The Inhibitory Effects of Auxin and Cytokinin on Dark- and ABA-induced Stomatal Closure in Broad Bean-文献传递-植物通论文库

摘要：以蚕豆(Vicia faba L.)为材料, 借助表皮条实验和激光扫描共聚焦显微镜技术研究合成和天然生长素、细胞分裂素吲哚乙酸(IAA)、萘乙酸(NAA)、2,4-二氯苯氧乙酸(2,4-D)、玉米素(ZT)、激动素(KT)和6-苄基腺嘌呤(6-BA)对黑暗和脱落酸(ABA)诱导气孔关闭的效应及其机制。结果表明, 黑暗和ABA均诱导气孔关闭, 与NO清除剂羧基-2-苯-4,4,5,5-四甲基咪唑-1-氧-3-氧化物(c-PTIO)、牛血红蛋白(Hb)和NO合酶(NOS)抑制剂N^G-N-L-精氨酸甲酯(L-NAME)以及过氧化氢(H₂O₂)清除剂抗坏血酸(AsA)、过氧化氢酶(CAT)和过氧化氢产生酶NADPH氧化酶抑制剂二苯基碘(DPI)一样, 供试合成和天然生长素、细胞分裂素均显著逆转黑暗和ABA诱导的气孔关闭, 暗示生长素、细胞分裂素可能通过降低NO和H₂O₂水平起作用。保卫细胞内源NO和H₂O₂检测结果显示, 合成和天然生长素、细胞分裂素确实均显著降低暗和ABA诱导的NO和H₂O₂水平。考虑到IAA、ZT分别为天然生长素、细胞分裂素的典型代表而且供试生长素、细胞分裂素均包括了合成和天然种类, 可以认为供试生长素、细胞分裂素逆转暗和ABA诱导的气孔关闭及降低NO、H₂O₂水平的作用可能是所有生长素类、细胞分裂素类植物生长物质均具备的效应。

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 confocal microscopy. Isolated epidermal strips of Vicia faba were incubated with IAA (10 mmol L^-1), NAA (10 mmol L^-1), 2,4-D (10 mmol L^-1), ZT (0.1 mmol L^-1), KT (0.2 mmol L^-1), 6-BA (0.2 mmol L^-1), NO scavenger c-PTIO (200 mmol L^-1), Hb (100 mmol L^-1), NOS inhibitor L-NAME (25 mmol L^-1), H₂O₂ scavenger AsA (100 mmol L^-1), CAT (100 U mL^-1), inhibitor of H₂O₂-generating enzyme NADPH oxidase DPI (10 mmol L^-1) for 3 h, in darkness or in light in the presence of ABA (1 mmol L^-1), 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. 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 fluorescence 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 lessen assuredly NO and H₂O₂levels induced by dark and ABA in guard cells. Considering synthetic auxin, cytokinin NAA, 2,4-D, KT, 6-BA and natural IAA, ZT were used in the present work and IAA, ZT are representative of endogenous auxin and cytokinin respectively, the effects of auxin, cytokinin tested on dark- and ABA-induced stomatal closure and NO, H₂O₂ level can be attributed to an universal effect of auxin or cytokinin.

全文： ACTA AGRONOMICA SINICA 2008, 34(6): 1034−1041 http://www.chinacrops.org/zwxb/
ISSN 0496-3490; CODEN TSHPA9 E-mail: xbzw@chinajournal.net.cn

:

(2003C101, 2005C112)
:

(1983–), , , !E-mail: zaoanbella@stu.snnu.edu.cn
*
#$%&(Corresponding author):

()!Tel: 029-85310265; E-mail: Shexiaoping@snnu.edu.cn
Received(*+,-): 2007-09-20; Accepted(./,-0: 2007-12-16.
DOI: 10.3724/SP.J.1006.2008.01034
ABA
*
( , 710062)
: (Vicia faba L.), !#$%&
()$*+,-(IAA)%.,-(NAA)%2,4-/012,-(2,4-D)%34$(ZT)%5$(KT) 6-6789:(6-BA)
;<=>?-(ABA)@ABCDEFGHIJKLMNO P, <= ABA Q@ABCDE, R NO STUV7
-2-1-4,4,5,5-WX7YZ-1-2-3-2[\(c-PTIO)%]^_`a(Hb) NO b(NOS)cLU NG-N-L-de-Xf
(L-NAME)Ig2[h(H2O2)STUij^-(AsA)%g2[hb(CAT)g2[hkb NADPH2[bcLU/
17l(DPI)mn, op !#$%&()$Qqrs<= ABA@AFBCDE, =t#$%&
()$uvwgxy NO H2O2z{|}~M& NO H2O2NOt, !#$%&
()$ Qqxy= ABA@AF NO H2O2z{M IAA%ZT( !#$%&()$F
op#$%&()$Q !, uop#$%&()$rs= ABA@A
FBCDEIxy NO%H2O2z{F}~uv#$%&()$\#\QFGHM
: #$; &()$; m2[; g2[h; =; >?-; BCDE
The Inhibitory Effects of Auxin and Cytokinin on Dark- and ABA-induced
Stomatal Closure in Broad Bean
ZHANG Bei, SHE Xiao-Ping*, ZHANG Guang-Bin, MENG Zhao-Ni, and SONG Xi-Gui
(College of Life Sciences, Shaanxi Normal University, Xi’an 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. Isolated epidermal strips of Vicia faba were incubated with IAA (10 μmol L−1), NAA (10 μmol L−1), 2,4-D (10
μmol L−1), ZT (0.1 μmol L−1), KT (0.2 μmol L−1), 6-BA (0.2 μmol L−1), NO scavenger c-PTIO (200 μmol L−1), Hb (100 μmol
L−1), NOS inhibitor L-NAME (25 μmol L−1), H2O2 scavenger AsA (100 μmol L−1), CAT (100 U mL−1), inhibitor of
H2O2-generating enzyme NADPH oxidase DPI (10 μmol L−1) for 3 h, in darkness or in light in the presence of ABA (1 μmol L−1),
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. 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 fluorescence
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 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 tested 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
6 : ABA 1035

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L−1 DAF-2DA 50 μmol L−1 H2DCF-DA Tris/KCl
cde(10 mmol L−1 Tris, 50 mmol L−1 KCl, pH 7.2),

1036 34

1
Table 1 Treatments for the effects of phytohormones on dark-induced stomatal closure

Treatment

Light intensity or
reagents concentration

Treatment

Reagents concentration

Treatment

Reagents concentration
Light 300 μmol m−2 s−1 ZT 0.1 μmol L−1 L-NAME 25 μmol L−1
Dark 0 μmol m−2 s−1 KT 0.2 μmol L−1 AsA 100 μmol L−1
IAA 10 μmol L−1 6-BA 0.2 μmol L−1 CAT 100 U L−1
NAA 10 μmol L−1 c-PTIO 200 μmol L−1 DPI 10 μmol L−1
2,4-D 10 μmol L−1 Hb 100 μmol L−1
Light Dark MES/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 ABA
Table 2 Treatments for the effects of phytohormones on ABA-induced stomatal closure

Treatment

Reagents concentration
(μmol L−1)

Treatment

Reagents concentration
(μmol L−1)

Treatment

Reagents concentration
(μmol L−1)
Control 0 L-NAME 25 ABA+KT 1 +0.2
IAA 10 AsA 100 ABA+6-BA 1 +0.2
NAA 10 CAT 100 ABA+ c-PTIO 1 +200
2,4-D 10 DPI 10 ABA+ Hb 1 +100
ZT 0.1 ABA 1 ABA+ L-NAME 1 +25
KT 0.2 ABA+IAA 1 +10 ABA+ AsA 1 +100
6-BA 0.2 ABA+NAA 1 +10 ABA+CAT 1 +100
c-PTIO 200 ABA+2,4-D 1 +10 ABA+DPI 1 +10
Hb 100 ABA+ZT 1 +0.1
(#(300 μmol m−2 s−1)$%&CAT)* U mL−1&
All the treatments are in the light (300 μmol m−2 s−1). The concentration unit of CAT is U mL−1.

(25) 60 min 10 min,
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gPhijklmno NOd H2O2pqrsT
UVW, tuvwxyz{|}[16])

1
Fig. 1 Effects of auxin and cytokinin on dark-induced stomatal closure
6 : ABA 1037

5 1~$g, u c-PTIO\Hb\L-NAME\AsACAT DPI |, `a\no8
$QefRSTUVW(P<0.05), g
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: IAA ZT, g\n
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2.2 ABA

5 2-C D , ABA $QRSTUVW(P
<0.05), NO\ H2O2 ]^_`abc_
c-PTIO\Hb\L-NAME d AsA\CAT\DPI
TU2u$Q(P>0.05), A$Qe
f ABATUVWRS(P<0.05), g ABAN
hijklmno NO\H2O2pqrsTUVW)
M5 2-A\BNO, \no8
TU2u$Q(P>0.05),
Au NO\H2O2]^_`abc_ c-PTIO\HbL-NAMEd AsA\CAT\DPI|$Qef ABA
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d ABA\no8C¤+7)
2.3 ABA
NO
2.3.1 NO M5
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2, 31), ¡u5 1 §¨|})u c-PTIO\Hb
L-NAME|, `a\no8
©ªARS NOpq(5¥ I-2~11, 31), ¡u
5 1 §¨|})«¢, IAA\ZT
RS NOpqYZg\no8h
i¬ NOpq,u EFTU)
2.3.2 ABA NO M
5¥ INO, ABA©ªjklmno NOpq(5¥
I-1, 12, 31), ¡u5 2-C §¨|})u c-PTIOHb L-NAME|, `a\no
8A ABA RS NO pq(5¥ I-12~30,
31), ¡u5 2-A\B §¨|})¡¢,
IAA\ZT ABARS NOpqYZg,
EFTU¦ ABA\no8
C¤+7ulmno NOpq@V)

2 ABA
Fig. 2 The effects of auxin and cytokinin on ABA-induced
stomatal closure
A: IAANAA2,4-D ABA ; B:
ZTKT6-BA ABA ; C: c-PTIO
HbL-NAME ABA ; D: AsACAT DPI
ABA
A+IA+NA+DA+ZA+KA+BA+PA+HA+LA+AsA
A+CAT A+DPI ABA IAANAA2,4-D
ZTKT6-BAc-PTIOHbL-NAMEAsACAT DPI MES/KCl
!#
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, 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 ABA
H2O2
2.4.1 H2O2 M
5¥ II NO, lmno H2O2 pqF¦k
1038 34

(5¥ II-1, 2, 31), ¡u5 1§¨|})u AsACAT DPI |, `a\no8
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})u AsA\CAT DPI |, `a
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\no8ef ABARSTUVWÂÃÄJ
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, ¡ÅÆ×Ø¼\no8ef
ABARSTUVWulmno NO\H2O2
pq@V, ~$g\no8YZ
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Ã, \no8rsTUç ABARS
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º[2,21])ìíz{î, TU\WuÇÙÚ
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òhióô H+-ATPïrsTUVW[22])Irving¸[12]
ÂÃ KT IAA rsTUçulmnooÛÖô
CVõ, Blatt¸[23]¼ª ABAhiRSoÛöôðô
¢÷ K+ hÃsrsTUVW)ìíz{TU
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q, ¡\ABA d±RSTUVWiâ¦
NO u H2O2V½Ê|ß4V )@Æ
æ$g, ABA RS NO » H2O2`a, NO
û¾S ABARS H2O2»[24-25])vw¯¼UV-BRSTUVW¦ H2O2rs NO», NO½R
S H2O2`a[16,26])Mìíz{NO, ABA u
\UV-BAhijk NO\H2O2pqrsTUVW,
± fS B)
/Ê¬TUK, å¶
·. /hi¬lmno`+7ø
\{Ö\K+ Cl–2sTU,
½@/¬ H+-ATPï\oÛ pHsøTU
ÂÃ[27], IÁûOÙÚ¾S/T
UÂÃ)ÅÆ¹º ABA ARSTUV
W\\no8ArsTUç,
grs ABAaRS\no
8», sTUW)M
úyÄ!#(+7lmnoÙÚ
pq, $/Ê%©ª¬lmnop
qÄ&¼, vwz{IJg:/hi
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4
ABA ARSTUVW , u NO ]^_
c-PTIO\Hb, NOS bc_ L-NAME, H2O2 ]^_
AsA\CAT, NADPHÔôïbc_ DPI|, `
a \no8A$Qef ABA
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6 : ABA !# 1039

ABAARSlmno NO\H2O2pqéê,
u NO]^_ c-PTIO\Hb NOSbc_ L-NAME
d H2O2]^_ AsA\CAT NADPHÔôïbc_
DPI|, `a \no8$Q
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1040 34

I ABA NO
Plate I The effects of auxin and cytokinin on dark- and ABA-induced NO level in guard cells
1~2 3 h; 3~11 10 μmol L−1 IAA10 μmol L−1 NAA10 μmol L−1 2,4-D0.1 μmol L−1 ZT0.2 μmol L−1
KT0.2 μmol L−1 6-BA200 μmol L−1 c-PTIO100 μmol L−1 Hb 25 μmol L−1 L-NAME 3 h; 12~30 1 μmol L−1 ABA
10 μmol L−1 IAA10 μmol L−1 NAA10 μmol L−1 2,4-D0.1 μmol L−1 ZT0.2 μmol L−1 KT0.2 μmol L−1 6-BA200 μmol L−1 c-PTIO100 μmol L−1 Hb
25 μmol L−1 L-NAME1 μmol L−1 ABA+10 μmol L−1 IAA1 μmol L−1 ABA+10 μmol L−1 NAA1 μmol L−1 ABA+10 μmol L−1 2,4-D1 μmol L−1 ABA+0.1
μmol L−1 ZT1 μmol L−1 ABA+0.2 μmol L−1 KT1 μmol L−1 ABA+0.2 μmol L−1 6-BA1 μmol L−1 ABA+200 μmol L−1 c-PTIO1 μmol L−1 ABA+100 μmol
L−1 Hb1 μmol L−1 ABA+25 μmol L−1 L-NAME 3 h; 311~30 , SE !1~30
#$ 1~30%&(!30)*$ 16 μm, +)*,- 1~30!30#)*$ 8 μm, +)*,- 1~30.#!
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 μmol L−1 IAA, (4)
10 μmol L−1 NAA, (5) 10 μmol L−1 2,4-D, (6) 0.1 μmol L−1 ZT, (7) 0.2 μmol L−1 KT, (8) 0.2 μmol L−1 6-BA, (9) 200 μmol L−1 c-PTIO, (10) 100 μmol L−1 Hb, (11)
25 μmol L−1 L-NAME respectively in the darkness for 3 h. Guard cells in image (12) were treated with 1 μmol L−1 ABA, (13) 10 μmol L−1 IAA, (14) 10 μmol L−1
NAA, (15) 10 μmol L−1 2,4-D, (16) 0.1 μmol L−1 ZT, (17) 0.2 μmol L−1 KT, (18) 0.2 μmol L−1 6-BA, (19) 200 μmol L−1 c-PTIO, (20) 100 μmol L−1 Hb, (21) 25
μmol L−1 L-NAME, (22) 1 μmol L−1 ABA+10 μmol L−1 IAA, (23) 1 μmol L−1 ABA+10 μmol L−1 NAA, (24) 1 μmol L−1 ABA+10 μmol L−1 2,4-D, (25) 1 μmol
L−1 ABA+0.1 μmol L−1 ZT, (26) 1 μmol L−1 ABA+0.2 μmol L−1 KT, (27) 1 μmol L−1 ABA+0.2 μmol L−1 6-BA, (28) 1 μmol L−1 ABA+200 μmol L−1 c-PTIO, (29)
1 μmol L−1 ABA+100 μmol L−1 Hb, (30) 1 μmol L−1 ABA+25 μmol L−1 L-NAME respectively in the light for 3 h. Fig.(31) shows the average fluorescent intensity
of guard cells in image (1–30) in three repeated experiments, data are means ± SE. The insets show the bright-field images corresponding to the fluorescence
image (1–30). Scale bar in image 30 represents 16 μm for image (1–30). The bar in inset of image (30) represents 8 μm for all insets.
6 : ABA !# 1041

II ABA H2O2
Plate II The effects of auxin and cytokinin on dark- and ABA-induced H2O2 level in guard cells
1~2 3 h; 3~11 10 μmol L−1 IAA10 μmol L−1 NAA10 μmol L−1 2,4-D0.1 μmol L−1 ZT0.2 μmol L−1
KT0.2 μmol L−1 6-BA100 μmol L−1 AsA100 U mL−1 CAT 10 μmol L−1 DPI 3 h; 12~30 1 μmol L−1 ABA10 μmol L−1
IAA10 μmol L−1 NAA10 μmol L−1 2,4-D0.1 μmol L−1 ZT0.2 μmol L-1 KT0.2 μmol L−1 6-BA100 μmol L−1 AsA100 U mL−1 CAT10 μmol L−1
DPI1 μmol L−1 ABA+10 μmol L−1 IAA1 μmol L−1 ABA+10 μmol L−1 NAA1 μmol L−1 ABA+10 μmol L−1 2,4-D1 μmol L−1 ABA+0.1 μmol L−1 ZT
1 μmol L−1 ABA+0.2 μmol L−1 KT1 μmol L−1 ABA+0.2 μmol L−1 6-BA1 μmol L−1 ABA+100 μmol L−1 AsA1 μmol L−1ABA+100 U mL−1 CAT1 μmol
L−1 ABA+10 μmol L−1 DPI 3 h311~30 , ±SE 1~30!# 1~30
$%&30()# 16 μm, *()+, 1~3030!()# 8 μm, *()+, 1~30-!
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 with 10 μmol L−1 IAA,
(4) 10 μmol L−1 NAA, (5) 10 μmol L−1 2,4-D, (6) 0.1 μmol L−1 ZT, (7) 0.2 μmol L−1 KT, (8) 0.2 μmol L−1 6-BA, (9) 100 μmol L−1 AsA, (10) 100 U mL−1 CAT,
(11) 10 μmol L−1 DPI respectively in the darkness for 3 h. Guard cells in image (12) were treated with 1 μmol L−1 ABA, (13) 10 μmol L−1 IAA, (14) 10 μmol
L−1 NAA, (15) 10 μmol L−1 2,4-D, (16) 0.1 μmol L−1 ZT, (17) 0.2 μmol L−1 KT, (18) 0.2 μmol L−1 6-BA, (19) 100 μmol L−1 AsA, (20) 100 U mL−1 CAT, (21)
200 μmol L−1 DPI, (22) 1 μmol L−1 ABA+10 μmol L−1 IAA, (23) 1 μmol L−1 ABA+10 μmol L−1 NAA, (24) 1 μmol L−1 ABA+10 μmol L−1 2,4-D, (25) 1 μmol
L−1 ABA+0.1 μmol L−1 ZT, (26) 1 μmol L−1 ABA+0.2 μmol L−1 KT, (27) 1 μmol L−1 ABA+0.2 μmol L−1 6-BA, (28) 1 μmol L−1 ABA +100 μmol L−1 AsA, (29)
1 μmol L−1 ABA+100 U mL−1 CAT, (30) 1 μmol L−1 ABA+ 10 μmol L−1 DPI respectively in the light for 3 h. Fig.(31) shows the average fluorescent intensity
of guard cells in image (1–30) in three repeated experiments, data are means ± SE. The insets show the bright-field images corresponding to the fluorescence
image (1–30). Scale bar in image (30) represents 16 μm for image (1–30). The bar in inset of image (30) represents 8 μm for all insets.

The Inhibitory Effects of Auxin and Cytokinin on Dark- and ABA-induced Stomatal Closure in Broad Bean

生长素、细胞分裂素对黑暗和ABA诱导蚕豆气孔关闭的抑制效应

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