Cloning and characterization of diamine oxidase and glutamate decarboxylase genes of mustard (brassica juncea) and their roles in shoot morphogenesis in vitro

5.2.5.4 Effects of exogenous glutamate and GABA 171 5.2.6 GAD member specific expression in different organs 177 5.2.7 Differential expression of GAD in response to external stimuli 182

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

2.1.2 PA catabolism 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

2.1.3 Modulation of PAs in transgenic plants 28

2.5.1 Stress response 50

2.6 Interaction between PAs, ethylene and GABA 56

3.11.3 Generation of full-length cDNA sequences 74

3.12.2 Promoter cloning by Genome Walking strategy 75

3.13.3 Sense, antisense and dominant-negative GAD 78

3.13.4 Generation of BjDAO promoter::GUS fusions 81

3.14.1 Mustard 84

3.14.2 Arabidopsis 85

3.15.1 GFP detection with confocal microscopy 87

3.15.2 Histochemical assays for the GUS activity 87

3.15.6 Histochemical detection of H2O2 89

3.16 Bioinformatics tools used for sequence analysis 90

4 Cloning and characterization of DAO gene and promoter 91

4.2.1 Molecular cloning of DAO gene and promoter 94

4.2.2 Subcellular localization of BjDAO 107

4.2.5 DAO expression during germination 113

4.2.6 Sequence analysis of DAO promoter 113

4.2.7 Functional analysis of DAO promoter 120

4.2.8 Gene expression conferred by different promoters in response to

4.3.6 Regulation of DAO expression in response to stress 138

5 Cloning and characterization of GAD genes 142

5.2.4 Spatial and temporal GAD expression 159 5.2.5 GAD expression in response to external stimuli 163

5.2.5.2 Effects of paraquat and H2O2 166

5.2.5.4 Effects of exogenous glutamate and GABA 171

5.2.6 GAD member specific expression in different organs 177 5.2.7 Differential expression of GAD in response to external stimuli 182

5.3.2 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

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

FAD flavin adenine dinucleotide

GA3 gibberellins acid

GABA γ-aminobutyric 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

NBT 4-nitro blue tetrazonium chloride

PAs polyamines

PIPES piperazine-N,N’-bis (2-ethanesulfonic acid)

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

CAT catalase

GABA-T γ-aminobutyric acid deaminase

GFP green fluorescence protein

GHBDH γ-hydroxybutyrate dehydrogenase

GUS β-glucuronidase

PDH ∆1Pyrroline dehydrogenase

PMT putrescine N-methyltransferase

SAMDC S-adenosyl methionine decarboxylase

SAMS S-adenosyl methionine synthetase

SSADH succinic semialdehyde dehydrogenase

3) Others

AOS active oxygen species

CaMV cauliflower mosaic virus

RACE rapid amplification of cDNA ends

TLC thin layer chromatography

List of Figures

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

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

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

18 Southern analysis of DAO in the mustard genome 114

19 Expression of DAO in etiolated mustard seedlings 115

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 H2O2 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

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

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 H2O2 169

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 CaCl2 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

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 H2O2 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

List of Tables

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 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

Summary

There is accumulating evidence showing that polyamines (PAs), including putrescine (Put), spermidine (Spd) and spermine (Spm), and its oxidative product, H2O2,

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 position396 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

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 H2O2 at the dosage-dependent manner, paraquat and salicylic acid (SA) These results suggest that DAO expression may be feedback regulated by H2O2 Furthermore, the inhibitory effect of paraquat and SA may be due to high levels of H2O2, as the former usually releases free radicals, including H2O2, while the latter inhibits H2O2-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

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 H2O2 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

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 H2O2 production is inhibitory

to regeneration

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 AgNO3) (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

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 H2O2 production in culture H2O2accumulation 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 H2O2and NH3 (Smith, 1985b) 4-Aminobutyraldehyde can be further oxidized to form γ-

aminobutyric acid (GABA) by the action of pyrroline dehydrogenase (PDH) In higher plants, GABA can also be formed by decarboxylation of glutamate (Glu) catalyzed by Glu decarboxylase (GAD) (Bown and Shelp, 1997) Results from several recent studies have shown that GABA is also involved in regulation of plant growth and development (Shelp et al., 1999)

To investigate whether shoot morphogenesis in vitro is associated with PA

catabolism, the objectives of this study are:

1 To clone and characterize the DAO gene from mustard, including the promoter;

2 To clone and characterize the GAD genes from mustard;

3 To manipulate the cellular content of PAs by overexpression and downregulation of the DAO gene in transgenic plants and its effects on

shoot regeneration in vitro;

4 To investigate the role of GAD on shoot regeneration in vitro by

overexpression and downregulation of a GAD gene in transgenic plants;

5 Functional analysis of the DAO promoter in transgenic plants by spatial and temporal expression and in response to external stimuli

2 Literature Review

Polyamines (PAs) are small molecules with polycations, which are found in all living organisms (Martin-Tanguy, 2001) The most common aliphatic PAs are the diamine putrescine (Put), triamine spermidine (Spd) and tetraamine spermine (Spm) Put and Spd are generally more abundant, while Spm is present in lower or trace amounts (Bagni and Tassoni, 2001) In addition, diamine cadaverine (Cad) is mainly found in members of the family Fabaceae (Federico and Angelini, 1988) PAs are positively charged at physiological pH value and can interact with proteins, acidic phospholipids and nucleic acid with negative charges (Tabor and Tabor, 1984; Tiburcio et al., 1990)

Plant PAs often exist in free form However, they can be conjugated with small molecules such as hydroxycinnamic acids by the formation of an amide linkage (conjugated form) Conjugated PAs appeared to be important for floral induction and reproduction (Martin-Tangury, 1997) PAs can also bind to various macromolecules such

as proteins (bound form), which is catalyzed by transglutaminase (TGase), resulting in changes in the conformation or the overall charge of the proteins (Serafini-Fracassini et al., 1995) For example, PAs could bind 14-3-3 proteins, which are ubiquitous binding proteins among eukaryotes and have been shown to be involved in several import cellular processes (van Hemert et al., 2001) The binding of PAs to 14-3-3 proteins resulted in a conformational change and facilitated the binding and inhibition of nitrate reductase by 14-3-3 (Athwal and Huber, 2002)

PAs have been shown to play an important role in a wide range of physiological processes during plant growth and development, including dormancy breaking of tubers

and seed germination (Bagni, 1989), stimulation and development of flower buds,

embryogenesis, fruit set, development and ripening (Bagni, 1993), in vitro organogenesis,

flower and leaf senescence (Evans and Malmberg, 1989) In addition, PAs are also involved in response to environmental stimuli such as osmotic stress, mineral deficiency, salinity (Bouchereau, et al., 1999) and pathogen infection (Walters, 2003)

2.1 Metabolism and regulation of PAs

2.1.1 PA biosynthesis

PAs are amino acid derivatives Cad arises from lysine (Lys) by the action of Lys decarboxylase (LDC; EC4.1.1.18) The carbon skeleton of Put, Spd and Spm is derived from arginine (Arg) and ornithine (Orn), while methionine supplies the aminopropyl moiety for the formation of Spd and Spm (Bagni and Tassoni, 2001) Although bacteria, fungi, animal and plants can synthesize PAs through Orn decarboxylase (ODC; EC 4.1.1.17) and/or Arg decarboxylase (ADC; EC 4.1.1.19) pathways, some variations also occur (Tiburcio et al., 1997) In some fungi, Put is formed only by Orn decarboxylation

Arg serves as an alternative source of Orn by arginase (EC 3.5.3.1) when de novo Orn

synthesis is feedback inhibited by Arg (Davis et al., 1992) In bacteria and other fungi, two synthetic pathways coexist Besides the direct decarboxylation of Orn by ODC, Put can also be produced indirectly by the ADC pathway In the ADC pathway, Arg is first decarboxylated by ADC to form agmatine (Agm) and the latter is converted to Put through removal of urea by agmatinase (EC 3.5.3.11) This pathway provides Put when abundant

Arg feedback inhibits de novo Orn biosynthesis (Davis et al., 1992) In mammals, it was

believed that the ODC pathway was exclusive for Put synthesis and Orn is derived from

exogenous Arg by arginase (Tabor and Tabor, 1984) However, with the reported ADC (Lortie et al., 1996; Regunathan and Reis, 2000) and agmatinase (Sastre et al., 1996; Sastre et al., 1998) activities and the isolation of the gene encoding human agmatinase (Iyer et al., 2002; Mistry et al., 2002), it is likely that an alternate ADC pathway also exists

in mammals Higher plants also possess two alternative pathways for Put synthesis including the direct ODC pathway and indirect ADC pathway (Fig 1) (Slocum et al., 1984) Different from ADC pathway for bacteria and mammals, Agm in plants is converted to N-carbamoylputrescine by Agm iminohydrolase (AIH; EC 3.5.3.12), which can be further converted to Put by N-carbamoylputrescine amidohydrolase (CPA; EC 3.5.1.53) (Tiburcio et al., 1990)

Among the three PAs, Put is the obligate precursor for Spd and Spm synthesis It

is now well established that the conversion from Put to Spd is catalyzed by Spd synthase (Put aminopropyltransferase, PAPT or SPDS; EC 2.5.1.16) through transferring an aminopropyl group from decarboxylated S-adenosylmethionine (dcSAM), which is derived from SAM by the action of SAM decarboxylase (SAMDC; EC 4.1.1.50) Spm is formed by transferring an aminopropyl group from dcSAM to Spd catalyzed by Spm synthase (spermidine aminopropyltransferase, SAPT or SPMS; EC 2.5.1.22) (Bouchereau

et al., 1999; Bagni and Tassoni, 2001) In alfalfa, the presence of PAPT, which was highly specific for Put as initial substrate, was associated with accumulation of several PAs such

as Spd, Spm, thermospermine, putative homocaldopentaime and homoaldohexamine (Bagga et al., 1997) However, it has been reported that Spd can be converted backward to Put in maize seedlings treated with exogenous Spd (de Agazio et al., 1995) but the underlying biochemical steps remain unknown

Arginine

Figure 1 Ethylene and PA biosynthetic pathways

ACC, 1-aminocyclopropane-1-carboxylate ACCO, ACC oxidase; ADC, arginine decarboxylase; DAO, diamine oxidase; dcSAM: decarboxylated-SAM; GABA, γ-aminobutyric acid; GABA-T, GABA transaminase; MET, methionine; ODC, ornithine decarboxylase; PAO, PA oxidase; PDH, pyrroline dehydrogenase; SAM,

S-adenosylmethionine; SAMDC, SAM decarboxylase; SAMS, SAM synthetase; SPDS,

Spd synthase; SPMS, Spm synthase; SSADH, succinic semialdehyde dehydrogenase

SAM and Put are not only the precursors for Spd and Spm synthesis, but are also involved in the synthesis of alkaloids Put N-methyltransferase (PMT; EC 2.1.1.53) transfers the methyl moiety of SAM to Put to form N-methyl Put (Hibi et al., 1994), which

is the precursor for nicotine and tropane Spd also participates in the formation of homo-Spd, the precursor for pyrrolizidine alkaloids, by transferring the aminobutyl moiety

of Spd to Put catalyzed by homo-Spd synthase (HSS; EC2.5.1.44) (Graser and Hartmann, 2000) Furthermore, the post-translational activation of the eukaryotic initiation factor 5A (eIF5A) also involved Spd by transferring the aminobutyl moiety of Spd to the Lys residue in the eIF5A preprotein, which is catalyzed by deoxyhypusine synthase (DHS; EC

2.5.1.46) (Ober et al., 2003a) HSS and DHS from Senecio vernalis shared 79% sequence

identity (Ober and Hartmann, 1999) and the former behaves as a DHS losing the binding function to the eIF5A precursor protein (Ober et al., 2003a), indicating that HSS evolved from DHS by gene duplication Moreover, as a side-reaction, DHS catalyzes the formation

of homo-Spd Thus DHS is suggested to be responsible for the assumed universal occurrence of homo-Spd (Ober et al., 2003b)

2.1.1.1 ODC

ODC catalyzes Orn decarboxylation to form Put Genes encoding for ODC have

been isolated from several plant species, including Datura (Michael et al., 1996), tobacco

(Imanishi et al., 1998), tomato (Alabadi and Carbonell, 1998; Kwak and Lee, 2001) and

Nicotiana glutinosa (Lee and Cho, 2001) However, it has been reported that Arabidopsis

lacks the ODC gene (Hanfrey et al., 2001) DL-α-difluoromethylornithine (DFMO), a

specific ODC inhibitor, could inhibit the ODC activity of N glutinosa completely and irreversibly (Lee and Cho, 2001) Tertiary structure of the N glutinosa ODC was similar

to ODC from mouse and human (Lee and Cho, 2001) However, plant ODCs generally lacked the 3’ terminal extension, which was postulated to be involved in rapid turnover of enzymes in mammals and thus they might be more stable with a longer half-life (Michael

et al., 1996)

Animal ODC cDNAs possess a long and highly conserved 5’- UTR (5’- untranslated region) containing a small uORF (upstream Open Reading Frame) Although the deduced uORFs showed no conserved amino acid (aa) sequences and contained variable lengths among different species, both the 5’ UTR and the presence of the small uORF have been shown to reduce translation efficiency (Shantz and Pegg, 1999) The regulatory role of uORF in plant ODC has also been reported in tomato (Kwak and Lee, 2001) The 96-bp 5’-UTR of tomato ODC possessed a uORF of 5 aa residues Functional analysis of uORF showed that elimination of uATG resulted in increased enzyme activity and translation level, whereas changes in nucleotides and aa residues had no effect, implying that translation repression by uORF is not sequence specific but relies on the presence of the uATG because ribosomes translating the uORF reinitiated inefficiently

ODC expression has been shown to be associated with active cell division In tomato, ODC mRNA could be detected in all organs tested, where transcript was most abundant in roots, shoot tips and whole flowers at anthesis, but lower in stems and lowest

in adult leaves, which were coincided with lower growth rate and limited cell division in these tissues (Alabadi and Carbonell, 1998) Similar result has also been reported in

Datura (Michael et al., 1996) Moreover, exogenous application of sucrose increased

ODC expression and enzyme activity in the tomato root (Kwak and Lee, 2001) The increase appeared to be dose-dependent and was detected only at the root apical meristem, indicating the possible implication of sucrose in regulating ODC expression

2.1.1.2 ADC

ADC is the first enzyme involved in Put synthesis through the ADC pathway Genes encoding ADC have been cloned from several plant species, including oat (Bell and Malmberg, 1990), tomato (Rastogi et al., 1993), soybean (Nam et al., 1997), pea

(Perez-Amador et al., 1995), Arabidopsis (Watson and Malmberg, 1996), rice

(Chattopadhyay et al., 1997), mustard (Mo and Pua, 1998) and grape (Primikirios and Roubelakis-Angelakis, 1999) No intron has been detected in all ADC genes studied so far (Galloway et al., 1998) ADC resembles ODC at both the protein and DNA levels Several conserved regions including PLP and DFMO binding sites and a decarboxylase motif are present in all eukaryotic ADCs and ODCs (Poulin et al., 1992)

ADC expression can be affected by different developmental stage and under stress During fruit ripening of tomato, the level of stable-state ADC mRNA appeared to increase from the immature green to breaker stage, but it declined in the ripe fruit (Rastogi et al., 1993) In soybean, the change in ADC activity during early development was preceded by

a corresponding change in transcript abundance (Nam et al., 1997) However, in

Arabidopsis, K+ deficiency resulted in increased ADC activity and Put content but there

was no change in the levels of transcript and protein (Walston and Mammberg, 1996) Similar results has also been reported in grape suspension culture in response to high NH4+ concentration (Primikirios and Roubelakis-Angelakis, 1999)

In Arabidopsis, the two ADC genes showed differential expression ADC1

expressed in all organs, where transcript was most abundant in siliques and rosette leaves, lower in flower and cauline leaves and lowest in stem and root (Soyka and Heyer, 1999)

Unlike ADC1, ADC2 transcript was accumulated predominantly in siliques and cauline

leaves Mutation of ADC2 resulted in reduced ADC activity or free Put content under normal growth conditions, suggesting that ADC2 plays a more important role in Put biosynthesis than ADC1 (Soyka and Heyer, 1999; Urano et al., 2004) ADC1 and ADC2 also expressed differentially in response to stress Stress induced ADC2 expression in wild type, whereas ADC1 expressed constitutively in wild type and ADC2 mutants, regardless

of the presence or absence of stress (Soyka and Heyer, 1999; Urano et al., 2004)

Compared to wild type plants, ADC activity in EN9 ADC2 mutant was 20-fold lower under osmotic stress (Soyka and Heyer, 1999) In addition, the free Put content in adc2-1

mutant remained unchanged under salt stress, which rendered the mutant more sensitive to salt stress (Urano et al., 2004) However, the deleterious effect of salt could be abolished

by exogenous application of Put These results indicate that ADC2 is responsible for

increased ADC activity, which may be regulated transcriptionally in response to stress

However, the level of increased ADC2 transcript was not high enough to account for the

Put accumulation under salt stress It was therefore proposed that post-transcriptional regulation might be involved (Urano et al., 2004) Apart from salt and osmotic stress,

ADC2 expression was also upregulated by mechanical wounding and application of MeJA and ABA, but ADC1 expression was not affected (Perez-Amador et al., 2002) Differential

expression of different ADC genes has also been reported in mustard, where exogenous

application of PAs upregulated expression of MADC2 and MADC3 but not MADC1 (Mo

and Pua, 2002)

2.1.1.3 AIH and CPA

In plants, AIH and CPA are the two successive enzymes involved in the conversion

of Agm, the product of ADC action, to Put Both AIH and CPA are C-N hydrolase C-N

hydrolase is a protein superfamily and the members of this superfamily are involved in the cleavage of C-N bonds but display only moderate homology (Pace and Brenner, 2001)

Genes encoding AIH and CPA were first isolated from bacteria Pseudomonas aeruginosa, where AIH and CPA are involved in Put synthesis as well as Agm utilization

as the sole carbon and nitrogen source (Nakada et al., 2001; Nakada and Itoh, 2003)

PaAIH has no homology with other C-N hydrolases (Nakada and Itoh, 2003) The genome

sequencing project has identified several proteins similar to PaAIH, which shared > 50%

similarity and appeared to form a small family of C-N hydrolase with a novel structure A

putative protein from Arabidopsis that exhibited 59% aa identity to PaAIH was shown to

possess the AIH activity (Janowitz et al., 2003)

CPA was included in the nitrilase family because of its high similarity with other members of the family (Bewley et al., 1999; Nakada and Itoh, 2003) However, the structural features and the strict substrate specificity of CPA suggested that CPA

constituted a new subfamily (Piotrowski et al., 2003) In Arabidopsis, three nitrilase-like

proteins (NLPs) with unknown function were detected, among which NLP1 exhibited high sequence homology with PaCPA and was shown to display CPA activity (Piotrowski et al., 2003) The level of NLP1 transcript was very low but was detected in all organs The expression of NLP1 in leaves did not change under osmotic stress, indicating that CPA was not the rate-limiting enzyme during osmotic-induced Put accumulation (Piotrowski et al., 2003) NLP1 homologs could be detected in several plant species, indicating that ADC pathway may be widely distributed among higher plants (Piotrowski et al., 2003)

2.1.1.4 SAMDC

SAMDC catalyzes the conversion of SAM to dcSAM, which is the key enzyme

irreversibly committing the pool of SAM for the synthesis of Spd and Spm (Flores et al., 1989) Genes encoding SAMDC have been characterized in several plant species, including potato (Mad Arif et al., 1994), periwinkle (Schroder and Schroder, 1995),

Tritordeum (Dresselhaus et al., 1996), spinach (Bolle et al., 1995), tomato, tobacco

(Kumar et al., 1997), carnation (Lee et al., 1997a), mustard (Lee et al., 1997b),

Arabidopsis (Franceschetti et al., 2001) and pea (Marco and Carrasco, 2002) SAMDCs

from various sources displayed sufficient functional similarity at the enzyme level and a proenzyme cleavage site (Schroder and Schroder, 1995) and a putative PEST sequence (Maric et al., 1992) are conserved in all SAMDCs

Plant SAMDCs differ from their mammalian counterparts in several aspects The activity of plant SAMDCs is not stimulated by Put but possesses a constitutively high activity similar to Put-activated human SAMDC, which may be attributed to the replacement of two positively charged Arg residues that are absent from human enzyme (Bennett et al., 2002) Moreover, plant SAMDCs also possess a highly conserved pair of overlapping tiny and small uORFs in the long 5’-UTR, which are flanked by introns present at the conserved positions The tiny uORF consists of 2-3 codons, while the small uORF encodes 50-54 residues (Franceschetti et al., 2001) The last nucleotide A of the stop codon in the former is the first nucleotide of the start codon for the latter, suggesting that the ATG for the small uORF may not be recognized if ATG for the tiny uORF is identified by the translation machinery However, the translation initiation sequence context is always poor for the tiny uORF but is invariably better for the small uORF, implying that leaky scanning of the tiny uORF can occur and the small uORF may be recognized (Hanfrey et al., 2003) It is speculated that the small uORF is responsible for the translational repression of plant SAMDC (Franceschetti et al., 2001) In tobacco,

overexpression of the wild type SAMDC did not significantly increase SAMDC activity, but elimination of the small uORF increased relative translational efficiency that, in turn, caused a more than 400-fold increase in dcSAM level together with depleted Put and reduced Spm content (Hanfrey et al., 2002) The response has been correlated with severe growth and development defects in transgenic tobacco, including stunted phenotype with reduced internode length, wrinkled and curled leaves, delayed flowering and abnormal

flower organs These results indicate that plant SAMDC is translationally repressed in planta and the uORF-mediated translational control of SAMDC is essential for PA

homeostasis and normal growth and development (Hanfrey et al., 2002)

SAMDC expression varies spatially and temporally during plant growth and development SAMDC expressed strongly in actively dividing and differentiating tissues

in potato (Mad Arif et al., 1994) but constitutively in all organs of pea, where higher transcript level was detected in stem and roots than leaves (Marco and Carrasco, 2002) Furthermore, SAMDC expression in pollinated ovaries was increased transiently in the 1stday after anthesis but declined thereafter when the ovary growth reached the full size (Marco and Carrasco, 2002) SAMDC expression has also been associated with stress SAMDC transcript accumulated in pea stems and leaves when exposed to ozone (Marco and Carrasco, 2002), which is in line with induced PA biosynthesis in ozone tolerance in tobacco (Langebartels et al., 1991)

(Hatanaka et al., 1999), pea (Alabadi and Carbonell, 1999), and apple (Zhang et al., 2003) The putative SAM or dcSAM-binding regions are present in all SPDSs Plant SPDSs display lower identity with the mammalian counterparts than that of the tobacco PMT, which catalyzes SAM-dependent N-methylation of Put (Hibi et al., 1994) It is therefore suggested that PMT may evolve from plant SPDS after plant and animal SPDS diverged (Hashimoto et al., 1998) However, SPDS and PMT expressed differentially in tobacco, where SPDS is expressed constitutively in root, stem and leaf, while PMT transcript accumulated predominantly in root (Hashimoto et al., 1998) The constitutive expression

of SPDS has also been reported in coffee, where transcript was most abundant in rapid growing tissues such as younger leaves and green stems (Hatanaka et al., 1999)

In pea, the two SPDS cDNAs, psSPDSYN1 and psSPDSYN2, expressed differentially (Alabadi and Carbonell, 1999) The psSPDSYN1 transcript was relatively

abundant at 0 and 1 DPA (day post anthesis) but decreased thereafter, whereas the level of

psSPDSYN2 transcript was relatively low at anthesis but increased at the later stage of

ovule development Moreover, their expression also varied with organs, among which the former generally expressed strongly in shoot tips and young leaves, whereas the latter predominantly expressed in fully elongated stem (Alabadi and Carbonell, 1999) These results indicate that the former is associated with actively growing tissues, while the latter

is related to more advanced stages of organ development Differential expression of SPDS

genes has also been reported in apple, where SPDS2a and SPDS2b were derived from the alternative splicing of SPDS2 (Zhang et al., 2003) SPDS1 and SPDS2a transcripts were shown to accumulate in young leaves and fruits, whereas SPDS2b transcript was detected

in mature leaves and shoot but not in young leaves and fruits, suggesting that SPDS expression may be regulated developmentally and splicing of SPDS2 may play an

important role in SPDS regulation It was also observed that SPDS expression remained high during fruit development, whereas the Spd content was highest at the early growth stage but decreased sharply later, suggesting the possible posttranscriptional regulation of SPDS during the apple fruit development (Zhang et al., 2003)

Four SPDS-related genes, including SPDS1-3 and ACL5, have been reported in Arabidopsis (Hanzawa et al., 2002) SPDS1-3 showed more than 60% identity with each

other and were thought to be the recent derivatives of a common ancestor, while there was only ~30% identity between them and ACL5 (Hanzawa et al., 2002) SPDS1 and SPDS2 were shown to possess SPDS activity (Hanzawa et al., 2002) Although transcripts of both

genes could be detected in all organs, they were higher in roots In addition, SPDS2

expression was upregulated by cytokinin (Hanzawa, et al., 2002)

2.1.1.6 SPMS

SPMS has been found only in eukaryotes It catalyzes the similar aminopropyl transferring reaction as SPDS by converting dcSAM and Spd to Spm The SPMS gene was first cloned from human (Korhonen et al., 1995) and later from yeast (Hamasaki-Katagiri et al., 1998) In contrast to the absolute requirement of Spd for growth, Spm was not essential for growth in yeast, although Spm was normally present in the wild type yeast The high identity between SPDS and SPMS in human and yeast suggested that SPMS evolved from SPDS by gene duplication, which probably occurred when the eukaryotes arose (Hamasaki-Katagiri et al., 1998)

An Arabidopsis mutant ACAULIS 5 (acl5) was characterized with a defect in stem

elongation due to the reduced cell expansion after transition to the reproductive stage

(Hanzawa et al., 2000) The ACL5 protein displayed high sequence similarity with SPDS

and SPMS from several organisms and was demonstrated to possess SPMS activity In

wild type flowering plants, ACL5 transcript accumulated in stem internodes and root,

while the transcript was lower in young seedlings and rosette leaves before flowering

Exogenous auxin upregulated ACL5 transcript accumulation (Hanzawa et al., 2000) and ACL5 mutation affected GA3-regulated gene expression in stem internodes (Hanzawa et al., 1997), which promoted the speculation that interactions of auxin and GA3 in shoot development might be, in part, mediated through PA metabolism (Hanzawa et al., 2000)

Arabidopsis SPDS3 was shown to complement the spe∆4 Spm deficiency in yeast, indicating that SPDS3 encoded a SPMS (Panicot et al., 2002) Similar to ACL5, SPMS

expressed predominantly in stem internodes, flower buds and roots (Hanzawa et al., 2002)

In addition, expression of SPMS could be upregulated by exogenous application of ABA (Hanzawa, et al., 2002) Recently, the SPMS mutant has been characterized in

Arabidopsis In spms-1 mutant, the Spm level decreased more than 90% and SPMS

transcript could not be detected, but the plant exhibited normal morphology (Imai et al.,

2004) In contrast, acl5-1 mutant showed unaltered PA content These results suggested that SPMS might be responsible mainly for Spm synthesis in Arabidopsis, whereas ACL5

might interact with unidentified molecules or possessed additional enzyme activity to

account for the observed phenotype in acl5 mutant (Imai et al., 2004) Although Spm was absent in the acl-5 spms-1double mutant, the mutant was viable, indicating that Spm was not essential for Arabidopsis survival as for yeast (Imai et al., 2004)

The recent cloning and characterization of genes encoding AIH and CPA indicates the complete description of PA biosynthetic pathway at the molecular level Sequence

comparison showed the diverse origins of PA biosynthetic genes in Arabidopsis It was

proposed that genes in the ADC pathway were inherited from the cyanobacterial ancestor