CLONING AND CHARACTERIZATION OF DIAMINE
OXIDASE AND GLUTAMATE DECARBOXYLASE
GENES of MUSTARD (BRASSICA JUNCEA) AND THEIR
ROLES IN SHOOT MORPHOGENESIS IN VITRO









JIAO YUXIA





NATIONAL UNIVERSITY OF SINGAPORE
2004


CLONING AND CHARACTERIZATION OF DIAMINE
OXIDASE AND GLUTAMATE DECARBOXYLASE
GENES of MUSTARD (BRASSICA JUNCEA) AND THEIR
ROLES IN SHOOT MORPHOGENESIS IN VITRO


JIAO YUXIA (M.Eng. )

A THESIS SUBMITTED
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
DEPARTMENT OF BIOLOGICAL SCIENCES
NATIONAL UNIVERSITY OF SINGAPORE
2004


Acknowledgements



I would like to express my deepest gratitude to my supervisor, Associate Professor
Pua Eng Chong for his invaluable advice, guidance, inspiration and patience, help and
support over the past 4 years.
I would also like to extend my sincere thanks to all my friends in the laboratory,
Carol Han Ping, Francis Tan Chee Kuan, Serena Lim Tze Soo, Emily Tay Boon Hui, Gong
Haibiao, Hu wenwei, Cheng Wei, Mo Hua, Xu Yifeng, Yang Shuhua, Wang Yu, Teo Lai
Lai and Dr. Yu Hao, for their help and encouragement during my staying in Singapore.
Their presence has created an enjoyable and productive working environment and made
my staying in Singapore an unforgettable memory.
Finally, I would like to thank my family members. My parents and siblings have
been an everlasting source of power throughout my life. Without their support and
encouragement, I could not go so far in my education and academic career. At last but not

the least, I should owe my thanks to my husband Liu Feng, to whom this thesis is
dedicated. Besides love, care, encouragement and patience, as a fellow, he also gives me
professional support, help and inspiration.

i

Table of Contents


Page
Acknowledgements
i
Table of Contents
ii
List of Abbreviations
x
List of Figures
xiii
List of Tables
xvii
Summary
xviii
1 Introduction 1


2 Literature Review 4
2.1 Metabolism and regulation of PAs 5

2.1.1 PA biosynthesis 5


2.1.1.1 ODC 8

2.1.1.2 ADC 10

2.1.1.3 AIH and CPA 11

2.1.1.4 SAMDC 12

2.1.1.5 SPDS 14

2.1.1.6 SPMS 16

ii

2.1.2 PA catabolism 18

2.1.2.1 DAO 18

2.1.2.1.1 Molecular features and catalytic mechanism 19

2.1.2.1.2 Expression during plant growth and development 21

2.1.2.1.3 Expression in response to external stimuli 22

2.1.2.2 PAO 25

2.1.3 Modulation of PAs in transgenic plants 28
2.2
Roles of PAs in plant morphogenesis in vitro 34


2.2.1 Somatic embryogenesis 34

2.2.2 Shoot morphogenesis 35

2.2.3 Rhizogenesis 38
2.3
Effects of PAs on oxidative stress 39
2.4 GABA shunt pathway 41

2.4.1 GAD 43
2.4.1.1 Sequence characteristics of plant GADs 43
2.4.1.2 GAD expression 44
2.4.1.3 Regulation of plant GADs 46
2.4.2 GABA-T 48
2.4.3 SSADH 48
2.5 Proposed roles of GABA 50

iii
2.5.1 Stress response 50
2.5.2 Plant defense to herbivory 52
2.5.3 Plant growth and development 53
2.6 Interaction between PAs, ethylene and GABA 56

3 Materials and Methods 59
3.1 Plant materials 59
3.1.1 Mustard 59
3.1.2 Arabidopsis 59
3.2 Chemical treatments 60
3.3 Shoot regeneration from cultured explants 60
3.4 Gene cloning 62

3.4.1 Cloning of PCR-amplified products 62
3.4.2 Bacterial transfection 62
3.4 3 Plasmid DNA isolation 63
3.4.4 DNA sequencing and analysis 64
3.5 Probe labeling 65
3.5.1 DNA probes 65
3.5.2 RNA probes 66
3.6 Isolation of cDNA clones 66

iv
3.6.1 Library titering 66
3.6.2 Library screening 67
3.6.3 In vivo excision 68
3.7 Genomic DNA isolation and Southern analysis 68
3.8 RNA isolation and northern blot analysis 70
3.9 cDNA synthesis by reverse transcription 71
3.10 Quantitative reverse transcription PCR (RT-PCR) 71
3.11 Cloning of full-length cDNA by RACE 72
3.11.1 5’-RACE 72
3.11.2 3’-RACE 74
3.11.3 Generation of full-length cDNA sequences 74
3.12 Cloning of the BjDAO promoter 74
3.12.1 Construction of libraries 74
3.12.2 Promoter cloning by Genome Walking strategy 75
3.13 Construction of chimeric genes 76
3.13.1 DAO-GFP fusion protein 76
3.13.2 Sense and antisense DAO 78
3.13.3 Sense, antisense and dominant-negative GAD 78
3.13.4 Generation of BjDAO promoter::GUS fusions 81
3.14 Genetic transformation of plants 84


v
3.14.1 Mustard 84
3.14.2 Arabidopsis 85
3.15 Biochemical analysis 87
3.15.1 GFP detection with confocal microscopy 87
3.15.2 Histochemical assays for the GUS activity 87
3.15.3 GUS fluorometric assay 87
3.15.4 Ethylene measurement 88
3.15.5 Assays for endogenous PAs 88
3.15.6 Histochemical detection of H
2
O
2
89
3.16 Bioinformatics tools used for sequence analysis 90

4 Cloning and characterization of DAO gene and promoter 91
4.1 Introduction 91
4.2 Results 94
4.2.1 Molecular cloning of DAO gene and promoter 94
4.2.2 Sequence analysis of DAO gene 97
4.2.2 Subcellular localization of BjDAO 107
4.2.4 Genomic Southern analysis 111
4.2.5 DAO expression during germination 113
4.2.6 Sequence analysis of DAO promoter 113

vi
4.2.7 Functional analysis of DAO promoter 120
4.2.8 Gene expression conferred by different promoters in response to

external stimuli
125
4.3 Discussion 128
4.3.1 Characteristics of DAO 128
4.3.2 Molecular characterization of the 5’-upstream regulatory
sequence of DAO
132
4.3.3 Light-regulated DAO expression 137
4.3.6 Regulation of DAO expression in response to stress 138

5
Cloning and characterization of GAD genes
142
5.1 Introduction 142
5.2 Results 145
5.2.1 Cloning of GAD genes 145
5.2.2 Sequence analysis of GAD 147
5.2.3 Genomic Southern analysis 159
5.2.4 Spatial and temporal GAD expression 159
5.2.5 GAD expression in response to external stimuli 163
5.2.5.1 Effects of phytohormones 163
5.2.5.2 Effects of paraquat and H
2
O
2
166
5.2.5.3 Effects of NaCl and mannitol 166

vii
5.2.5.4 Effects of exogenous glutamate and GABA 171

5.2.5.5 Effects of CaCl
2
and pH 171
5.2.5.6 Effects of temperature 177
5.2.6 GAD member specific expression in different organs 177
5.2.7 Differential expression of GAD in response to external stimuli 182
5.3 Discussion 182
5.3.1 Characteristics of GAD genes 184
5.3.2 Spatial and temporal expression of GAD 186
5.3.3 GAD expression in response to exogenous stimuli 188

6
Effects of overexpression and downregulation of DAO and GAD RNAs
on shoot regeneration in vitro
192
6.1 Introduction 192
6.2 Results 194
6.2.1 Construction of sense and antisense DAO and GAD genes 194
6.2.2 Production of transgenic plants 194
6.2.3 Characterization of transgenic plants 200
6.2.4 Ethylene production in transgenic plants 203
6.2.5 Cellular PA content in transgenic plants 206
6.2.6 H
2
O
2
production in transgenic plants 209
6.2.7 Shoot regeneration response of transgenic plants 212

viii

6.3 Discussion 216
6.3.1 Modulation of PA levels and ethylene by genetic engineering 216
6.3.2 Regulation of shoot regeneration in vitro by PAs, ethylene and
H
2
O
2
220

7 General discussion and conclusion
225
8 References
235


ix
List of Abbreviations
1) Chemicals and reagents

2,4-D 2,4-dichlorophenoxyacetic acid
ABA 2-cis-4-trans-abscisic acid
ACC 1-aminocyclopropane-1-carboxylic acid
AG amino-guandine
AS acetosyringone
AVG 2-aminoethoxyvinyl glycine
BA Benzyladenine
BCIP 5-bromo-4-chloro-2-indolyl-phosphate
CHA cyclohexylamine
DAB 3,3-diaminobenzidine
Dap diaminopropane

dcSAM decarboxylated S-adenosyl methionine
DEPC diethyl-pyrocarbonate
DFMA difluoromethylarginine
DFMO difluoromethylornithine
DIG digoxigenin
DMSO dimethyl sulfoxide
FAD flavin adenine dinucleotide
GA
3
gibberellins acid
GABA
γ-aminobutyric acid
IBA indole-3-butyric acid
MeJA methyl jasmonic acid
MES 2N-morpholino ethanesulfonic acid
MGBG methylgloxal-bis-guanylhydrazone
MS murashige and Skoog
MU 4-methylumbelliferone
MUG 4-methylumbelliferyl glucuronide
NAA naphthaleneacetic acid

x
NBT 4-nitro blue tetrazonium chloride
PAs polyamines
PCA perchloric acid
PEG polyethylene glycol
PIPES piperazine-N,N’-bis (2-ethanesulfonic acid)
PLP pyridoxal phosphate
Put putrescine
SA salicylic acid

SAM S-adenosyl methionine
SDS sodium dodecylsulphate
Spd spermidine
Spm spermine
SSC standard saline citrate
Tris tris (hydromethyl)-aminomethane
x-Gluc
5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid

2)
Enzymes
ACCO 1-aminocyclopropane-1-carboxylic acid oxidase
ACCS 1-aminocyclopropane-1-carboxylic acid synthase
ADC arginine decarboxylase
APX ascorbate peroxidase
CAT catalase
DAO diamine oxidase
GABA-T
γ-aminobutyric acid deaminase
GAD glutamate decarboxylase
GFP green fluorescence protein
GHBDH
γ-hydroxybutyrate dehydrogenase
GUS
β-glucuronidase
ODC ornithine decarboxylase
PAO polyamine oxidase

xi
PDH

∆1
Pyrroline dehydrogenase
PMT putrescine N-methyltransferase
SAMDC S-adenosyl methionine decarboxylase
SAMS S-adenosyl methionine synthetase
SOD superoxide dismutase
SSADH succinic semialdehyde dehydrogenase
SPDS spermidine synthase
SPMS spermine synthase


3)
Others
AOS active oxygen species
UTR untranslated region
SE somatic embryogenesis
RM rooting medium
CIM callus inducing medium
SIM shoot inducing medium
CaMV cauliflower mosaic virus
RACE rapid amplification of cDNA ends
TLC thin layer chromatography


xii

List of Figures

Figure Description Page
1. Ethylene and PA biosynthetic pathways 7

2. GABA shunt pathway in relation to TCA cycle 42
3. Schematic diagram of GFP and DAO fusion constructs in pGreen-
GFP
77
4. Construction of DAO-AS and DAO-S 79
5. Construction of chimeric genes consisting of GADS, GAD-AS and
tGAD
80
6. Schematic representation of constructs carrying the gene fusions of
the GUS coding sequence under 35S promoter with DAO 5’UTR
behind
82
7. Schematic representation of constructs carrying the gene fusions of
the GUS coding sequence under different BjDAO promoters
83
8 DNA amplification of DAO from mustard leaves treated with
chemicals using RT-PCR
95
9 DNA amplification of DAO using 5’ and 3’ RACE 96
10 Gel electrophoresis of Genome walker PCR to generate the
sequence of DAO promoter
98
11 Genomic nucleotide and deduced amino acid sequences of BjDAO 101
12 Comparison of the deduced amino acid sequence between BjDAO
and other plant DAO homologs
104
13 Comparison of the deduced amino acid sequence between BjDAO
and other non-plant DAO homologs
106
14 Hydropathy profile of BjDAO amino acid sequence derived from

cDNA
108
15 Signal peptide prediction for BjDAO using Signal-IP software 109
16 Phylogenetic analysis of DAO homologs from different sources 110
17 Subcellular localization of the DAO-GFP fusion protein 112

xiii
18 Southern analysis of DAO in the mustard genome 114
19 Expression of DAO in etiolated mustard seedlings 115
20 The nucleotide sequence of BjDAO promoter 117
21 Histochemical detection of GUS activity conferred by different
DAO promoters in transgenic Arabidopsis seedlings (14 day-old)
grown in light and in the dark
121
22 GUS activity conferred by different DAO promoters in 3 week-old
transgenic Arabidopsis seedlings
123
23 Relative GUS activity in different organs of transgenic Arabidopsis
conferred by DAOPF promoter
124
24 Comparison of the GUS activity in different organs of transgenic
plants conferred by DAO PF and PDF2 promoter
126
25 Effects of external stimuli on relative GUS activities in plants
conferred by different DAO promoters
127
26 Effect of external H
2
O
2

on relative GUS activities in plants
conferred by different DAO promoters
129
27 Amplification of GAD cDNA fragments from mustard 146
28 Nucleotide and deduced amino acid sequences of mGAD2 149
29 Comparison of the deduced amino acid sequence between mGADs
and other plant GAD homologs.
152
30 Comparison of the deduced amino acid sequence between mGAD2
and GADs from non-plant species
154
31 Alignment of nucleotide sequence of the 5’- and 3’-termini of
mustard GADs
156
32 Prediction of the secondary structure of C-terminal of mGADs with
PSIPRED software
158
33 Hydropathy profile of cDNA-derived amino acid sequence and
signal peptide prediction for mGAD2 using Signal-IP software
160
34 Phylogenetic analysis of GAD from different plant species 161
35 Southern analysis of the GAD gene in mustard 162

xiv
36 Expression of GAD in mustard plants 164
37 GAD expression in response to exogenous 2,4-D 165
38 GAD expression in response to different growth regulators 167
39 GAD expression in response to exogenous paraquat 168
40 GAD expression in response to exogenous H
2

O
2
169
41 GAD expression in response to NaCl 170
42 GAD expression in response to mannitol 172
43 GAD expression in response to exogenous glutamate 173
44 GAD expression in response to exogenous GABA 174
45 GAD expression in response to exogenous CaCl
2
175
46 GAD expression in response to pH change 176
47 GAD expression under different temperatures 178
48 Dot blot analysis for gene-specific probes hybridized to members of
the mustard GAD genes
180
49 PCR amplification of the mustard GAD genes using member-
specific primers
181
50 Differential expression of GAD members in mustard leaves in
response to different treatments
183
51 Southern blot analysis of putative transformants of mustard with
sense and antisense DAO cDNAs
196
52 Southern blot analysis of putative Arabidopsis transformants with
sense and antisense DAO cDNAs
198
53 Southern blot analysis of putative mustard transformants bearing
sense, antisense and truncated GAD cDNAs
199

54 Southern analysis of putative Arabidopsis transformants bearing
sense, antisense and truncated GAD cDNAs
200
55 Transcript levels of DAO in wild type and transgenic Arabidopsis
plants expressing sense and antisense DAO cDNAs
202

xv
56 Transcript levels of GAD in wild type and transgenic Arabidopsis
plants expressing sense, antisense and truncated GAD cDNAs
204
57 Ethylene production in cultured explants of wild type and transgenic
Arabidopsis plants expressing antisense and sense DAO cDNAs
205
58 Ethylene production in cultured explants of wild type and
transgenic Arabidopsis plants expressing antisense, sense and
truncated GAD cDNAs
207
59 Free PA content in cultured explants of wild type (WT) and
transgenic Arabidopsis (AtDAO-AS26 and AtGAD-S50) during
shoot regeneration
208
60 Localization of H
2
O
2
production in leaf of wild type and transgenic
Arabidopsis expressing sense DAO cDNA (AtDAO-S)
211
61 Shoot regeneration from foliar explants of WT and transgenic plants 215



xvi

List of Tables

Table Description Page
1 Chemicals used for treatment of mustard leaves and Arabidopsis. 24
2 Summary of bioinformatics programs used for sequence analysis 90
3 Putative regulatory elements in the BjDAO promoter 119
4 The ratios of Put/Spd and Put/Spd+Spm in WT and transgenic
explants expressing antisense (AtDAO-AS26) and sense (AtDAO-
S50) DAO cDNAs during culture
210
5 Shoot regeneration from cultured explants of different lines of
AtDAO-AS and AtDAO-S plants
213
6 Shoot regeneration from cultured explants of different lines of
AtGAD-AS, AtGAD-S and AttGAD plants
214



xvii
Summary

There is accumulating evidence showing that polyamines (PAs), including
putrescine (Put), spermidine (Spd) and spermine (Spm), and its oxidative product, H
2
O

2
,
are implicated in shoot morphogenesis in vitro and/or somatic embryogenesis in several
plant species. However, the mechanism of PA action is not clear. The main aim of this
study is to investigate whether PA oxidation and its downstream catabolic pathway are
involved in shoot regeneration in vitro. This was achieved by the cloning and
characterization of diamine oxidase (DAO) and glutamine decarboxylase (GAD) genes
from mustard.
The genomic (gBjDAO) and cDNA (BjDAO) clones of DAO were isolated from
mustard. gBjDAO consisted of 5’ upstream regulatory sequences and 4 exons interrupted
by 3 introns, and the protein-coding sequence was identical to the open reading frame
(ORF) of BjDAO that encoded a polypeptide of 649 amino acid residues. BjDAO was
shown to possess a long (489-bp) 5’-untranslated region (UTR), in which eight putative
upstream ORF (uORF) were identified. The cDNA was highly homologous (90%) to
Arabidopsis DAO but considerably less homologous (44-47 %) to DAOs from other plant
species. Sequence analysis of BjDAO revealed the presence of several conserved residues
for all DAOs. These residues included Tyr at the position 396 that was modified into TPQ
cofactor and the copper-binding His residues at 451, 453 and 617. Function analysis of
35S::DAO-GFP expression in transgenic Arabidopsis indicated that DAO was an
extracellular protein that targeted to the cell wall.
Southern analysis revealed that there might be 1-2 DAO genes present in the
mustard genome. DAO expression was low and barely detectable in mustard. This

xviii
prompted the cloning of the 1630-bp DAO promoter. The DAO promoter might possess
several silencer or repressor elements; the activity was functionally analyzed in transgenic
Arabidopsis expressing the GUS gene under the control of different lengths of the DAO
promoter region in the presence or absence of DAO 5’-UTR. The promoters were PU (-
1629 to +500, inclusive of DAO 5’-UTR), PDF1 (-1629 to +1), PDF1 (-1091 to +1),
PDF2 (-478 to +1) and PSF (-207 to +1). These upstream regulatory sequences were

tested by using the GUS gene as the reporter. Histochemical assays for the GUS activity
revealed a strong promoter activity that was detected constitutively in all organs of
PU::GUS plants. However, deletion of the 5’-UTR greatly decreased the activity in
PF::GUS plants and no activity was detected in the root. The deletion at the 5’-end of the
promoter up to -479 increased the activity but the increase was confined only to the
aboveground part of the plant, as shown in PDF1::GUS and PDF2::GUS plants. These
results indicate that the presence of 5’UTR is important for DAO expression, especially in
the root. Further deletion of the promoter to -208, as shown in PSF::GUS plants, abolished
the promoter activity.
Effect of external stimuli on the DAO promoter activity was also investigated. The
promoter activity was downregulated by exogenous application of H
2
O
2
at the dosage-
dependent manner, paraquat and salicylic acid (SA). These results suggest that DAO
expression may be feedback regulated by H
2
O
2
. Furthermore, the inhibitory effect of
paraquat and SA may be due to high levels of H
2
O
2
, as the former usually releases free
radicals, including H
2
O
2

, while the latter inhibits H
2
O
2
-degrading enzyme catalase.
In addition to DAO, four members (mGAD1, mGAD2, mGAD4a and mGAD4b) of
the GAD gene, with high sequence similarity (62-89 %), were cloned from mustard. The
presence of multiple GAD gene family in mustard was also confirmed by Southern

xix
analysis. mGAD2 was shown to encode a polypeptide of 494 amino acid residues, which
was highly homologous (68-91%) to other plant GADs. All members of the mustard
GADs possessed the conserved Lys residue for PLP-binding and Trp residue for CaM
binding.
GAD expressed constitutively in all organs of the plants but transcripts were
accumulated predominantly in the young leaf tissue. The four GAD members were
expressed differentially in all organs examined. mGAD1, mGAD4a and mGAD4b
expressed predominantly in the root, while mGAD2 transcript was most abundant in
flowers and leaves. These results indicate that GAD expression is regulated spatially in a
gene-specific manner. Similar differential gene expression was also observed in mustard
leaves in response to various external stimuli. Among GAD members, mGAD4a was most
responsive to exogenous glutamate, low pH, mannitol, NaCl, SA, paraquat and H
2
O
2
that
upregulated expression. Chilling upregulated mainly mGAD1 and mGAD2 expression,
while low pH also induced mGAD1 transcript accumulation.
The role of PA catabolism in shoot regeneration in vitro was investigated by
overexpression and downregulation of DAO and GAD genes under the control of CaMV

35S promoter in transgenic Arabidopsis. DAO downregulation was shown to decrease
ethylene production in cultured AtDAO-AS tissues that possessed lower ratios of Put/Spd
and Put/Spd+Spm compared to cultured AtDAO-S tissues that overexpressed DAO.
AtDAO-AS and wild type (WT) tissues were equally highly regenerative, whereas the
regenerability of AtDAO-S tissue was considerably lower. With respect to transgenic
plants expressing sense (AtDAD-S), antisense (AtGAD-AS) and truncated GAD
(AttGAD) cDNAs, the capacity of ethylene production in cultured tissues of AtGAD-S
and AtGAD-AS was comparable to that produced in WT, but the amount of ethylene

xx
produced in AttGAD tissues was significantly higher than that of WT and other transgenic
plants. However, there was no difference in shoot regeneration between different
transgenic tissues, whose regenerability was highly variable.
Results of this study indicate that PA oxidation that is feedback-regulated by DAO
is involved in shoot regeneration in vitro and the increased H
2
O
2
production is inhibitory
to regeneration.



xxi
1 General Introduction
Polyamines (PAs) are ubiquitous in all living organisms. In plants, the most
abundant PAs are spermidine (Spd), spermine (Spm) and their diamine precursor
putrescine (Put) (Kumar et al., 1997), which have been shown to play the important
roles in various physiological processes/responses during plant growth and
development in vivo (Evans and Malmberg, 1989). There has been increasing evidence

showing that PAs are also implicated in shoot organogenesis in vitro and somatic
embryogenesis in several plant species (Pua, 1999). The presence of higher levels of
cellular PAs has been associated with increased shoot regeneration from maize callus
(Guregue et al., 1997), somatic embryogenesis of eggplant (Sharma and Rajam, 1995;
Yadav and Rajam, 1998), carrot (Noh and Minocha, 1994) and rice (Bajaj and Rajam,
1995) and rhizogenesis of tobacco (Altamura, 1994) and poplar (Hausman et al.,
1997a, 1997b).
In addition to PAs, results from several lines of study have shown that shoot
regeneration and somatic embryogenesis can be enhanced by inhibition of synthesis or
action of ethylene (Pua, 1999; Pua and Gong, 2004), which is a gaseous plant
hormone. In this laboratory, we have previously shown that shoot regeneration from
cultured tissues of several Brassica genotypes could be greatly enhanced by inhibition
of ethylene production or action using inhibitors (aminoethoxyvylglycine (AVG) and
AgNO
3
) (Chi and Pua, 1989; Chi et al., 1990, 1991; Pua and Chi, 1993) or
downregulation of gene expression of 1-aminocyclopropane-1-carboxylate (ACC)
oxidase (Pua and Lee, 1995) and ACC synthase (Cheng, 2002), which are the key

1
enzymes of the ethylene biosynthesis. However, enhancement of shoot regeneration
could also be achieved by exogenous applications of PAs (Chi et al., 1994), which was
also shown to abolish the inhibitory effect of difluoromethyarginine (DFMA), a potent
inhibitor of Put synthesis, on ethylene inhibitor-induced shoot regeneration (Pua et al.,
1996). The possible involvement of PAs in shoot regeneration has been supported by
the results of our recent study, in which highly regenerative tissues originated from
transgenic plants that expressed antisense ACC synthase cDNA accumulated
significantly higher levels of PAs, whereas poorly regenerative tissues from transgenic
plants overexpressing ACC synthase gene accumulated higher ethylene and Put but
lower Spd and Spm. (Cheng, 2002). These findings have prompted the speculation that

increased shoot regeneration may be attributed to increased levels of cellular PAs,
especially Spd and Spm, rather than inhibition of ethylene synthesis or action.
However, the mechanism of PA action is not clear.
Recently, somatic embryogenesis of Lycium barbarum (Cui et al., 1999) and
Astragalus adsurgens (Luo et al., 2001) and shoot regeneration from strawberry callus
(Tian et al., 2003) have been associated with H
2
O
2
production in culture. H
2
O
2

accumulation in culture has also been shown to be important for the regeneration
potential of tobacco protoplasts (de Marco and Roubelakis-Angelakis, 1996). These
findings indicate that the promoting effect of PAs on plant morphogenesis in vitro
might be mediated through PA oxidation and/or downstream of the PA catabolic
pathway. DAO catabolizes Put to produce 4-aminobutyraldehyde together with H
2
O
2

and NH
3
(Smith, 1985b). 4-Aminobutyraldehyde can be further oxidized to form γ-

2