生長素、細(xì)胞分裂素對黑暗和ABA誘導(dǎo)蠶豆氣孔關(guān)閉的抑制效應(yīng)

1、 ACTA AGRONOMICA SINICA 2008, 34(6): 10341041ISSN 0496-3490; CODEN TSHPA9 /zwxb/ E-mail: xbzw DOI: 10.3724/SP.J.1006.2008.01034ABA * (, 710062) : (Vicia faba L.), (IAA)(NAA)2,4-(2,4-D)(ZT)(KT)6-(6-BA)(ABA), ABA, NO-2-4,4,5,5-1-3-(c-PTIO)(Hb)NO(NOS)NG-N-L-(L-NAME)(H2O2)(AsA)(C

2、AT)NADPH(DPI), ABA, NOH2O2NOH2O2, ABANOH2O2IAAZT, ABANOH2O2 : ; ; ; ; ; ; The Inhibitory Effects of Auxin and Cytokinin on Dark- and ABA-induced Stomatal Closure in Broad BeanZHANG Bei, SHE Xiao-Ping*, ZHANG Guang-Bin, MENG Zhao-Ni, and SONG Xi-Gui(College of Life Sciences, Shaanxi Normal University

3、, Xian 71006, Shaanxi, China)Abstract: In the present studies, the effects and mechanisms of natural and synthetic auxin IAA, NAA, 2,4-D, cytokinin ZT, KT, 6-BA on dark- and ABA-induced stomatal closure were investigated by means of stomatal bioassay and using laser-scanning con-focal microscopy. Is

4、olated epidermal strips of Vicia faba were incubated with IAA (10 mol L1), NAA (10 mol L1), 2,4-D (10 mol L1), ZT (0.1 mol L1), KT (0.2 mol L1), 6-BA (0.2 mol L1), NO scavenger c-PTIO (200 mol L1), Hb (100 mol L1), NOS inhibitor L-NAME (25 mol L1), H2O2 scavenger AsA (100 mol L1), CAT (100 U mL1), i

5、nhibitor of H2O2-generating enzyme NADPH oxidase DPI (10 mol L1) for 3 h, in darkness or in light in the presence of ABA (1 mol L1), respectively. The results showed that auxin, cytokinin, as well as c-PTIO, Hb, L-NAME, AsA, CAT, and DPI, reversed dark- and ABA-induced stomatal closure significantly

6、. Epidermal strips treated with auxin and cytokinin were loaded with NO-fluorescent dye DAF-2DA or H2O2-fluorescent dye H2DCF-DA. The results indicated that darkness and ABA could induce an intense DAF-2DA or H2DCF-DA fluorescence in guard cells. However, dark- and ABA-induced DAF-2DA and H2DCF-DA f

7、luorescence were largely prevented by auxin and cytokinin tested. Similarly, the treatments of c-PTIO, Hb, L-NAME and AsA, CAT, DPI also substantially suppressed dark- and ABA-induced DAF-2DA and H2DCF-DA fluorescence, respectively. These results provide the evidence that auxin and cytokinin tested

8、lessen assuredly NO and H2O2 levels induced by dark and ABA in guard cells. Consider-ing synthetic auxin, cytokinin NAA, 2,4-D, KT, 6-BA and natural IAA, ZT were used in the present work and IAA, ZT are repre-sentative of endogenous auxin and cytokinin respectively, the effects of auxin, cytokinin t

9、ested on dark- and ABA-induced stomatal closure and NO, H2O2 level can be attributed to an universal effect of auxin or cytokinin.Keywords: Auxin; Cytokinin; Nitric oxide; Hydrogen peroxide; Dark; ABA; Stomatal closure-: (2003C101, 2005C112): (1983), , , E-mail: zaoanbella* (Corresponding author): T

10、el: E-mail: ShexiaopingReceived(): 2007-09-20; Accepted( : 2007-12-16.6 : ABA 1035, (ABA)1-220803-4, , H+-ATP5, (IAA)(NAA)pHK+6-89, H+-cAMP10-11Irving12, (KT)IAApHABA, ABAABA, ABACa2+5,13(NO)(H2O2), ABANOH2O2, 14-17KT6-(6-BA)NAAIAANOH2O2, NOH2O2, NOH2O218-19, NOH2O2(1)ABANOH2O2(2)NOH2O

11、2, , 18-19IAA, /, /NOH2O2, , , (ZT)IAANO(NOS)NONADPHH2O2ABANOH2O2 1 1.1 (Vicia faba L. 10, )24 h, 253 d, , 25300 mol m2 s1/14 h/10 h80%1, 4 1.2 NO-4,5-(DAF-2DA)(Hb)NG-N-L-(L-NAME)-2-4,4,5,5-1-3-(c-PTIO)(CAT)(DPI)2-(N-)(MES)Sigma, H2O2-2,7-(H2DCF-DA)Biotium, (DMSO)Amresco 1.3 4,1.0 cm×0.5 cm12ME

12、S/KCl(10 mmol L1 MES/KOH, 50 mmol L1 KCl, 100 mol L1 CaCl2, pH 6.15), 253 h, McAinsh205, 6, 3, 1.4 NOH2O2NOH2O2 MES/KCl(10 mmol L1 MES/KOH, 50 mmol L1 KCl, 100 mol L1 CaCl2, pH 6.15)25(300 mol m2 s1) 3 h, 10 mol L1 DAF-2DA50 mol L1 H2DCF-DATris/KCl(10 mmol L1 Tris, 50 mmol L1 KCl, pH 7.2),1036 1 Tab

13、le 1 Treatments for the effects of phytohormones on dark-induced stomatal closure Treatment Light Dark IAA NAA 2,4-D Light intensity or reagents concentration300 mol m2 s1 0 mol m2 s1 10 mol L1 10 mol L1 10 mol L1 Treatment ZT KT 6-BA c-PTIO HbReagents concentration0.1 mol L1 0.2 mol L1 0.2 mol L1 2

14、00 mol L1 100 mol L1 Treatment L-NAME AsA CAT DPI34Reagents concentration25 mol L1 100 mol L1 100 U L1 10 mol L1LightDarkMES/KCl, Light and Dark means that the strips are incubated in MES/KCl buffer without any reagent in light or in darkness, all the other treat-ments are in darkness.2 ABATable 2 T

15、reatments for the effects of phytohormones on ABA-induced stomatal closure TreatmentReagents concentration(mol L1) TreatmentReagents concentration(mol L1) TreatmentReagents concentration(mol L1)Control 0 LABA+KT 1 +0.2 AsA 1 +0.2 NAA 10 CAT 100 ABA+ c-PTIO 1 +200 2,4-D 10 DPI 10 ABA+ Hb 1 +100ABA 1A

16、BA+ L-NAME1 +25 KT 0.2 ABA+IAA 1 +10 ABA+ AsA 1 +100 ABA+NAA 1 +10 ABA+CAT 1 +100 ABA+2,4-D 1 +10 ABA+DPI 1 +10 ABA+ZT (300 mol m2 s1)CATU mL1All the treatments are in the light (300 mol m2 s1). The concentration unit of CAT is U mL1.(25)60 min 10 min, Tris/KCl, (Leica, TCS SP2)Ex=488 nm, Em=505530

17、nm, Power=10%, Zoom=4, Frame= 512h512, Leica Image SoftwarePhotoshop software, 32 2.1 1, (P <0.05),NOH2O2c-PTIOHbL-NAMEAsACATDPI(P <0.05), NOH2O2, 161 Fig. 1 Effects of auxin and cytokinin on dark-induced stomatal closure6 : ABA 10371, c-PTIOHbL-NAMEAsACATDPI, (P<0.05), NOH2O2IAAZT, 2.2 ABA

18、2-CD, ABA(P <0.05), NOH2O2c-PTIOHbL-NAMEAsACATDPI(P>0.05), ABA(P<0.05), ABANOH2O22-AB, (P>0.05), NOH2O2c-PTIOHbL-NAMEAsACATDPIABA(P <0.05), NOH2O2ABA, ABAIAAZTABA2.3 ABANO2.3.1 NO I, NO(I-1, 2, 31), 1c-PTIOHbL-NAME, NO(I-211, 31), 1, IAAZTNONO2.3.2 ABANO I, ABANO(I-1, 12, 31), 2-Cc-PT

19、IOHbL-NAME, ABANO(I-1230,31), 2-AB, IAAZTABANO, ABANO2 ABA Fig. 2 The effects of auxin and cytokinin on ABA-induced stomatal closure A: IAANAA2,4-DABA; B: ZTKT6-BAABA; C: c-PTIOHbL-NAMEABA; D: AsACATDPIABA A+IA+NA+DA+ZA+KA+BA+PA+HA+LA+AsAA+CATA+DPIABAIAANAA2,4-DZTKT6-BAc-PTIOHbL-NAMEAsACATDPIMES/KCl

20、 A: effects of auxin IAA, NAA, 2, 4-D on ABA-induced stomatal closure; B: effects of cytokinin ZT, KT, 6-BA on ABA-induced stomatal closure; C: effects of c-PTIO, Hb, and L-NAME on ABA-induced stomatal closure; D: effects of AsA, CAT, and DPI on ABA-induced stomatal closure. A+I, A+N, A+D, A+Z, A+K,

21、 A+B, A+P, A+H, A+L, A+ AsA, A+ CAT, and A+DPI means epidermal strips were treated in MES/KCl buffer with IAA, NAA, 2,4-D, ZT, KT, 6-BA, c-PTIO, Hb, L-NAME, AsA, CAT, or DPI in the presence of ABA , respectively. 2.4 ABAH2O2 2.4.1 H2O2 II, H2O21038 34(II-1, 2, 31), 1AsACATDPI, H2O2(II-211, 31),1, IA

22、AZTH2O2H2O2 2,21, H+-ATP5,10-11, ABAH+-ATP22Irving12KTIAA, Blatt23ABAK+ 2.4.2 ABAH2O2 H+-ATPII, , ABAH2O2(II-1, 12, 31), 2-DAsACATDPI, ABAH2O2(II-1230, 31), 2-AB,IAAZTABAH2O2, ABAH2O23 ABA, Neill14Garcia-Mata15ABANO, Zhang17ABAH2O2, NOH2O2/16IAANAAKT6-BA,IAANAAKT6-BANOH2O218-19, ABA21, IAAZT, IAANAA

23、2,4-D, ABANOH2O2, , , ABA1,3-4, ABApH, ABANOH2O2NOH2O2ABA, ABANOH2O2, ABANOH2O2, ABANOH2O2, NOABAH2O224-25UV-BH2O2NO, NOH2O216,26, ABAUV-BNOH2O2, /, /K+Cl, /H+-ATPpH27, /ABA, ABA, , /, / 4 ABA, NOc-PTIOHb, NOSL-NAME, H2O2AsACAT, NADPHDPI, ABA6 : ABA 1039ABANOH2O2, NOc-PTIOHbNOSL-NAMEH2O2AsACATNADPHD

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40、 mol L1 IAA10 mol L1 NAA10 mol L1 2,4-D0.1 mol L1 ZT0.2 mol L1 KT0.2 mol L1 6-BA200 mol L1 c-PTIO100 mol L1 Hb25 mol L1 L-NAME3 h; 12301 mol L1 ABA10 mol L1 NAA10 mol L1 2,4-D0.1 mol L1 ZT0.2 mol L1 KT0.2 mol L1 6-BA200 mol L1 c-PTIO100 mol L1 Hb10 mol L1 IAA25 mol L1 L-NAME1 mol L1 ABA+10 mol L1 IA

41、A1 mol L1 ABA+10 mol L1 NAA1 mol L1 ABA+10 mol L1 2,4-D1 mol L1 ABA+0.11 mol L1 ABA+0.2 mol L1 KT1 mol L1 ABA+0.2 mol L1 6-BA1 mol L1 ABA+200 mol L1 c-PTIO1 mol L1 ABA+100 mol mol L1 ZT1 mol L1 ABA+25 mol L1 L-NAME3 h; 31130, eSE130 L1 Hb1303016 m, 130308 m, 130 Guard cells shown in image (1) and (2) were treated for 3 h in light and in darkness, respectively. Guard cells in image (3) were treated with10 mol L1 IAA, (4) 10 mol L1 NAA, (5) 10 mol L1 2,4-D, (6) 0.1