Plant Cell Physiol. 38(9): 1087-1090 (1997)
J S P P © 1997
Short Communication
Zinc Deficiency Affects the Levels of Endogenous Gibberellins in Zea mays
L.
Hitoshi Sekimoto ', Mitsuo Hoshi', Takahito Nomura' and Takao Yokota 2
1
2
Department of Plant Science, Faculty of Agriculture, Utsunomiya University, Utsunomiya, 321 Japan
Department of Biosciences, Teikyo University, Utsunomiya, 320 Japan
Zinc deficiency in Zea mays L. markedly reduced the
level of GA,, but not GA M , suggesting blockage of 3^-hydroxylation. The level of IAA was also decreased although
not as markedly. Castasterone was affected less than IAA
by zinc deficiency.
Key words: Brassinosteroids — Gibberellins — 3/?-Hydroxylation — IAA — Zea mays L. — Zinc deficiency.
Zinc (Zn) deficiency causes growth retardation, such
as the reduction of internode elongation or leaf area expansion. This is similar to the dwarfisra caused by the biosynthetic inhibition of gibberellins (GAs). Thus Zn is
speculated to have important roles in biosynthesis and metabolism of GAs. In most studies, the role of Zn in plant
growth has been discussed in relation to IAA. Skoog (1940)
and Tsui (1948) noted that the level of IAA was lowered in
Zn-deficient plants, but the mechanism of this regulation is
not clear (Takaki and Kushizaki 1970, Salami and Kenefick
1970).
Recently Cakmak et al. (1989) detected low levels of
IAA in Zn-deficient plants of Phaseous vulgaris L. by immunoassay and an increase in tryptophan content as well as
other amino acids in the leaves. This might indicate a role
for Zn in the biosynthesis of IAA from tryptophan, however, since the increase in amino acids induced by Zn
deficiency is not specific to tryptophan, the lower IAA level
is not always caused by the impairment of the biosynthesis
of IAA from tryptophan. They speculated the lower IAA
level is more likely the result of enhanced oxidative degradation of IAA, based on the decrease in superoxide dismutase, a Zn-enzyme, and catalase activities in Zn-deficient
plants (Cakmak and Marschner 1988).
Suge et al. (1986) noted a significantly low level of gibberellin in Zn-deficient plants of Zea mays L. by bioassay
of the total biological activity. The comparative effects of
Zn deficiency on the levels of GAs and IAA as well as relevant quantitative data have not been reported. We quantified both GAs and IAA by GC-MS using internal standards. Brassinosteroids (BRs), which have recently been
found to be important in plant growth, were also exam-
ined.
Zea mays L. (Pioneer Hybrid 3358) was grown in vermiculite in a greenhouse. Seedlings were transferred to
plastic vats with a 70-liter nutrient solution composed
of 3.3 mM KNO3, 2.5 mM Ca(NO3)2.4H2O, 1.0 mM
NH 4 H 2 PO 4 , 2.0 mM MgSO4.7H2O, 100//M Fe-EDTA, 9
fiM MnCl2-4H2O, 45fiM H3BO3, 0.3//M CuSO4-5H2O
and 0.5 /xM Na2Mo4>2H2O in deionized water! ZnSO47H2O as Zn was supplied at sufficient (0.77 fiM), critical
(0.12//M) and deficient (OpM) levels. The nutrient solutions were adjusted to pH 5.8 by 1.0 M HC1 or NaOH and
replaced every two days. Then, after transplanting, shoots
which had not been seriously damaged by Zn deficiency,
were removed (Table 1). Plant culture for analysis of GAs,
IAA and BRs in Zn-deficient Zea mays L. was repeated
three times. The shoots were soaked in methanol (MeOH)
immediately after weighing.
The tissue was homogenized and extracted three times
with excess MeOH. To the extract were added labeled internal standards, [2H5]GA, (50 ng), fHJGAa, (200 ng), ["CJIAA (100 ng), [2H6]brassinolide (1 /jg) and [2H6]castasterone (1 fig). The extract was concentrated in vacuo and
fractionated by the usual method (Endo et al. 1989) to give
an acidic ethyl acetate (AE) and neutral ethyl acetate (NE)
fraction. The AE fraction was pre-purified with a polyvinylpyrrolidone column, a Sep-Pak (ODS) cartridge and a
Sepralyte (diethylaminopropyl) column as reported by
Nakayama et al. (1989). A fraction containing GAs and
IAA was dissolved in 100 fA of 50% aqueous MeOH, subjected to ODS-HPLC (Senshu-Pak ODS 4253D; 250x10
mm i.d.), and eluted at a flow rate of 3 ml min" 1 with 30%
aqueous MeOH containing 0.1% acetic acid (0-2 min) and
then a linear gradient of 30-100% MeOH (2-30 min). The
retention time was 13-15 min for GA,, 15-17 min for IAA
and 20-23 min for GAa,.
The NE fraction was purified in a column of silica gel.
Elution was carried out step wise with chloroform (CHC13),
and CHC13 containing 1, 2.5, 5, 7, 10, 20 and 100% MeOH
(90 ml each). To monitor the biological activity of BRs,
sample aliquots were assayed by the rice lamina inclination
test (Arima et al. 1984). Eluates obtained with 5 to 1% of
MeOH in CHC13 were combined, dissolved in MeOHCHCI3 (4 : 1) and loaded onto a column of Sephadex LH-
1087
Endogenous gibberellins in Zn-deficient Zea mays L.
1088
Table 1 Data on Zea mays shoots analyzed for endogenous GAs, IAA and Brs at Zn-deficient (0//M), Zn-critical (0.12
and Zn-sufficient (0.77 uM) levels
Zn supply
0/M)
No. of plants
Shoot height
(cm)
Total weight
(g)
Averaged weight
per segment (g)
Exp. 1
0
0.12
0.77
117
94
100
23.4±2.0
24.7 ±3.0
27.2±2.9
140
150
170
1.2±0.2
1.6±0.4
1.7±0.5
Exp. 2
0
0.12
0.77
106.
88
76
27.6±2.3
35.4±5.1
35.5±3.6
255
300
260
2.4±0.5
3.4±0.8
3.4±0.6
Exp. 3
0
0.77
98
87
19.7±0.8
21.7±1.0
118
122
1.2±0.1
1.4±0.1
12, 13 and 14-d old seedlings were cultured for 7 d (Exp. 1), 11 d(Exp. 2) and 8 d (Exp. 3), respectively. Data are means±SE of 20 plants
randomly selected.
20 (bed vol., 500 ml) using MeOH-CHCl 3 (4:1) as a
mobile phase. Ten ml fractions were collected. Fractions
No. 35-39 were combined and subjected to ODS-HPLC
(Senshu-Pak ODS 3251D; 250x8 mm i.d.) and eluted at
a flow rate of 2.5mlmin~ 1 as follows: 0-20 min, 45%
aqueous acetonitrile; 20-40 min, with a linear gradient of
45-100% acetonitrile; 40-45 min, 100% acetonitrile. The
retention time was 12-15 min for brassinolide and 20-24
min for castasterone.
The fractions thus obtained were analyzed by GC-SIM
after methylation and trimethylsilylation for GAs and IAA
or after methaneboronation for BRs. GC-SIM was conducted using a JEOL JMS AX 505 instrument equipped
with DB-1 for GAs and IAA and DB-5 for BRs (J&W Scientific; 0.25 mm x 15 m; 0.25 mm film thickness). For GA
samples, the injection temperature was 220°C. The column
temperature was held at 130°C for the first 2 min, and then
was programmed to increase at 32°C min" 1 to 220°C with
a 4 min isothermal hold at 220°C, and subsequently at 8°C
min" 1 to 270°C with a 10 min isothermal hold at the end of
the program. For IAA samples, the injection temp, was
200°C. The column temp, was held at 80°C for 1.5 min,
and increased to 130°C at 32°C min" 1 , followed by a further increase to 220°C at 8°C min" 1 . The analytical conditions for BR samples were as described by Yokota et al.
(1994).
The following ions were monitored to identify compounds: GA./fHsJGA,, m/z 511/506 and 496/491; GA*/
[ 2 HJGA M , m/z 420/418 and 405/403; IAA/^QJIAA,
m/z 267/261 and 208/202; brassinolide/pHdbrassinolide,
m/z 534/528 and 161/155; castasterone/[2H6]castasterone,
m/z 518/512 and 161/155. The concentrations of GA,,
GA20, IAA, brassinolide and castasterone in the samples
were calculated from the ratios of peak areas at m/z 506/
511, 434/437, 418/420, 202/208, 155/161 and 512/518, respectively.
To confirm the recovery of plant growth from Zn
deficiency by GAs, GA3 (20 and 40 fig liter"1) was added to
the Zn-deficient nutrient solution in 4-liter pots. At 13 days
after addition of GA3 (short-term group), the growth of
Zn-deficient plants appeared the same as that of Zn-critical
plants (Table 2). The plant height was more
Table 2 Effect of GA3 on the shoot growth of Zn-deficient Zea mays
Zn supply
0/M)
GA3 supply
Og liter l )
0 .
0
0
0.12
0.77
0.77
0
20
40
0
0
40
Plant height (cm)
After 13 days
After 20 days
28.7 d"
37.5 be
37.3 be
36.5 c
40.4 b
46.8 a
(100)'*
031)
(130)
(127)
(141)
(163)
32.5 d (100)
32.0 d (98)
41.0 c (126)
40.6 c (125)
52.9 b (163)
60.3 a (186)
Shoot dry weight (mg plant " )
After 20 days
After 13 days
431 b
512 a
501 a
514 a
585 a
579 a
(100)
(119)
(116)
(119)
(136)
(134)
641 be
442 c
741 b
930 ab
1,091 a
1,069 a
(100)
(69)
(116)
(145)
(170)
(167)
Eleven-d-old seedlings were cultured for 13 and 20 d with or without GA3. Data are presented as means of five plants.
" Within colums means followed by the same letter are not significantly different at the S% level by Duncan's multiple range test.
* Numbers in parentheses show % of the average of Zn-deficient plants (Zn 0/iM, GA3 0^g liter"1).
Endogenous gibberellins in Zn-deficient Zea mays L.
1089
Table 3 Endogenous levels of GAs, IAA and Brs in shoots of Zea mays at Zn-deficient (0 fiM), Zn-critical (0.12 pM) and
Zn-sufficient (0.77 ^M) levels
Exp. 1
Exp. 2
Exp. 3
Zn supply
GA,
GA•20
0
0.12
0.77
0
0.12
0.77
0
0.77
0.35 (26)
0.70 (52)
1.34 (100)
0.16 (16)
0.84 (83)
1.01 (100)
0.08 (38)
0.21 (100)
3.66 (98)
3.72 (100)
3.72 (100)
IAA
4.44(151)
3.68 (125)
2.94 (100)
0.82 (85)
0.96 (100)
Data are expressed as MS (kg fr wt) ' and numbers in parentheses show
" Not detectable.
* Not analyzed.
affected by Zn deficiency and GA3 than the shoot dry
weight. The recovery of plant height in Zn-deficient plants
at 20 days after addition of GA3 (long-term group) was less
than that of plants at 13 days. The shoot dry weight of
plants showed little effect of GA3 treatment at 20 days. The
fact that GA3 enhanced plant growth under Zn-deficient
conditions in the short-term group implies that the growth
retardation caused by Zn deficiency is partly attributable to
the reduction of the levels of endogenous GAs. Since Zn
is involved in the regulation of gene expression of zinc finger protein (Coleman 1992), RNA polymerase (Prask and
Plocke 1971), ribosomes (Kitagishi et al. 1987, Obata and
Umebayashi 1988) and RNAase (Johnson and Simons
1979, Sharma et al. 1981), the growth retardation which
was not recovered by GA3 under the long-term Zn-deficient
condition may depend on the impairment of protein synthesis more than that of GAs biosynthesis.
GA|, GA20, IAA, and castasterone were identified and
the quantitative data are shown in Table 3. Brassinolide
was not present at detectable levels. In Zn deficient plants,
the levels of phytohormones except for GA20 were clearly
lower than those in Zn-sufficient plants. The reduction in
GA, level with Zn deficiency was more marked than for
IAA. The level of GA, was 2.6 to 6.3-fold lower in Zndeficient plants than in Zn-sufficient plants. The level of
castasterone was affected less than that of IAA by zinc
deficiency.
Shoot growth retardation under Zn deficient conditions accompanied by lower levels of IAA have been found
in several plants (Skoog 1940, Tsui 1948, Garg et al. 1986,
Cakmak et al. 1989). Since Zn affects not only the endogenous levels of phytohormones but also metabolic activities
related to shoot growth, shoot growth retardation in Zndeficient plants may not attribute to the reduction of phytohormones alone. However, these findings indicate that
72.48 (72)
82.10 (82)
100.18 (100)
n.a.*
n.a.
n.a.
64.36 (83)
77.23 (100)
Brassinolide
n.d.°
n.d.
n.d.
n.a.
n.a.
n.a.
n.a.
n.a.
Castasterone
1.64 (81)
1.76 (87)
2.02 (100)
n.a.
n.a.
n.a.
n.a.
n.a.
of the levels of Zn-sufficient (0.77 jiM) plants.
symptoms of Zn deficiency such as growth retardation are
at least partially attributable to reduction of GA,.
In contrast, the levels of GA20, a precursor of GA,,
were changed less than those of GA, in Zn-deficient plants.
This suggested that Zn affects the conversion of GA20 to
GA,, 3/?-hydroxylation, in Zea mays L. However, since gibberellin 3y?-hydroxylase requires iron as a cofactor not Zn
(Kwak et al. 1988), Zn may be required for the transcription and/or translation steps of gibberellin 3/?-hydroxylase
synthesis.
We wish to thank Dr. Yamaguchi, The University of Tokyo,
for providing deuterated GAs and 13C-labeled IAA, and Dr.
Takatsuto, Joetsu University of Education, for providing deuterated BRs.
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(Received February 24, 1997; Accepted June 24, 1997)