 DAP) and the fertilized ovules (3 DAP). The level of cell of the embryo, there was almost no extensin
detected extensins was low in ovules with early (5 DAP) immunofluorescence (data not shown), but the integument
and middle (6 DAP) globular embryos, but gradually and nucellus tissue of ovules with a zygote showed
increased in ovules with late globular embryos (7 DAP), a strong signal (Fig. 1D). A faint signal was detected in
heart embryos (8 DAP), and torpedo-shaped embryos the two-celled embryos at the bottom of the based cell
(9 DAP), and the molecular weight was found to be from (Fig. 2A). And the signal gradually became stronger and
140 kDa to over 160 kDa. more evenly distributed in the early globular embryos
The JIM20-labelled extensins in ovules with an egg cell (Fig.2B).Inthelateglobularembryos,theextensinsignal
(Fig. 1C) and zygote (Fig. 1D) were examined by an was concentrated mainly in the top of the embryo proper
immunoenzyme technique. A strong extensin signal was (EP) and in the suspensor (Fig. 2C). When the embryos
detected in the integument and nucellus tissue of ovules began to differentiate, the signal was stronger in the
with an egg cell (Fig. 1C) and zygote (Fig. 1D). The suspensorofearlyandlateheartembryos(Fig.2D,E)and
control samples without the reaction with the JIM20 the shoot apical meristem of the torpedo embryos (Fig. D
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antibody showed no signal (Fig. 1D). 2F). In the control, no signal was detected in the embryos n
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not labelled with JIM20 (see Supplementary Fig. 1E at a
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Immunolocalization ofextensins in different JXB online). ed
developmentalstages of embryos To detect the subcellular localization of extensins, fro
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The expression of extensin epitopes was detected by immunogold labelling and TEM techniques were used. h
JIM20 during tobacco embryo development. In the first Numerous gold particles labelled with JIM20 were ttp
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Fig. 2. Immunofluorescence localization of extensins in tobacco embryos. The extensins in the embryos at different development stages were
immunostained with JIM20 monoclonal antibody. (A–F) Fluorescent images and (a–f) the corresponding bright-field micrographs. The embryo
properandsuspensorwereindicatedrespectivelybyanarrowandanarrowheadin(d).(A)Afaintsignalwasdetectedinthetwo-celledembryosat
thebottomofthebasedcell.(B)Anearlyglobularembryowithuniformfluorescencelabelling.(C)Alateglobularembryoshowingfluorescencelabelling
intheapexofembryoproper(EP)andsuspensorcells.(D)Thefluorescencelabellingisstronginthesuspensorcells,butdisappearsintheapexofembryo
properin an early heart-shaped embryo. (E) In a late heart-shaped embryo, thefluorescence is strong,especially in the shortened suspensor. (F) In the
torpedo-shapedembryo,thefluorescenceismainlypresentintheshootapicalmeristem.Bar¼20lm.
4050 Zhangetal.
detected at the top of the EP in the late globular embryos cultured ovules increased when the 3,4-DHP concentra-
(Fig. 3A), especially in the protruding cell walls of the EP tionwas100–200lM,butthesemainlyresultedfromthe
(Fig. 3B). In the suspensor of late globular embryos (Fig. augmentation of abnormal embryo differentiation, nor-
3A), gold particles were also found to be distributed mal embryo differentiation showed no visible contribu-
mainly in the cytoplasm (Fig. 3C). The control sections tion to the total differentiation frequencies. However, the
without JIM20 showed no gold particles (Fig. 3D). These 50 lM 3,4-DHP concentration showed a remarkably
results were consistent with those in the immunofluores- inhibitory effect on the normal embryo differentiation
cence experiments. and resulted in the decrease of the total differentiation
frequency, which was compared with the untreated
Effects of 3,4-DHP on the embryo development in control. In general, the normal differentiated embryos
ovule culture had two symmetrical cotyledon primordia (Fig. 4A), but
To investigate the role of extensins in embryogenesis, the abnormal differentiated embryos had asymmetry
ovules were chosen with early globular embryos at 5 (Fig. 4B), supernumerary (Fig. 4C) and single cotyledon D
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DAP and cultured in MS medium supplemented with primordia (Fig. 4D). These results indicate that extensins n
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different concentrations of 3,4-DHP. After 10 d of are important for embryo differentiation, and when their a
d
culture, the embryos in the ovules developed into three concentrations are lower than the natural level, the ed
types: undifferentiated, normal differentiated, and abnor- abnormal differentiated embryos increased. fro
m
mal differentiated. In 3,4-DHP treated ovules, the When the cultured ovules were transferred into half- h
frequencies of abnormal differentiated embryos obvi- strength MS medium and continuously cultured until ttp
s
ously increased (Table 1) and appeared in a concentra- germination, the fate of embryos was observed longitudi- ://a
c
tion-dependent manner (from 0 to 100 lM). But when nally. The resultshowed that the frequenciesof germinated a
d
the concentration was increased to 200 lM, there was no seedlings with normal cotyledons decreased compared em
change in the frequency of abnormal differentiated with the control, but the frequencies of seedlings with ic.o
u
embryos compared with the treatment with 100 lM. The abnormal cotyledons increased when the ovules were p
.c
total differentiation frequencies of embryos in the treated with 3,4-DHP (50, 100, and 200 lM; Table 2). o
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Fig.3. ImmunogoldlocalizationofextensinepitopesrecognizedbyJIM20inthelateglobularembryosat7DAPoftobacco.(A)Ultrathinsectionof
alateglobularembryo.(B)Amagnifiedimagesoftheareaindicatedbysquare1in(A).Numerousgoldparticlesappearedintheprotrudingcellwall
atthetopoftheembryoproper.(C)Amagnifiedimageoftheareaindicatedbysquare2in(A).Thegoldparticleswereseeninthecytoplasm.(D)A
control image showed no gold particles. (A) Bar¼10 lm; (B–D) bar¼0.2 lm; C, cytoplasm; CW, cell wall; EP, embryo proper; S, suspensor; V,
vacuole.
RolesinextensinsincotyledonandshootapicalmeristeminNicotiana 4051
Table1. Effectsof3,4-DHPontobaccoembryodevelopmentincultureof5DAPovules
Treatmentof No.ofisolated Frequencyof Frequencyofdifferentiatedembryos(%)a
3,4-DHP(lM) embryos undifferentiatedembryos(%)
Total Normal Abnormal
0 284 56.4364.54 43.5664.54 41.3865.76 2.1961.55
50 338 68.02611.96 31.98611.96** 19.4068.23** 12.5866.06**
100 292 33.0368.01** 66.9768.01** 39.8362.21 27.1468.46**
200 179 33.1466.04** 66.8666.40** 40.7664.65 26.161.57**
aThe results are shown by a value with a standard deviation (SD).** P<0.01 indicates a significant difference compared with the zero-DHP
control.
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Fig.4. Effectsof3,4-DHPonthedevelopmentofembryosin5DAPovulesafter10dofinvitrocultureandthephenotypesofgerminatedseedlings /5
in tobacco. (A, E) Embryos isolated from ovules and seedlings cultured on MS medium without 3,4-DHP. (B–D, F–I, K) Embryos isolated from 52
ovulesandseedlingsculturedonMSmediumwith3,4-DHP.(A)Anormaltorpedo-shapedembryowithtwosymmetricalcotyledonprimordia.(B) 00
Anabnormal torpedo-shaped embryowithtwoasymmetric cotyledonprimordia. (C)An abnormalembryo withthreecotyledonprimordia. (D)An 7 b
abnormal embryo with a single cotyledon primordium. (E) A normal germinating seedling with two symmetrical cotyledons. (F) An abnormal y
g
seedlingwithtwoasymmetriccotyledons.(G)Anabnormalseedlingwiththreecotyledons.(H)Anabnormalseedlingwithasinglecotyledon.(I)An u
e
abnormalseedlingwithacup-shapedcotyledon.(J)Acontrolseedlingcamefromtheovulesculturedwithout3,4-DHP(arrow)andtwocup-shaped s
seedlings from ovules cultured with 3,4-DHP (arrowheads) after being transplanted into soil for 20 d. The control seedling grew, while the cup- t o
n
shapedseedlingsstoppedgrowingandgraduallydied.(K)Amagnifiedimageofacup-shapedseedlingin(J).(A–D)Bar¼20lm;(E–I)bar¼2mm. 1
2
A
p
However, 3,4-DHP had no obvious effects on the total After the seedlings were transplanted into soil for 20 d, ril 20
1
frequencies of seedling germination (Table 2). In the the control seedlings developed into normal plantlets (Fig. 9
control, most seedlings had two normal symmetric 4J, indicated by an arrow), flowered, and set seeds. The
cotyledons (Fig. 4E), but in the tests, the germinated seedlings with cotyledon defects in Fig. 4F, G, and H
seedlings displayed various cotyledon defects, including could continuously develop into plantlets which had no
two asymmetric (Fig. 4F), supernumerary (Fig. 4G), obvious difference with the control plantlets (data not
single (Fig. 4H), and cup-shaped (Fig. 4I) cotyledons. shown). However, seedlings with cup-shaped cotyledons
These results indicated that adding 3,4-DHP to the culture (Fig. 4I) stopped growing and finally died (Fig. 4J, K,
medium probably inhibits the biosynthesis of extensins, indicated by arrowheads). Therefore, it is speculated that
thereby caused the defects in cotyledons. This suggests the seedlings with cup-shaped cotyledons which did not
that extensins may play a role in the cotyledon differen- grow were the result of a defect in the shoot apical
tiation of embryos. meristem.
4052 Zhangetal.
Effects of 3,4-DHP on the developmentof isolated the control. The Hyp in cultured ovules treated with or
embryos inculture without 3,4-DHP was 1.5 lg mg(cid:1)1 dry weights and
Inordertoinvestigatefurthertheeffectof3,4-DHPonthe 0.731 lg mg(cid:1)1 dry weights, respectively (Fig. 5A). The
differentiation of embryos, late globular embryos at 7 distribution of extensins in embryos isolated from
DAP were chosen and cultured in KM8p medium with cultured ovules was also detected by immunolocalization
differentconcentrationsof3,4-DHP. After10dof culture, usingtheJIM20antibody.Theresultshowedthatthesignal
the embryos also developed into three types: undifferenti- in the heart-embryos (Fig. 5B) cultured without 3,4-DHP
ated, normal differentiated, and abnormal differentiated was similar to the embryos in vivo (Fig. 2E), whereas only
(Table 3). In 3,4-DHP-treated embryos, the frequencies of a faint signal was detected in the embryos cultured with
the abnormal differentiated embryos significantly in- 3,4-DHP(seeSupplementary Fig. 1C, D atJXB online).
creased compared with the untreated control, and the
configurationoftheseedlingswithdefectivecotyledonsin
Effectsof 3,4-DHP on the activity ofshootapical D
the embryo culture was similar to that of the seedlings in o
meristem and the formation of the cotyledon w
the ovule culture (data not shown). The 5–100 lM 3,4- n
vasculature lo
DHP concentrations could improve the total differentiation a
d
The characteristics of shoot apical meristems (SAM, e
frequencies of embryos compared with the treated control, d
but these increases mainly come from the augmentation of indicated by arrows in Fig. 6) in various types of fro
m
germinated seedlings after treatment with 100 lM
abnormal embryo differentiation, the normal embryo h
differentiation had a weak contribution to the increase. 3,4-DHP were investigated using the semi-section tech- ttps
However,the2lM3,4-DHPshowedthereverseeffectand nique. The position and morphology of the SAM in ://a
inhibited total embryo differentiation, specifically normal the supernumerary cotyledons (Fig. 6B) was similar to cad
the control, which was located at the base of two e
embryo differentiation. These results further indicated that m
separated cotyledons (Fig. 6A). The SAM in the cup- ic
extensins are important for embryo differentiation, which .o
shaped (Fig. 6C), single (Fig. 6D), and asymmetrical u
agrees with the resultsofovule culture. p
cotyledon (Fig. 6E) plantlets had some dissimilarity .co
m
compared with the control. The position of the SAM /jx
Hydroxyproline level in thecultured ovules in the single (Fig. 6D) and asymmetrical (Fig. 6E) b/a
TotalHyp levelinthe culturedovules treatedwith100 lM cotyledon plantlets was inclined towards the abnormal rtic
le
3,4-DHP was significantly reduced as compared with cotyledon. And the SAM in the cup-shaped cotyledon -a
b
s
tra
Table2. Effectsof3,4-DHPonthegerminationofculturedovulesintobacco ct/5
9
DHP(lM) Frequenciesofgermination(%) /1
4
/4
Total Normal Abnormal Typeofabnormalcotyledon 04
5
/5
SC AC SupC CC 5
2
0
0
0 46.3669.06 45.6769.13 0.6960.43 0 0.3560.47 0.3460.38 0 7
b
50 49.5367.39 38.4663.64* 11.0760.56** 0.9261.44** 4.6261.42 4.9260.92 0.6160.37** y
100 40.4962.71 31.2563.49** 9.2462.84** 3.2660.31** 2.4561.59 1.3660.35 2.1760.37** gu
200 44.3165.53 37.5066.01** 6.8160.79** 3.4161.09** 2.2760.29 0.2860.30 0.8560.80** es
t o
n
Theresultswerelistedbyavaluewithastandarddeviation(SD).*0.01<P<0.05indicatesdistinctdifference;**P<0.01indicatessignificantdistinct 1
difference compared with the zero-DHP treated control. SC, Single cotyledon; AC, Asymmetry cotyledon; SupC, Supernumerary cotyledon; 2 A
CC,Cup-shapecotyledon.Thenumberofovulesculturedisabout280-380. p
ril 2
0
1
9
Table3. Effectsof3,4-DHPontobaccoembryodevelopmentinculture
Treatmentof No.ofisolated Frequencyof Frequencyofdifferentiatedembryos(%)a
3,4-DHP(lM) embryos undifferentiatedembryos(%)
Total Normal Abnormal
0 217 82.4960.48 18.4560.48 17.0561.40 0.4661.00
2 181 90.4860.72 9.5260.72** 4.9761.25** 3.8761.45**
5 185 76.7663.80 23.2463.80 19.9263.39 3.7861.02**
10 211 76.3067.02 23.767.02 19.4368.16 4.2763.33**
50 168 78.5767.79 21.4367.79 17.2665.20 4.1762.61**
100 219 77.1665.01 22.8465.01 18.7364.08 4.1161.00**
aTheresultsareshownbyavaluewithastandarddeviation(SD).**P<0.01indicatesasignificantdifferencecomparedwithzero-DHPcontrol.
RolesinextensinsincotyledonandshootapicalmeristeminNicotiana 4053
(Fig. 7A). In the DHP-treated seedlings, a single-fused
cotyledon contained two main veins (Fig. 7B), which
indicates that it comes from the incomplete fusion of two
cotyledons. The venation pattern of another single
cotyledon type, in which only one cotyledon primordium
could develop into a cotyledon, was similar to the
untreated control (Fig. 7C). In seedlings with two
asymmetrical cotyledons, the larger cotyledon had three
main veins (indicated by three arrowheads), whereas the
smaller one had a similar vasculature to the normal
cotyledons (Fig. 7D). The cup-shaped cotyledon was
completely fused and its vascular tissue was made up of
more than two main veins and a large number of small D
o
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branched veins (Fig. 7E). The venation of the supernu- n
lo
merary cotyledon (Fig. 7F) was similar to the normal a
d
cotyledons (Fig. 7A). ed
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Discussion ttp
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://a
Establishment ofan effectivesystem to study the ca
d
function ofextensins by using 3,4-DHP e
m
Hydroxyproline-rich glycoproteins (HRGPs), of which ic.o
u
extensins are a strongly basic protein presented in the p
.c
plant cell wall, are characterized by a high level of the o
m
comparatively rare amino acid hydroxyproline (Hyp) /jx
b
(Chen and Varner, 1985). Because of the high abundance /a
in cell walls and the extremely high level of Hyp, the use rtic
le
of specific inhibitors of prolyl hydroxylase which hydrox- -a
b
ylated ProintoHyp couldprovide a valuableapproachfor stra
studying the function of extensins among all types of c
t/5
HRGPs. Unlike most other proline analogues, the potent 9
/1
inhibitor 3,4-DHP functions at micromole concentrations 4
Fig. 5. Level of detection of extensins in the ovules treated with or /4
without 3,4-DHP by hydroxyproline assays and the localization of to inactivate prolyl hydroxylase in vivo rapidly and 04
5
extensinsinembryosisolatedfromthetreatedovulesdetectedbyJIM20 irreversibly (Cooper and Vamer, 1983). Studies with an /5
antibody. (A) Comparison of total Hyp content in the cultured ovules Escherichia coli proline auxotroph demonstrated that 52
withorwithout100lM3,4-DHPtreatment.Measurementsweretaken 00
in triplicate and the standard deviation (SD) was shown. **: P<0.01 3,4-DHP was the only Pro analogue tested that could 7 b
indicated a significant difference compared with zero-DHP control. replace proline in the synthesis of enzymically active y
Fluorescent images of immunolocalization were shown in (B) and the acid phosphatase (Morris and Schlesinger, 1972). For gu
corresponding bright-field micrograph in (b). (B) A heart-shaped es
embryo isolated from the ovules treated without 3,4-DHP showed 3,4-DHP offers many advantages over other inhibitors, t o
strongfluorescenceinthesuspensor.Bar¼50lm. so it becomes a useful tool for studying the physiological n 1
2
and developmental functions of extensins among all A
p
seedlings was encircled by one conjoint cotyledon t3y,4p-eDsHofPHsRigGniPfisc.aInntloyurretdeustc,etdhethtereatotmtaelnhtywdirtohxy1p0r0ollinMe ril 20
1
(Fig. 6C), leading to a functional defect and no growth level in the cultured ovules about 51.3% (Fig. 5A). 9
(Fig. 4J, K, indicated by arrowheads). However, the A similar result was observed in Allium cepa L. roots
shape of the SAM in the single (Fig. 6D), asymmetrical that were treated with 3,4-DL-dehydroproline (DP),
(Fig. 6E), and the cup-shaped (Fig. 6C) cotyledon a specific inhibitor of peptidyl-prolyl hydroxylase (Tullio
plantlets was flatter than that of the untreated control et al., 1999). These results indicate that the use of 3,4-
which looked like a cone (Fig. 4J, K, indicated by DHP or 3,4-DL-DP can offer good methods to study the
arrowheads), so it is presumed that the activity of the functions of hydroxyproline-rich glycoproteins in spe-
SAM was dependent on its position. cific tissues and development stages. So, using 3,4-DHP
The transparency technique showed that the normal in culture tests primarily to inhibit the synthesis of
structure of the cotyledon venation in control seedlings extensins is an effective way to study their functions in
was made up of one main vein and some branched veins embryo development.
4054 Zhangetal.
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Fig.6. Effectsof3,4-DHPonthedevelopmentofthetobaccoshootapicalmeristem(SAM,indicatedbyblackarrows).(A)TheSAMfromanormal rticle
seedlingculturedinMSmediumwithout3,4-DHPasacontrol.(B–E)TheSAMfromabnormalseedlingsculturedinMSmediumwith100lM3,4- -a
DHP (A). The SAM of a control seedling located at the base of two separated cotyledons. (B) The SAM of an abnormal seedling with three bs
cotyledonswas similar to thecontrol. Oneofthethree cotyledonscould notbe shown inthe picture. (C)TheSAM of acup-shaped seedlingwas tra
e(inncdiirccaleteddbbyyaacbolnacjokinatrrcoowtyhleeaddo)n,.w(hDil)eTthheeoStAheMroinneadseignegnleercaotetdyle(idnodnicpatleadntbleyt.aOwnhlyiteonaerrocwothyeleaddo).n(Epr)imThoerdSiuAmMdoefvaelsoepeeddliningtwheithnotwrmoaalscyomtymleedtroinc ct/59
cotyledons,thesmalleronedevelopedmoreslowlyandstayedatanincompletelydevelopedstate(indicatedbyawhitearrowhead).C,cotyledon;P, /1
4
leafprimordium.Bar¼50lm. /4
0
4
5
/5
Roles of extensinsin embryo development removal of cell wall proteins increased extensibility in 52
0
Embryogenesis is a critical stage of sporophytes in the Avena sativa coleoptiles (Ruesink, 1969). In Allium cepa, 07
plant life cycle, in which many genes and proteins play the treatment of roots with a peptidylprolyl hydroxylase by
important roles. In our study, the dramatic change of inhibitor caused a reduction in the level of hydroxypro- gu
e
extensins during tobacco embryo development was line-rich glycoproteins that led to the early elongation of st o
detected by Western blot and immunolocalization, and the developing root cells (Tullio et al., 1999). Conversely, n 1
the results indicated that extensins may play roles in increasing the level of extensins resulted in obviously 2 A
eemxtbenrysoingse,ntehseis.inIhniboitrodreroftoprionlvyelsthigyadtreoxtyhleasfeu,n3c,t4io-DnsHoPf, s2h0o0r6te).rWinflhoyredsoceesncreedsuincinAgratbhiedolepvseisl(oRfocbeelrltswaanlldpSrhoitresiants, pril 20
1
was applied to the in vitro culture of ovules and embryos. promote cell growth? According to the previous studies, 9
Theresultsshowedthatadditionof100–200lM3,4-DHP the cell wall comprised three interpenetrating networks:
to the ovule culture increased the total embryo differenti- cellulose, pectins, and extensins and our results suggested
ation frequencies, and the enhancement mainly reflected that the changes to the extensins network may cause the
the increase of the abnormal differentiation frequencies. A cell wall to loosen and promote cell growth. Similarly, the
similar result was also observed in embryo culture when decrease of the cellulose density stimulated pollen tube
the 3,4-DHP concentration was 5–100 lM. As 3,4-DHP growthinToreniafournieriandcausedthemtobeslender
inhibited the biosynthesis of Hyp and decreased the level and straighter (Wu et al., 2008).
of extensins in our tests, the in vitro culture results Somewhat surprisingly in our work the frequency of
suggested that a low level of extensins could promote cell total differentiated embryos decreased when treated with
growth, which was in agreement with the result that low concentration of 3,4-DHP (50 lM in ovule culture