植物細胞壁合成與加厚調(diào)控課件

1、植物細胞壁合成與加厚調(diào)控Robert Hooke觀察到的植物細胞壁細胞壁是最早觀察到的生物微觀結(jié)構(gòu)Concrete Forest Natural Forest Common feature : Cell WallsAbundant Cell Wall Material 細胞壁的結(jié)構(gòu)和組成Hemicellulose(20%)Lignin (25%) Cellulose (50%)Extract(5%)植物細胞壁結(jié)構(gòu)和組成 Nature Materials (2003)Cellulose Hemicellulose Pectin圖12-7 植物細胞壁電鏡圖。A：水稻厚壁細胞電鏡圖。B：A圖方框區(qū)域

2、的放大。ML：中膠層；PCW：初生壁；SCW：次生壁。S1，S2，S3：次生細胞壁的三層。標尺，A：50m；B：500nm。圖片修改自Cell Wall GenomicsLittle Is Known For cell wall formation:About 15% of the plant genome, possibly 3000 5000 genes.Only a small part of them characterized細胞壁纖維素的合成Cellulose Fiber StructurePear, et al. 1996, PNAS, 93: 12637-12642From m

3、icrobial cellulose to plant cellulose synthase gene (CelA -CesA)Acetobacter xylinum is Natures most prolific cellulose-producing bacterium. Arioli, T. et al. 1998. Science 279:717-720 Identification of CesA genes in primary wall formationTaylor, et al. (1999) Plant Cell 11:769-779 Identification of

4、CesA genes in secondary wall formationCesA gene superfamily in Plants: Cellulose synthase (CesA); Cellulose synthase-like (Csl) Proposed CesA gene structure Kimura, et al. 1999. Plant Cell 11: 2075-2086.Observation of Cellulose synthase complex -“rosette” complexes. Paredez, et al., (2006) Science 3

5、12, 1491“Rosette” complex moving on the track of microtubules 纖維素由纖維素合酶復(fù)合體（CSC）合成microfibrilCesA-(1,4)-Glucan chain“rosette” complexcomplex subunitIn Arabidopsis:3 CesAs in primary cell wall 3 CesAs in secondary cell wallNature Materials (2003)LeafShoot TipPhloem XylemPtiCesA1-BPtiCesA1-BPtiCesA6-DP

6、tiCesA4PtiCesA6-CPtiCesA6-APtiCesA7-APtiCesA8-APtiCesA6-BPtiCesA1-APtiCesA3-APtiCesA3-CPtiCesA3-BPtiCesA6-FPtiCesA7-BPtiCesA8-BPtCslA1PtCslA2PtCslA3PtCslA5PtCslC1PtCslC2PtCslC3PtCslC4PtCslC5PtCslD1PtCslD2PtCslD4PtCslD6PtCslD8PtCslD9PtCslD10PtCslE1PtCslE2PtCslE3PtCslG4PtCslG50.0200.0400.0600.0800.010

7、00.01200.0楊樹中至少6個CesA基因參與次生壁纖維素合成 (x104 copies/g total RNA)In Populus 18 CesAs30 Csls identified In Arabidopsis 10 CesA31 Csls identified6 CesA genes expressed in secondary cell wall formationcomplex subunit?CesAs 如何形成CSC？Precipitating Antibodies纖維素合酶復(fù)合體分離Immunobloting Antibodiescomplex subunitAnti-

8、PtiCesA7-AAnti-PtiCesA1-AAnti-PtiCesA3-CAnti-PtiCesA8-BPre-immuneIgGCSC type CesA nameGene model Type IPtiCesA4eugene3.00002636PtiCesA7-AestExt_Genewise1_v1.C_LG_VI2188PtiCesA7-Bgw1.XVIII.3152.1PtiCesA8-Agw1.XI.3218.1PtiCesA8-Beugene3.00040363Type IIPtiCesA1-AestExt_fgenesh4_pm.C_LG_XVIII0125PtiCesA

9、1-Bfgenesh4_pg.C_LG_VI001789PtiCesA3-CestExt_fgenesh4_pg.C_LG_IX0979PtiCesA3-DestExt_Genewise1_v1.C_LG_I1792PtiCesA6-Efgenesh4_pm.C_LG_XIII000084PtiCesA6-Ffgenesh4_pg.C_scaffold_133000012CSCs 的蛋白組學(xué)鑒定實驗結(jié)果(b)(a)(c)(d)實驗結(jié)果Anti-PtiCesA7-AcaCesA7A in the secondary cell wall formation of differentiating x

10、ylem cells實驗結(jié)果Anti-PtiCesA1-AcaCesA-1A in both the secondary and primary wall formation of differentiating xylem CesA constituents of two type CSCsPtiCesA7-APtiCesA7-BPtiCesA8-BPtiCesA8-APtiCesA4PtiCesA3-CPtiCesA3-DPtiCesA1-APtiCesA6-EPtiCesA6-FType I complexType II complexRaven et al. 2005 微纖絲纖維素的結(jié)

11、構(gòu)可能與CSC調(diào)控相關(guān)Song, et al. New Phytologist，2010木質(zhì)素的合成How is lignin synthesized ?G “condensed lignin”difficult to break downS “relax lignin”, relatively easy to breakdownS/G, different in plants (G in softwood S/G hardwood) 43% efficiency improvement of lignin breakdown per S/G ratio unit increaseCH2OHO

12、HOCH3CH2OHOHH3COOCH3CH2OHOHp-coumarylalcoholConiferylalcoholSinapyl alcoholG ligninH ligninS ligninPhenylalanine5-Hydroxyferulate 11OHOOHOHOHOHOOCH3OHOHOH3COOCH3OHOHOOCH3OHOHOOHSinapate 124-Coumarate 1Caffeate 2Ferulate 10OSHOOCH3OHCoA5-HydroxyferuloylCoA 14OSOCH3OHCoAH3COSinapoylCoA 15OSOCH3OHCoAFe

13、ruloylCoA 4OSOHOHCoACaffeoylCoA 3OSOHCoA4-CoumaroylCoA 13HHOOCH3OHO5-Hydroxyconifer-aldehyde 7OHOCH3OHH3COSinap-aldehyde 8OHOCH3OHConifer-aldehyde 5OHOHOHCaffe-aldehyde 17OHOH4-Coumar-aldehyde 16OCH3OHOHH3COSinapylalcohol 9Syringyl monolignolOCH3OHOHConiferylalcohol 6Guaiacyl monolignolOCH3OHOHHO5-H

14、ydroxyconiferylalcohol 20OHOHOHCaffeylalcohol 19OHOH4-Coumarylalcohol 18Hydroxyphenyl monolignolOHOPALC4HCinnamateC3H? OMT F5H OMT 4CL 4 CL 4CL 4CL 4CL?C3H? CoAOMT F5H? CoAOMT CCR CCR? CCR CCR? CCR CAD CAD? CAD CAD? CAD ? ? ? ? ? ? ? ?Guaiacyl-syringyl lignin GSSSSSGGGSSGGGSSSSSSSPossible Monolignol

15、 Biosynthesis PathwaysLi et al., 1997, PNAS, 94:5493-5466; Li et al., 2006, Critical Review in Plant Science. 25:213-233. Li et al., 1999, PMB, 40:555-563; Hu et al.,1999, Nat. Biotechnol., 17: 808-812Li et al., 2000, JBC, 275: 6537-6545; Osakabe et al., 1999, PNAS, 96: 8955-8960; Li et al., 2005, P

16、C&P 46: 1234-1245 Li et al., 2001, the Plant Cell, 13:1567-1585OHOHOOCH3OHHOOCH3OHHOOCH3OHHOOCH3OHOHOHOOCH3OHHOOCH3OHHOOCH3OHHOOCH3OH5-Hydroxyferulate 11OHOH4-Coumar-aldehyde 16OHOOH4-Coumarate 1OHOOH4-Coumarate 1OHOOHOHCaffeate 2OHOOCH3OHFerulate 10OHOOCH3OHFerulate 10HOOCH3OHFerulate 10HOOCH3OHFer

17、ulate 10OHOH3COOCH3OHSinapate 12HOH3COOHSinapate 12OHOH3COOCH3OHSinapate 12HOH3COOHSinapate 12OSOHCoA4-CoumaroylCoA 13OSOHOHCoACaffeoylCoA 3OSOCH3OHCoAFeruloylCoA 4OSHOOCH3OHCoA5-HydroxyferuloylCoA 14OSOCH3OHCoAH3COSinapoylCoA 15OHOCH3OHH3COSinap-aldehyde 8OHOHOHCaffe-aldehyde 17OHOCH3OHConifer-alde

18、hyde 5HHOOCH3OHO5-Hydroxyconifer-aldehyde 7OCH3OHOHH3COSinapylalcohol 9S-MonolignolOHOH4-Coumarylalcohol 18OHOHOHCaffeylalcohol 19OCH3OHOHConiferylalcohol 6G-monolignolOCH3OHOHHO5-Hydroxyconiferylalcohol 20PhenylalanineOHOPALCinnamateC4HCald5HAldOMTCoAOMT4CLCCRCADSADHou and Li, 2011, J I P B Lu et a

19、l., 2010. GM Crops宋東亮等，2010，植物生理通訊沈君輝等，2010，植物生理通訊 宋東亮等，2008， 植物生理通訊Horvath et al., 2010, Forest Products Horvath et al., 2010, Wood ResearchHorvath et al., 2010, IAWA JSuzuki et al., 2010, J. Wood Sci Lu et al., 2008, J Exp BotNakatsubo et al., 2007, Cellulose Chem Technol Kasal et al., 2007, Holzf

20、orschungCell wall materialPretreatment hydrolysis fermentation Ethanol purification Lignin modification for biofuel production 020406080100120140Enzyme consumptionHydrolysisefficiencyConcentration of ethanolChange (%) ControlTransgenic-33%+21%+30%Hydroxyphenyl monolignolLignin Biosynthesis Pathways

21、in DicotsSyringyl monolignolGuaiacyl monolignol C3H? COMT F5H COMT CCR CAD CAD CAD? SADFeruloyl CoA Caffeoyl CoA 4-Coumaroyl CoA SOOCH3OHCoAOSOHOHCoAOSOHCoA5-Hydroxyconifer-aldehyde Sinap-aldehyde Conifer-aldehyde Caffe-aldehyde 4-Coumar-aldehyde OHOHHHOOCH3OHOOHOCH3OHH3COOHOCH3OHOHOHOHSinapylalcoho

22、l Coniferylalcohol 5-Hydroxyconiferylalcohol 4-Coumarylalcohol OCH3OHOH H3COOCH3OHOHOCH3OHOHHOOHOHC4H5-Hydroxyferulate Sinapate 4-Coumarate Caffeate Ferulate OHOHOOCH3OHO HO H3COOCH3OHOHOOCH3OHOHO O HOOHOHCinnamate NH2 O HOPhenylalaninePALHCT/C3HOMTOHOOH4CLCCRCCoAOMTF5HRice 4CLs form a different gro

23、up from those of dicotsPtr: Populus trichocarpa, At: Arabidopsis thaliana Os: Oryza Japonica, Pp: Physcomitrella patens (Moss)0123456783 Weeks5 Weeks10 WeeksRelative expressionRootStemLeafSheathSpike4CL500.511.522.53Relative expression3 Weeks5 Weeks10 Weeks4CL1RootStemLeafSheathSpike00.10.20.30.40.5

24、0.60.70.80.93 Weeks5 Weeks10 WeeksRelative expression4CL4RootStemLeafSheathSpike00.050.10.150.20.250.30.353 Weeks5 Weeks10 WeeksRelative expression4CL2RootStemLeafSheathSpike0204060801001203 Weeks5 Weeks10 WeeksRelative expressionRootStemLeafSheathSpike4CL3Expression of 4CLs in Various Tissues durin

25、g Development Catalytic Properties of the Os4CLs0200040006000CIp-CACAFASAKenz (M-1S-1)4CL502004006008001000Kenz (M-1S-1)CIp-CACAFASA4CL104000800012000Kenz (M-1S-1)CIp-CACAFASA4CL40400080001200016000CIp-CACAFASAKenz (M-1S-1)4CL2020000400006000080000100000CIp-CACAFASAKenz (M-1S-1)4CL3Morphological Cha

26、nge of 4CL Transgenic Rice (T3)Suppression of lignin biosynthesis in rice 4CL3AS transgenic plantsMonolignol (%)HGSWT1070204CL3AS87517Monolignol composition in rice Absorbance at 310nmWTTransgenicsp-CoumarateFerulate2x35S:Anti-4CL3 2x35S:4CL3oxFerulatep-Coumarate10 15 20 25 30/10 15 20 25 30 Phenoli

27、c Compounds in Rice Stem4CLHCT/C3H Syringyl monolignolGuaiacyl monolignolHydroxyphenyl monolignol COMT F5H COMT 4CL CCR CCR? CCR CCR? CCR CAD CAD? CAD CAD? SAD 5-Hydroxyferuloyl CoA SinapoylCoA FeruloylCoA CaffeoylCoA 4-CoumaroylCoA OSHOOCH3OHCoAOSOCH3OH CoA H3COSOOCH3OHCoAOSOHOHCoAOSOHCoA5-Hydroxyc

28、onifer-aldehyde Sinap-aldehyde Conifer-aldehyde Caffe-aldehyde 4-Coumar-aldehyde OHOHHHOOCH3OHOOHOCH3OHH3COOHOCH3OHOHOHOHSinapylalcohol Coniferylalcohol 5-Hydroxyconiferylalcohol Caffeylalcohol 4-Coumarylalcohol OCH3OHOH H3COOCH3OHOHOCH3OHOHHOOHOHOHOHOH C4H5-Hydroxyferulate Sinapate 4-Coumarate Caff

29、eate Ferulate OHOHOOCH3OHO HO H3COOCH3OHOHOOCH3OHOHO O HOOHOHOHOCinnamate NH2 O HOPhenylalaninePAL CCoAOMT CAld5H AldOMT? COMT F5H? COMTPossible 4CL-Mediated Pathways in RiceC3H?Gui et al，2011 Plant Physiology圖12-2 擬南芥花序軸莖橫切示不同類型細胞及其分布。不同細胞類型-形成不同結(jié)結(jié)構(gòu)和組成的細胞壁？不同結(jié)構(gòu)與組成的細胞壁-決定細胞的不同分化？Cell elongation and

30、wall thickening Cell elongationWall thickeningCoordination ?實驗結(jié)果Secondary growth - A system of wall thickening differentiationSong et al. 2010, New Phytol.Song et al. 2011, Plant Mol. Biol. Xu et al, 2013, BMC GenomicsPlasma membrane proteomics of the wall thickening cells Cell WallPM?TransporterRLK

31、CSC：10 CesAsPtrMAN6 localized in expanding vessels of xylemPtrMAN6 localized in plasma mebraneActivity assay of the recombinant PtrMAN6 & PtrMAN4 in E. coliActivity of recombinant PtrMAN6 in plantN-glycosylation required for PtrMAN6 activityCK Endo HN1 N2 N3 N4 N5Glutamine (Q)Asparagine (N)Multiple

32、sites of N-glycosylation on PtrMAN6GGM is a main substrate of PtrMAN6 Stable oligosaccharides generated from PtrMAN6 activity Dimerization of PtrMAN6 required for the activity Protein from E. coliProtein from plantCysteineAlanineDimerization of PtrMAN6 through S-S linkages 4 5 6PtrMAN6LeMAN4aPtrMAN6

33、LeMAN4aPtrMAN6LeMAN4aPtrMAN6LeMAN4aPtrMAN6LeMAN4aSecondary xylem differentiation is regulated by PtrMAN6PtMAN6 regulated both cell expansion and wall thickening Plants RhamnoseFucoseArabinoseXyloseMannoseGalactoseGlucoseWT6.590.131.660.05 13.350.93216.937.0117.450.8713.340.4337.471.04PtrMAN6ASLine55

34、.600.20*1.220.04*6.910.59*280.557.32*19.870.76*9.590.41*34.691.12PtrMAN6ASLine65.190.19*1.160.03*6.560.38*288.675.23*20.190.14*8.850.37*34.440.66*PtrMAN6OELine89.720.13*3.20.07*35.960.96*165.691.83*11.830.28*31.530.61*45.940.87*PtrMAN6OELine38.780.15*2.690.04*24.560.60*178.645.29*11.320.29*22.200.49*38.321.12AIR: Alcohol-insoluble residues; Value: mean (g mg-1 AIR) SE of 4 replicates. Single asterisk and double asterisks represent P 0.05 and P 0.01, respectively, in Students t test. Cell wall sugar composition regulated by PtrMA