Shikimate and phenylalanine biosynthesis in the green lineage · REVIEW ARTICLE published: 27 March...
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REVIEW ARTICLE published: 27 March 2013 doi: 10.3389/fpls.2013.00062 Shikimate and phenylalanine biosynthesis in the green lineage TakayukiTohge*, MutsumiWatanabe, Rainer Hoefgen and Alisdair R. Fernie Max-Planck-Institute of Molecular Plant Physiology, Potsdam-Golm, Germany Edited by: Kazuki Saito, RIKEN Plant Science Center and Chiba University, Japan Reviewed by: Gad Galili, The Weizmann Institute of Science, Israel Hiroshi Maeda, University of Wisconsin-Madison, USA *Correspondence: TakayukiTohge, Max-Planck Institute of Molecular Plant Physiology, Am Muehlenberg 1, 14476 Potsdam-Golm, Germany. e-mail: [email protected] The shikimate pathway provides carbon skeletons for the aromatic amino acids l- tryptophan, l-phenylalanine, and l-tyrosine. It is a high flux bearing pathway and it has been estimated that greater than 30% of all fixed carbon is directed through this pathway. These combined pathways have been subjected to considerable research attention due to the fact that mammals are unable to synthesize these amino acids and the fact that one of the enzymes of the shikimate pathway is a very effective herbicide target. However, in addition to these characteristics these pathways additionally provide important precursors for a wide range of important secondary metabolites including chlorogenic acid, alkaloids, glucosinolates, auxin, tannins, suberin, lignin and lignan, tocopherols, and betalains. Here we review the shikimate pathway of the green lineage and compare and contrast its evo- lution and ubiquity with that of the more specialized phenylpropanoid metabolism which this essential pathway fuels. Keywords: shikimate pathway, aromatic amino biosynthesis, evolution, gene copy number, gene duplication, plant secondary phenolic metabolite INTRODUCTION The shikimate pathway is closely interlinked with those of the aromatic amino acids (L-tryptophan, l-phenylalanine, and L- tyrosine) and in land plants bears very high fluxes with estimates of the amount of fixed carbon passing through the pathway varying between 20 and 50% (Weiss, 1986; Corea et al., 2012; Maeda and Dudareva, 2012). Considerable research focus has been placed on this pathway since the aromatic amino acids are not produced by humans and monogastric livestock and are therefore an impor- tant dietary component (Tzin and Galili, 2010). Furthermore, one of the enzymes of the pathway – 5-enol pyruvalshikimate-3- phosphate synthase (EPSP) – is one of the most widely employed herbicide target sites (see, Duke and Powles, 2008). Moreover, as we have recently described, plant phenolic secondary metabo- lites and their precursors are synthesized via the pathway of shikimate biosynthesis and its numerous branchpoints (Tohge et al., 2013). The shikimate pathway is highly conserved being found in fungi, bacteria, and plant species wherein it operates in the biosynthesis of not just the three aromatic amino acids described above but also of innumerable aromatic secondary metabolites such as alkaloids, flavonoids, lignins, and aromatic antibiotics. Many of these compounds are bioactive as well as playing important roles in plant defense against biotic and abi- otic stresses and environmental interactions (Hamberger et al., 2006; Maeda and Dudareva, 2012), and as such are highly physio- logically important. It is estimated that under normal conditions as much as 20% of the total fixed carbon flows through to shiki- mate pathway (Ni et al., 1996), with greater carbon flow through the pathway under times of plant stress or rapid growth (Corea et al., 2012). Given its importance it is perhaps not surpris- ing that all members of biosynthetic genes and corresponding enzymes involved in shikimate pathway have been characterized in model plants such as Arabidopsis . Cross-species comparison of the shikimate biosynthetic enzymes has revealed that they share sequence similarity, divergent evolution, and commonal- ity in reaction mechanisms (Dosselaere and Vanderleyden, 2001). However, all other species vary considerably from fungi which has evolved a complex system with a single pentafunctional polypep- tide known as the AroM complex which performs five consecutive reactions (Lumsden and Coggins, 1977; Duncan et al., 1987). In this review we will summarize current knowledge concerning the genetic nature of this pathway focusing on cross-species compar- isons bridging a wide range of species including algae (Chlamy- domonas reinhardtii, Volvox carteri, Micromonas sp., Ostreococcus tauri, Ostreococcus lucimarinus ), moss (Selaginella moellendorf- fii, Physcomitrella patens ), monocots (Sorghum bicolor, Zea mays, Brachypodium distachyon, Oryza sativa ssp. japonica and Oryza sativa ssp. indica ), and dicots (Vitis vinifera, Theobroma cacao, Carica papaya, Arabidopsis thaliana, Arabidopsis lyrata, Populus tri- chocarpa, Ricinus communis, Manihot esculenta, Malus domestica, Fragaria vesca, Glycine max, Lotus japonicus, Medicago truncatula ) species (Table 1). Finally, we compare and contrast the evolution of this pathway with that of the more specialized pathways of phenylpropanoid biosynthesis. SHIKIMATE BIOSYNTHESIS AND PHENYLALANINE DERIVED SECONDARY METABOLISM IN PLANTS Given that phenolic secondary metabolites which are derived from phenylalanine via shikimate biosynthesis are widely distrib- uted in plants and other eukaryotes, genes encoding shikimate biosynthetic enzymes are generally highly conserved in nature. Eight and two reactions are involved in shikimate and phenylala- nine biosynthesis, respectively. Both members of all gene families and the corresponding biosynthetic enzymes involved in these www.frontiersin.org March 2013 |Volume 4 | Article 62 | 1
Transcript of Shikimate and phenylalanine biosynthesis in the green lineage · REVIEW ARTICLE published: 27 March...
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REVIEW ARTICLEpublished: 27 March 2013
doi: 10.3389/fpls.2013.00062
Shikimate and phenylalanine biosynthesis in the greenlineageTakayukiTohge*, Mutsumi Watanabe, Rainer Hoefgen and Alisdair R. Fernie
Max-Planck-Institute of Molecular Plant Physiology, Potsdam-Golm, Germany
Edited by:Kazuki Saito, RIKEN Plant ScienceCenter and Chiba University, Japan
Reviewed by:Gad Galili, The Weizmann Institute ofScience, IsraelHiroshi Maeda, University ofWisconsin-Madison, USA
*Correspondence:Takayuki Tohge, Max-Planck Instituteof Molecular Plant Physiology, AmMuehlenberg 1, 14476Potsdam-Golm, Germany.e-mail: [email protected]
The shikimate pathway provides carbon skeletons for the aromatic amino acids l-tryptophan, l-phenylalanine, and l-tyrosine. It is a high flux bearing pathway and it hasbeen estimated that greater than 30% of all fixed carbon is directed through this pathway.These combined pathways have been subjected to considerable research attention due tothe fact that mammals are unable to synthesize these amino acids and the fact that oneof the enzymes of the shikimate pathway is a very effective herbicide target. However, inaddition to these characteristics these pathways additionally provide important precursorsfor a wide range of important secondary metabolites including chlorogenic acid, alkaloids,glucosinolates, auxin, tannins, suberin, lignin and lignan, tocopherols, and betalains. Herewe review the shikimate pathway of the green lineage and compare and contrast its evo-lution and ubiquity with that of the more specialized phenylpropanoid metabolism whichthis essential pathway fuels.
Keywords: shikimate pathway, aromatic amino biosynthesis, evolution, gene copy number, gene duplication, plantsecondary phenolic metabolite
INTRODUCTIONThe shikimate pathway is closely interlinked with those of thearomatic amino acids (L-tryptophan, l-phenylalanine, and L-tyrosine) and in land plants bears very high fluxes with estimates ofthe amount of fixed carbon passing through the pathway varyingbetween 20 and 50% (Weiss, 1986; Corea et al., 2012; Maeda andDudareva, 2012). Considerable research focus has been placed onthis pathway since the aromatic amino acids are not produced byhumans and monogastric livestock and are therefore an impor-tant dietary component (Tzin and Galili, 2010). Furthermore,one of the enzymes of the pathway – 5-enolpyruvalshikimate-3-phosphate synthase (EPSP) – is one of the most widely employedherbicide target sites (see, Duke and Powles, 2008). Moreover, aswe have recently described, plant phenolic secondary metabo-lites and their precursors are synthesized via the pathway ofshikimate biosynthesis and its numerous branchpoints (Tohgeet al., 2013). The shikimate pathway is highly conserved beingfound in fungi, bacteria, and plant species wherein it operatesin the biosynthesis of not just the three aromatic amino acidsdescribed above but also of innumerable aromatic secondarymetabolites such as alkaloids, flavonoids, lignins, and aromaticantibiotics. Many of these compounds are bioactive as well asplaying important roles in plant defense against biotic and abi-otic stresses and environmental interactions (Hamberger et al.,2006; Maeda and Dudareva, 2012), and as such are highly physio-logically important. It is estimated that under normal conditionsas much as 20% of the total fixed carbon flows through to shiki-mate pathway (Ni et al., 1996), with greater carbon flow throughthe pathway under times of plant stress or rapid growth (Coreaet al., 2012). Given its importance it is perhaps not surpris-ing that all members of biosynthetic genes and correspondingenzymes involved in shikimate pathway have been characterized
in model plants such as Arabidopsis. Cross-species comparisonof the shikimate biosynthetic enzymes has revealed that theyshare sequence similarity, divergent evolution, and commonal-ity in reaction mechanisms (Dosselaere and Vanderleyden, 2001).However, all other species vary considerably from fungi which hasevolved a complex system with a single pentafunctional polypep-tide known as the AroM complex which performs five consecutivereactions (Lumsden and Coggins, 1977; Duncan et al., 1987). Inthis review we will summarize current knowledge concerning thegenetic nature of this pathway focusing on cross-species compar-isons bridging a wide range of species including algae (Chlamy-domonas reinhardtii, Volvox carteri, Micromonas sp., Ostreococcustauri, Ostreococcus lucimarinus), moss (Selaginella moellendorf-fii, Physcomitrella patens), monocots (Sorghum bicolor, Zea mays,Brachypodium distachyon, Oryza sativa ssp. japonica and Oryzasativa ssp. indica), and dicots (Vitis vinifera, Theobroma cacao,Carica papaya, Arabidopsis thaliana, Arabidopsis lyrata, Populus tri-chocarpa, Ricinus communis, Manihot esculenta, Malus domestica,Fragaria vesca, Glycine max, Lotus japonicus, Medicago truncatula)species (Table 1). Finally, we compare and contrast the evolutionof this pathway with that of the more specialized pathways ofphenylpropanoid biosynthesis.
SHIKIMATE BIOSYNTHESIS AND PHENYLALANINE DERIVEDSECONDARY METABOLISM IN PLANTSGiven that phenolic secondary metabolites which are derivedfrom phenylalanine via shikimate biosynthesis are widely distrib-uted in plants and other eukaryotes, genes encoding shikimatebiosynthetic enzymes are generally highly conserved in nature.Eight and two reactions are involved in shikimate and phenylala-nine biosynthesis, respectively. Both members of all gene familiesand the corresponding biosynthetic enzymes involved in these
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Tohge et al. Shikimate and phenylalanine biosynthesis
Table 1 | Summary of the species used in the study.
Species name ID Common name Classification Species
1 Chlamydomonas reinhardtii CR Green algae Chlorophyte Chlamydomonadaceae
2 Volvox carteri VC Algae Chlorophyte Volvoceae
3 Micromonas sp. RCC299 MRC Micromonas Chlorophyta Prasinophyceae
4 Ostreococcus tauri OT Microalgae Prasinophyte Prasinophyceae
5 Ostreococcus lucimarinus OL Microalgae Prasinophyte Prasinophyceae
6 Selaginella moellendorffii SM Spike moss Lycophytes Selaginellaceae
7 Physcomitrella patens PP Moss Lycophytes Funariaceae
8 Sorghum bicolor SB Sorghum Monocot Poaceae
9 Zea mays ZM Corn Monocot Poaceae
10 Brachypodium distachyon BD Purple false brome Monocot Poaceae
11 Oryza sativa ssp. japonica OS Japonica rice Monocot Poaceae
12 Oryza sativa ssp. indica OSI Indica rice Monocot Poaceae
13 Vitis vinifera VV Grapevine Dicot Vitaceae
14 Theobroma cacao TC Cacao Dicot Malvaceae
15 Carica papaya CP Papaya Dicot Caricaceae
16 Arabidopsis thaliana AT Arabidopsis Dicot Brassicaceae
17 Arabidopsis lyrata AL Lyrata Dicot Brassicaceae
18 Populus trichocarpa PT Poplar Dicot Salicaceae
19 Ricinus communis RC Castor oil plant Dicot Euphorbiaceae
20 Manihot esculenta ME Cassava Dicot Euphorbiaceae
21 Malus domestica MD Apple Dicot Rosaceae
22 Fragaria vesca FV Strawberry Dicot Rosaceae
23 Glycine max GM Soybean Dicot Fabaceae
24 Lotus japonicus LJ Lotus Dicot Fabaceae
25 Medicago truncatula MT Medicago Dicot Fabaceae
Coding genes is estimated by Plaza (http://bioinformatics.psb.ugent.be/plaza/). Relationships among the species considered are presented on the Plaza website
(http://bioinformatics.psb.ugent.be/plaza/).
pathways have been characterized in model plants such as Ara-bidopsis (Figure 1A). In contrast, phenolic secondary metabolitesderived from phenylalanine display considerable species-specificdistribution with the phenolic secondary metabolites have beenfound in plant kingdom such as coumarin derivatives, monolig-nal, lignin, spermidin derivatives, flavonoid, tannin being presentin specific families within the green lineage (Figure 1B). Thisdiversity has arisen by the action of diverse evolutionary strate-gies for example gene duplication and cis-regulatory evolution inorder to adapt to prevailing environmental conditions. Given theirspecies-specific distribution, the genes involved in plant pheno-lic secondary metabolism such as phenylammonia-lyase (PAL),polyketide synthase (PKS), 2-oxoglutarate-dependent deoxyge-nases (2ODDs), and UDP-glycosyltransferases (UGTs) are fre-quently used as case studies of plant evolution (Tohge et al., 2013).Despite the fact that shikimate-phenylalanine biosynthetic genesare well conserved in all species including algae species, phe-nolic secondary metabolism related orthologous genes were notdetected in all algae species (Table 2, Tohge et al., 2013). This resultsuggests a considerably more ancient origin of the shikimate-phenylalanine pathways. In the next sections, we will discuss theevolution of shikimate-phenylalanine pathways focusing on cross-species comparisons for each gene encoding on of the constituentenzymes of either pathway.
3-DEOXY-D-ARABINO-HEPTULOSONATE 7-PHOSPHATESYNTHASEThe first enzymatic step of the shikimate pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS), cat-alyzes an aldol condensation of phosphoenolpyruvate (PEP), andD-erythrose 4-phosphate (E4P) to produce 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) (Figure 1). According to theirprotein structure, DAHPSs can be clustered into two distincthomology classes. The microbe derived class I DAHPS containa bifunctional chorismate mutase (CM)-DAHPS domains, forthat reason microbial DAHPSs, for example, E. coli (AroF, G,and H) and S. cerevisiae (Aro3 and 4), are classified as class IDAHPSs. By contrast, class II DAHPS were previously thoughtto be present only in plant species, but have subsequently beenreported in certain microbes such as Streptomyces coelicolor, Strep-tomyces rimosus, and Neurospora crassa (Bentley, 1990; Maeda andDudareva, 2012). The DAHPS (AroA) and CM (AroQ) activ-ities of B. subtilis DAHPS are, however, separated by domaintruncation. Detailed sequence structure analysis of the bacterialAroA and AroQ families, enzymatic studies with the full-lengthprotein and the truncated domains of AroA and AroQ of B.subtilis, and comparison with fusion proteins of Porphyromonasgingivalis in which the AroQ domain was fused to the C termi-nus of AroA, suggest that “feedback regulation” may indeed be
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Tohge et al. Shikimate and phenylalanine biosynthesis
FIGURE 1 |The shikimate and phenylalanine derived secondarymetabolite biosynthesis in plants. (A) Shikimate biosynthesis starting fromphosphoenolpyruvate (PEP) and D-erythrose 4-phosphate is described withcharacterized genes and reported intermediate metabolites. (B) phenylalaninederived major phenolic secondary mebolite biosynthesis in the green lineage.Arrow indicates enzymatic reaction, circle indicates metabolite. Abbreviation:DAHPS, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase; DQS,3-dehydroquinate synthase; DHQD/SD, 3-dehydroquinate dehydratase; SK,shikimate kinase; ESPS, 3-phosphoshikimate 1-carboxyvinyltransferase; CS,chorismate synthase; CM, chorismate mutase; PAT, prephenateaminotransferase; ADT, arogenate dehydratase. PAL, phenylalanineammonia-lyase; C4H, cinnamate-4-hydroxylase; 4CL, 4-coumarate CoA ligase;
CAD, cinnamoyl-alcohol dehydrogenase; F5H, ferulate 5-hydroxylase; C3H,coumarate 3-hydroxylase; ALDH, aldehyde dehydrogenase; CCR,cinnamoyl-CoA reductase; HCT, hydroxycinnamoyl-Coenzyme Ashikimate/quinate hydroxycinnamoyltransferase; CCoAOMT,caffeoyl/CoA-3-O-metheltransferase; CHS, chalcone synthese; CHI, chalconeisomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid-3′-hydroxylase;F3GT, flavonoid-3-O-glycosyltransferase; FS, flavone synthase; FOMT,flavonoid O-methyltransferase; FCGT, flavone-C -glycosyltransferase; FLS,flavonol synthese; F3GT, flavonoid-3-O-glycosyltransferase; DFR,dihydroflavonol reductase; ANS, Anthocyanidin synthese; AGT,Flavonoid-O-glycosyltransferase; AAT, anthocyanin acyltransferase; BAN,oxidoreductase|dihydroflavonol reductase like; LAC, laccase.
the evolutionary link between the two classes which are evolvedfrom primitive unregulated member of class II DAHPS (Wu andWoodard, 2006). Class II plant DAHPSs have been reported fromcarrot roots (Suzich et al., 1985) and potato cell culture (Pintoet al., 1986; Herrmann and Weaver, 1999). DAHPS is encoded bythree genes in the Arabidopsis genome (AtDAHPS1, AT4G39980;AtDAHPS2, At4g33510; AtDAHPS3, At1g22410). Orthologousgene search queries using the Arabidopsis DAHPSs, revealed asingle gene in algae species (Chlamydomonas reinhardtii, Volvoxcarteri, Micromonas sp., and Ostreococcus tauri) and Lotus japon-ica but two to eight isoforms in other higher plant species(Table 2). AtDAHPS1-type and AtDAHPS2 type genes displaydifferential expression in Arabidopsis thaliana, Solanum lycoper-sicum, and Solanum tuberosum (Maeda and Dudareva, 2012).AtDAHPS1-type genes, which are additionally subject to redoxregulation by the ferredoxin-thioredoxin system, exhibit signifi-cant induction by wounding and pathogen infection (Keith et al.,
1991; Gorlach et al., 1995; Maeda and Dudareva, 2012), whereasAtDAHPS2 type genes display constitutive expression (Gorlachet al., 1995). A phylogenetic analysis of DAHPS genes reveals fourmajor clades, (i) a microphyte clade, (ii) a bryophyte duplica-tion clade, (iii) monocot and dicot woody species clade, (iv) aAtDAHPSs clade (Figure 2Aa). Furthermore, major clade iv hasfour sub-groups, (iv-a) AtDAHPS2 group, (iv-b) monocot, (iv-c)AtDAHPS1 group and (iv-d) AtDAHP3 group. This result indi-cates that the constitutively expressed AtDAHPS1 and the stressresponsive AtDAHPS 3 type genes display well conserved sequencebetween species (clade iv-c and iv-d), whereas the second con-stitutively expressed AtDAHPS2 type genes are clearly separatedbetween monocot and dicot species (clade iv-a).
3-DEHYDROQUINATE SYNTHASEThe second step of the shikimate pathway is catalyzed by 3-dehydroquinate synthase (DHQS), an enzyme which promotes
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Tohge et al. Shikimate and phenylalanine biosynthesis
Tab
le2
|Sh
ikim
ate
and
ph
enyl
alan
ine
bio
syn
thet
icge
nes
and
ho
mo
log
sin
each
spec
ies
wit
h/w
ith
ou
tta
nd
emd
up
licat
edge
nes
.
No.
ID1
CR
3M
RC
C29
94
OT
8S
B9
ZM
10B
D11
OS
12O
Sin
dic
a
DH
SC
r17g
0646
0M
rcc0
2g07
760
Ot0
6g03
510
Sb0
1g02
8770
Zm02
g392
00B
d1g2
1330
Os0
3g27
230
Osi
07g3
5030
Sb0
1G03
3590
Zm04
g315
50B
d1g6
0750
Os0
7g42
960
Osi
08g3
6090
Sb0
2G03
9660
Zm05
g069
90B
d3g
3365
0O
s08g
3779
0O
si10
g318
30
Sb0
7G02
9080
Bd
3g38
670
Os1
0g41
480
DQ
SC
r08g
0224
0M
rcc0
1g05
190
Ot0
5g01
830
Sb0
2G03
1240
Zm02
g343
20B
d4g3
6507
Os0
9g36
800
Osi
09g2
9080
DH
QD
Cr0
8g04
550
Mrc
c01g
0358
0O
t12g
0266
0S
b08G
0169
70Zm
03g1
7940
Bd4
g058
97O
s12g
3487
4O
si12
g233
10
Zm10
g051
40
SK
Cr1
0g04
010
Mrc
c13g
0250
0O
t14g
0318
0S
b06G
0302
60Zm
02g0
2970
Bd3
g592
37O
s04g
5480
0O
si02
g496
80
Zm04
g278
40B
d5g2
3460
Zm05
g405
30
SK
L1S
b08G
0186
30Zm
01g2
6660
Bd2
g036
80O
s01g
0130
2
SK
L2M
rcc0
2g03
490
Ot0
7g01
450
Sb0
1G02
7930
Zm01
g226
40B
d3g3
4245
Os1
0g42
700
ES
PS
Cr0
3g06
830
Mrc
c13g
0110
0O
t14g
0243
0S
b10G
0022
30Zm
09g0
5500
Bd1
g516
60O
s06g
0428
0O
si06
g031
90
CS
Cr0
1g12
390
Mrc
c05g
0143
0O
t02g
0602
0S
b01G
0407
90Zm
01g1
0020
Bd1
g677
90O
s03g
1499
0O
si03
g133
40
Zm09
g245
40
CM
Cr0
3g01
600
Mrc
c08g
0506
0O
t08g
0286
0S
b03G
0354
60Zm
03g3
1000
Bd2
g508
00O
s01g
5587
0O
si01
g528
50
Sb0
4G00
5480
Zm05
g212
70B
d3g0
6050
Os0
2g08
410
Osi
02g0
8160
Zm
08g
3432
0O
s12g
3890
0
Zm
08g
3433
0
PAT
Cr0
2g15
900
Mrc
c06g
0086
0O
t16g
0069
0S
b03G
0411
80Zm
03g2
5600
Bd2
g243
00O
s01g
6509
0O
si01
g617
00
Sb0
9G02
1360
Zm08
g152
10B
d2g5
6330
AD
TC
r06g
0276
0M
rcc0
1g05
870
Ot0
1g01
250
Sb0
1G03
8740
Zm01
g120
20B
d5g
0902
0O
s04g
3339
0O
si03
g163
50
Sb0
6G01
5310
Zm02
g163
20B
d5g
0903
0O
s03g
1773
0O
si04
g254
40
Zm10
g160
00B
d1g1
6517
Os0
7g49
390
Osi
07g4
1390
Bd1
g658
00
(Con
tinue
d)
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Tohge et al. Shikimate and phenylalanine biosynthesis
Tab
le2
|Co
nti
nu
ed
No.
ID13
VV
14T
C16
AT
17A
L18
PT
21M
D22
FV23
GM
24LJ
25M
T
DH
SV
v00g
0920
0Tc
01g0
0859
0A
t1g2
2410
Al1
g239
30P
t01g
1486
0M
d00g
0007
30Fv
0g22
320
Gm
02g3
7080
Lj1g
0025
20M
t2g0
0908
0
Vv0
0g17
890
Tc01
g012
940
At4
g335
10A
l7g0
2250
Pt0
2g09
760
Md0
0g36
1080
Fv5g
1961
0G
m06
g106
70M
t5g0
6450
0
Vv1
8g03
830
Tc02
g011
250
At4
g399
80A
l7g0
7720
Pt0
5g07
260
Md0
1g00
1320
Gm
14g3
5370
Tc03
g024
120
Pt0
5g16
320
Md0
4g00
2280
Gm
15g0
6020
Tc08
g008
780
Pt0
7g04
970
Md0
5g02
1570
Md0
5g02
5390
Md1
0g00
3880
Md1
1g02
1260
DQ
SV
v04g
0035
0Tc
01g0
0136
0A
t5g6
6120
Al8
g345
60P
t05g
1111
0M
d00g
0898
50Fv
1g13
270
Gm
01g3
6890
Lj2g
0224
20M
t5g0
2258
0
Gm
11g0
8350
DH
QD
Vv0
5g03
610
Tc04
g027
300
At3
g063
50A
l3g0
6450
Pt1
0g01
690
Md0
0g19
6450
Fv1g
1950
0G
m01
g207
60Lj
4g00
5930
Mt4
g090
620
Vv1
4g04
450
Tc0
5g02
4340
Pt1
3g02
880
Md0
0g19
9470
Fv6g
0723
0G
m20
g374
00
Vv1
4g04
460
Tc0
5g02
4370
Md0
0g20
8810
Fv6g
0724
0
Md
01g
0141
10
Md
01g
0141
30
Md0
4g01
7400
Md1
5g02
6460
SK
Vv0
0g22
160
Tc01
g010
070
At2
g219
40A
l4g0
1190
Pt0
2g06
000
Md0
0g39
6950
Fv6g
0158
0G
m04
g39
700
Lj1g
0148
90
Vv0
7g06
350
At4
g395
40A
l7g0
1530
Pt0
5g08
460
Md0
2g00
9820
Gm
04g
3971
0
Pt0
7g06
400
Gm
05g3
1730
Gm
08g1
4980
SK
L1V
v14g
1400
0Tc
04g0
0471
0A
t3g2
6900
Al5
g056
50P
t17g
0878
0Fv
6g51
520
Gm
02g0
8050
Lj1g
0084
80
Gm
16g2
7060
SK
L2V
v02g
0194
0Tc
03g0
2993
0A
t2g3
5500
Al4
g208
70P
t03g
0857
0M
d00g
0615
70Fv
0g29
740
Gm
01g0
1890
Lj3g
0209
70M
t1g0
0945
0
Md0
0g43
2830
Fv2g
1808
0Lj
3g02
0980
Mt5
g029
550
Md0
6g00
2680
ES
PS
Vv1
5g09
330
Tc01
g037
810
At1
g488
60A
l1g4
2610
Pt0
2g14
550
Md0
0g03
0870
Fv7g
1142
0G
m01
g336
60Lj
3g02
5840
Mt4
g024
620
Vv1
5g09
350
At2
g453
00A
l4g3
3160
Pt1
4g06
200
Md0
0g27
1560
Gm
03g0
3190
CS
Vv0
6g05
280
Tc10
g005
370
At1
g488
50A
l1g4
2550
Pt0
8g03
850
Md0
0g35
5380
Fv4g
1866
0G
m10
g355
60Lj
0g03
8950
Mt1
g09
5160
Vv1
3g03
240
Al3
g198
80P
t10g
2170
0M
d01g
0089
50Fv
4g18
670
Gm
20g3
1980
Lj0g
2845
50M
t1g
0952
40
Md0
8g00
5430
Fv7g
2395
0M
t1g
0952
50
Fv7g
2404
0
(Con
tinue
d)
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Tohge et al. Shikimate and phenylalanine biosynthesis
Tab
le2
|Co
nti
nu
ed
No.
ID13
VV
14T
C16
AT
17A
L18
PT
21M
D22
FV23
GM
24LJ
25M
T
CM
Vv0
1g02
110
Tc02
g032
570
At1
g693
70A
l2g1
7620
Pt1
0g15
830
Md0
0g25
0450
Fv0g
0469
0G
m13
g058
30Lj
5g00
5890
Mt1
g013
820
Vv0
4g13
080
Tc04
g009
770
At3
g292
00A
l5g0
8750
Pt1
7g12
090
Md0
0g32
9990
Fv2g
5232
0G
m14
g118
70Lj
5g00
5900
Mt5
g043
210
Vv1
4g02
700
Tc09
g001
490
At5
g108
70A
l6g1
0610
Pt1
8g02
330
Md0
1g02
0010
Fv6g
4368
0G
m17
g339
40
Md1
6g00
3330
Gm
19g0
3290
Md1
7g00
4580
PAT
Vv0
7g05
790
Tc01
g009
420
At2
g222
50A
l4g0
1710
Pt0
5g07
910
Md0
0g13
5490
Fv6g
0044
0G
m05
g314
90Lj
6g00
3720
Mt8
g091
280
Vv1
8g03
130
Pt0
7g05
690
Md0
0g24
6930
Gm
08g1
4720
Md0
0g30
4630
Gm
11g
3619
0
Gm
11g
3620
0
AD
TV
v06g
0479
0Tc
02g0
3499
0A
t1g0
8250
Al1
g121
00P
t00g
1369
0M
d00
g09
9570
Fv3g
0112
0G
m11
g157
50Lj
3g02
9800
Mt2
g088
130
Vv1
0g00
970
Tc06
g019
290
At1
g117
90A
l3g0
8080
Pt0
4g01
150
Md
00g
0995
80Fv
3g16
180
Gm
11g1
9430
Lj4g
0017
80M
t4g0
5531
0
Vv1
2g10
860
Tc09
g026
620
At2
g278
20A
l4g1
2300
Pt0
4g18
820
Md0
0g45
6520
Fv3g
2994
0G
m12
g077
20M
t4g0
6107
0
Tc09
g028
840
At3
g076
30A
l5g1
2520
Pt0
8g19
820
Md0
5g00
1400
Gm
12g0
9050
Mt4
g132
250
At3
g447
20A
l6g2
2310
Pt0
9g14
910
Md1
5g01
9040
Gm
12g3
0660
At5
g226
30G
m12
g319
40
Gm
17g0
1610
Ort
holo
gous
gene
sw
ere
estim
ated
byB
LAST
sear
chin
Pla
zaw
ebsi
te.B
old
indi
cate
sta
ndem
gene
dupl
icat
ion.
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Tohge et al. Shikimate and phenylalanine biosynthesis
the intramolecular exchange of the DAHP ring oxygen with car-bon 7 to convert DAHP into 3-dehydroquinate. Unlike the fungalsituation detailed above, the plant DHQS gene is monofunctionaland only found as a single copy in all species with the exceptionGlycine max which harbors two genes in its genome (Figure 2Ab).Phylogenetic analysis of DHQS genes reveals three major cladesconsisting of (i) microphyte (ii) bryophyte, (iii) monocot, (iv)Brassicaceae, and (v) dicot species. Intriguingly, by contrast toother shikimate biosynthetic genes, gene expression of DHQSgene is not well correlated to phenylpropanoid production inArabidopsis (Hamberger et al., 2006).
3-DEHYDROQUINATE DEHYDRATASE/SHIKIMATEDEHYDROGENASE3-Deoxy-d-arabino-heptulosonate 7-phosphate is converted to3-dehydroquinate by the bifunctional enzyme 3-dehydroquinate
dehydratase/shikimate dehydrogenase (DHQD/SD), which cat-alyzes firstly the dehydration of DAHP to 3-dehydroshikimate andconsequently the reversible reduction of this intermediate to shiki-mate using NADPH as co-factor. DHQD/SD exists in three forms;bacterial specific class I shikimate dehydrogenases (AroE type),class II shikimate/quinate dehydrogenases (YdiB type), and class IIIof shikimate dehydrogenase-like (SHD-l type) (Michel et al., 2003;Singh et al., 2005). In plants class IV, enzymatic activity of DHQDis 10 times higher than SD activity indicating that the amount of3-dehydroshikimate will be more than sufficient to support fluxthrough the shikimate pathway (Fiedler and Schultz, 1985). Thisbifunctional enzyme plays an important role in regulating metab-olism of several phenolic secondary metabolic pathways (Bentley,1990; Ding et al., 2007). In general, seed plants contain a singleDHQD/SD gene which contains a sequence encoding a plastictransit peptide in their genome (Maeda et al., 2011, Table 2).
FIGURE 2 | Continued
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FIGURE 2 | Phylogenetic tree analysis of shikimate and phenylalaninebiosynthetic genes in 25 species. Amino acid sequence phylogenetictrees of (A) shikimate pathway: (a), DAHPS, (b) DHS, (c) DHQD/SD, (d)SK, (e) ESPS, and (f) CS, (B) phenylalanine related genes, (a) CM and (b)PAT. Amino acid sequences of shikimate biosynthetic genes are obtainedfrom Plaza database (http://bioinformatics.psb.ugent.be/plaza/).Relationships among the species considered are presented on the Plaza
website. The phylogenetic tree was constructed with the aligned proteinsequences by MEGA (version 5.10; http://www.megasoftware.net/;Kumar et al., 2004) using the neighbor-joining method with the followingparameters: Poisson correction, complete deletion, and bootstrap(1000 replicates, random seed). The protein sequences were alignedby Plaza. Values on the branches indicate bootstrap support inpercentages.
However, an exception to this statement is Nicotiana tabacumwhich contains two genes in its genome. Intriguingly, silencingof NtDHD/SHD-1 results strong growth inhibition and reductionof the level of aromatic amino acids, chlorogenic acid, and lignincontents (Ding et al., 2007), however, a second cytosolic isoformcan compensate for the production of shikimate but not at thephenotypic level. On a more general basis phylogenetic analysisreveals that microphytes also contain a low number of DHQD/SDgenes (between one and two), whilst clear separation between (i)the microphyte clade, (ii) bryophyte clade, (iii) monocot clade,(iv) woody species-specific tandem gene duplication clade, and (v)dicot clades could be observed (Figure 2Ac; Table 2). Interestingly,the observation of the woody species-specific tandem gene dupli-cation clade suggests that these species evolved after DHQD/SD
gene duplication. The cytosolic localization of NtDHD/SHD-2 isintriguing since the presence of DAHP synthase, ESPS synthaseand CM isoforms lacking N-terminal plastid targeting sequenceshas been reported (d’Amato, 1984; Mousdale and Coggins, 1985;Ganson et al., 1986). Furthermore, the findings that both ESPS syn-thase and shikimate kinase (SK) are active even when they retaintheir target sequences (Dellacioppa et al., 1986; Schmid et al., 1992)suggests that they could also potentially be constituents of a cytoso-lic pathway. Finally, experiments in which isolated and highly puremitochondria were supplied with 13C labeled glucose to investi-gate the binding of the cytosolic isoforms of glycolysis (Giege et al.,2003) also revealed 13C enrichment in shikimate (Sweetlove andFernie, 2013), indicating that a full cytosolic pathway is likely alsoin this species.
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Tohge et al. Shikimate and phenylalanine biosynthesis
SHIKIMATE KINASEThe fifth reaction of the shikimate pathway is catalyzed by SKwhich catalyzes the ATP-dependent phosphorylation of shikimateto shikimate 3-phospate (S3P). E. coli has two SKs, one of classI (AroL type) and one of II (AroK type) which share only 30%sequence identity (Griffin and Gasson, 1995; Whipp and Pittard,1995; Herrmann and Weaver, 1999). In plants, different numbersof SK isoforms are found in several species; only one in greenalgae, lycophytes, and bryophytes but between one and three inmonocot and dicot plants (Table 2). A phylogenetic analysis ofSK genes presents five major clades consisting of (i) microphyte,(ii) bryophyte, (iii) dicot woody species-specific clade, (iv) mono-cot clade, and (v) dicot species clade (Figure 2Ad). Anaylsis ofthe SK protein of Spinacia olerancea revealed that it was mod-ulated by energy status and is therefore similar to bacterial SKprotein and other ATP-utilizing enzymes (Pacold and Anderson,1973; Huang et al., 1975; Schmidt et al., 1990). For this reason ithas recently been postulated that SK may link to energy requir-ing shikimate pathway to the cellular energy balance (Maeda andDudareva, 2012), however, direct experimental support for thishypothesis is currently lacking. In Arabidopsis, homologous genesnamed SKL1 and SKL2,which are functionally required for chloro-plast biogenesis have been demonstrated to have arisen from SKgene duplication (Fucile et al., 2008). SKL1 and SKL2 orthologshave been found in several seed plant species, but not in greenalgae (Table 2).
5-ENOLY PYRUVYLSHIKIMATE 3-PHOSPHATE SYNTHASEThe 5-enolypyruvylshikimate 3-phosphate synthase (EPSPS, 3-phosphoshikimate 1-carboxyvintltransferase) is the sixth stepand here a second PEP is condensed with S3P to form 5-enolpyruvylshiukimate 3-phosphate (EPSP). Since EPSPS is theonly known target for the herbicide glyphosate (Steinrucken andAmrhein, 1980), isoforms of this enzyme are often classifiedaccording to their sensitivity of glyphosate, glyphosate sensitiveEPSPS class I is present in bacteria and plant species, whilstglyphosate insensitive EPSPS class II which has been reportedin certain bacteria such as Agrobacterium (Fucile et al., 2011).In plants, different number of EPSPS isoforms is found in sev-eral species; only a single isoform in green algae, lycophytes, andbryophytes, but either one or two are found in monocot anddicot species (Table 2). Phylogenetic analysis of EPSPS genesrevealed, atypically for genes associated with shikimate metabo-lism, that five major groups could be observed; (i) microphyte, (ii)bryophyte, (iii) Brassicaceae specific clade, (iv) monocot species,and (v) dicot species clade (Figure 2Ae). There are clear indica-tions that duplicated EPSPS genes in Arabidopsis, apple, grapevine,soybean, and poplar are the result of independent duplicationevents within their lineages with both copies being maintainedin Arabidopsis (Hamberger et al., 2006), however, the reason forthe unique divergence in this gene of the pathway is currentlyunclear.
CHORISMATE SYNTHASEChorismate, the final product of the shikimate pathway, is subse-quently formed by chorismate synthase (CS) which catalyzes thetrans-1,4 elimination of phosphate from EPSP. CSs are categorized
within one of two functional groups (i) fungal type bifunctionalCS which are associated with NADPH-dependent flavin reductaseor (ii) bacterial and plant type monofunctional CSs (Schaller et al.,1991; Maeda and Dudareva, 2012). The reaction catalyzed by CSrequires flavin mononucleotide (FMN) and its overall reaction isredox neutral (Ramjee et al., 1991; Macheroux et al., 1999; Macleanand Ali, 2003). The FMN represents supplies an electron donor forEPSP which facilitates the cleavage of phosphate. The first clonedplant CS gene was that from C. sempervirens (Schaller et al., 1991)which contains a sole CS in its genome. Given that this gene has a5′ plastid import signal sequence, these results indicate that theremay be no CS outside of the plastid this species. Surveying otherspecies revealed that one to two CS genes were present in greenalgae, lycophytes, and bryophytes as well as dicot specie but thatone to three are present in the genomes of apple and leguminousspecies (Table 2). A phylogenetic analysis of CS genes reveals threemajor clades constituted by (i) microphyte, (ii) monocot, (iii) dicotspecies (Figure 2Af).
CHORISMATE MUTASEChorismate mutase catalyzes the first step of phenylalanine andtyrosine biosynthesis and additionally represents a key step oftoward the branch split of tryptophan biosynthesis. CM catalyzesthe transformation of chorismate to prephenate via a Claisenrearrangement. The bacterial minor CM proteins (AroQ type, classI CM) display monofunctional enzymatic activity whilst severalbifunctional CMs such as CM-PDT, CM-PDH, and CM-DAHPhave been additionally been found in fungi and bacteria (class IICM, Euverink et al., 1995; Romero et al., 1995; Chen et al., 2003;Baez-Viveros et al., 2004). In spite of the fact of only one CM geneis present in algae and lycophyte genomes, more a single gene copy(two to five) are found in bryophytes as well as monocot and dicotspecies (Table 2). In seed plants, the CM1 bears a putative plastidtransit peptide,but CM2 does not and is additionally usually insen-sitive to allosteric regulation by aromatic amino acids (Benesovaand Bode, 1992; Eberhard et al., 1996; Maeda and Dudareva, 2012).Several plant species, especially dicot plants, have an additionalCM3 family gene which displays high sequence similarity to CM2yet bears a putative plastid transit peptide. For example, Ara-bidopsis has three isozymes named AtCM1 (At3g29200), AtCM2(At5g10870), and AtCM3 (At1g69370) (Mobley et al., 1999; Tzinand Galili, 2010). Phylogenetic analysis of the CS genes revealsthree major clades constituting of (i) AtCM2 clade, (ii) microphyteand bryophyte clade, and (iii) AtCM2 clade (Figure 2Ba). Addi-tionally, clade iii shows two sub-groups, (iii-a) AtCM3 sub-groupsand (iii-b) AtCM1 sub-group (Figure 2Ba) (Eberhard et al., 1996).In spite of that the CM2 sub-group contains all species of seedplants, monocot species are not contained into AtCM3 sub-group.Recently the importance of CM has been extended beyond intra-cellular metabolism, In Zea mays, the chorismate mutase Cmu1secreted by Ustilago maydis, a widespread pathogen character-ized by the development of large plant tumors and commonlyknown as smut, is a virulence factor. The uptake of the UstilagoCMu1 protein by plant cells allows rerouting of plant metabo-lism and changes the metabolic status of these cells via metabolicpriming (Djamei et al., 2011). It now appears that secreted CMsare found in many plant-related microbes and this form of host
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manipulation would appear to be a general weapon in the arsenalof plant pathogens.
PREPHENATE AMINOTRANSFERASE AND AROGENATEDEHYDRATASEPrephenate aminotransferase (PAT) and arogenate dehydratase(ADT) catalyze the final steps for production of phenylalanine.Whilst ADT was first cloned in 2007 (Cho et al., 2007; Huang
et al., 2010), it is only more recently that PAT was cloned. Paperspublished in 2011 identified PAT in Petunia hybrid, Arabidopsisthaliana, and Solanum lycopersicum (Dal Cin et al., 2011; Maedaet al., 2011) and established that it directs carbon flux fromprephenate to arogenate but also that it is strongly and co-ordinately upregulated with genes of primary metabolism andphenylalanine derived flavor volatiles. In plant species, a differentnumber of PAT isoforms have been found. Although green algae
FIGURE 3 | Heat map for isoforms of shikimate-phenylalaninebiosynthetic genes in plant genomes and hypothetical schemefor the evolution of phenylalanine derived phenolic secondarymetabolism. (A) Heap map overview of number of
shikimate-phenylalanine biosynthetic gene isoforms in 25 species. (B)Hypothetical schematic figure for shikimate-phenylalaninebiosynthetic genes and their evolution of phenolic secondarymetabolism.
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only contain single PAT and ADT genes, monocot species havebetween one and two PATs and between two and four ADTs whilstdicot plants genomes contain the same number of PATs but twoto eight ADTs (Table 2). Phylogenetic analysis of PAT genes showsthree major clades of (i) microphyte, (ii) monocot, and (iii) dicotspecies (Figure 2Bb).
GENES INVOLVED IN PLANT PHENOLIC SECONDARYMETABOLISMSPhenolic secondary metabolism displays an immense chemicaldiversity due to the evolution of enzymatic genes which areinvolved in the various biosynthetic and decorative pathways.Such variation is caused by diversity and redundancy of sev-eral key genes of phenolic secondary metabolism such as PKSs,cytochrome P450s (CYPs), Fe2+/2-oxoglutarate-dependent dioxy-genases (2ODDs), and UDP-glycosyltransferases (UGTs). On theother hand, there are other general phenylpropanoid relatedbiosynthetic genes, phenylalanine ammonia-lyase (PAL), cinna-mate 4-hydroxylase (C4H), and 4-coumarate:coenzyme A ligase(4CL), which are required in order to differentiate various classesof phenolic secondary metabolism. All of these core genes encodeimportant enzymes which activate a number of hydroxycinnamicacids to provide precursors for the biosynthesis of lignins, mono-lignals, and indeed all other major phenolic secondary metabolitesin higher plants (Lozoya et al., 1988; Allina et al., 1998; Hu et al.,1998; Ehlting et al., 1999; Lindermayr et al., 2002; Hamberger andHahlbrock, 2004). Since phenolic secondary metabolism displayconsiderable species-specificity, investigation of the genes encod-ing the responsible biosynthetic enzymes are frequently used asan example of chemotaxonomy for understanding plant evolu-tion. However, considering the evolution of these genes in iso-lation is rather restrictive a deeper understanding is providedby combining this with investigation of the evolution of theshikimate-phenylalanine biosynthetic genes in the green lineage.
CONCLUSIONDuring the long evolutionary period covered from aquatic algaeto land plants, plants have adapted to the environmental nicheswith the evolutionary strategies such as gene duplication and
convergent evolution by the filtration of natural selection. Genes ofplant shikimate biosynthesis have evolved accordingly (Figure 3).In this review, we demonstrated that biosynthetic genes ofaromatic amino acid primary metabolism are well conservedbetween algae and all land plants. However, in contrast to algaespecies which have neither isoforms nor duplicated genes in theirgenomes, all land plants harbor gene duplications including tan-dem gene duplications which are particularly prominent in thecases of DAHPS, DHQD/SD, CS, CM, and ADT (Figure 3A;Table 2). Our phylogenetic analysis revealed clear separationbetween algae, monocots, dicots, woody species, and leguminousplants. Analysis of the presence and copy number of key genesacross these species gives several hints as to how to improveour understanding of the scaffold from which these genes haveevolved. However, the exact evolutionary pressures on genes ofshikimate biosynthesis including the unique occurrence of theArom complex will require considerable further studies. That saidit is intriguing to compare and contrast biosynthetic genes ofthose downstream of them in the production of plant phenolics(Figure 3B). Interestingly, shikimate pathway genes are ubiquitousacross the green lineage whilst this cannot be said for all down-stream genes of phenylpropanoid biosynthesis. Furthermore, thereis a much greater gene duplication within phenylpropanoid thanshikimate biosynthesis (Figure 3A; Table 2). This fact also reflectedin the level of chemical diversity of the respective pathways with theessentiality of the shikimate pathway preventing much diversity,but phenylpropanoid species often being redundant in function toone another. It would seem likely that the phenylpropanoid path-way initially arose via mutations accumulating in the shikimatepathway genes. However, whilst these were potentially beneficialin land plants for reasons we discuss in our recent review of thesecompounds (Tohge et al., 2013) they do not appear to share theessentiality of shikimate across the entire green lineage.
ACKNOWLEDGMENTSResearch activity of Takayuki Tohge is supported by the Alexan-der von Humboldt Foundation. Funding from the Max-Planck-Society (to Takayuki Tohge, Mutsumi Watanabe, Rainer Hoefgen,Alisdair R. Fernie) is gratefully acknowledged.
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Conflict of Interest Statement: Theauthors declare that the research wasconducted in the absence of any com-mercial or financial relationships thatcould be construed as a potential con-flict of interest.
Received: 21 January 2013; accepted: 04March 2013; published online: 27 March2013.
Citation: Tohge T, Watanabe M, Hoef-gen R and Fernie AR (2013) Shikimateand phenylalanine biosynthesis in thegreen lineage. Front. Plant Sci. 4:62. doi:10.3389/fpls.2013.00062This article was submitted to Frontiers inPlant Metabolism and Chemodiversity, aspecialty of Frontiers in Plant Science.Copyright © 2013 Tohge, Watan-abe, Hoefgen and Fernie. This is anopen-access article distributed underthe terms of the Creative Com-mons Attribution License, which per-mits use, distribution and reproduc-tion in other forums, provided the orig-inal authors and source are creditedand subject to any copyright noticesconcerning any third-party graphicsetc.
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Shikimate and phenylalanine biosynthesis in the green lineageIntroductionShikimate biosynthesis and phenylalanine derived secondary metabolism in plants3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase3-Dehydroquinate synthase3-Dehydroquinate dehydratase/shikimate dehydrogenaseShikimate kinase5-Enolypyruvylshikimate 3-phosphate synthaseChorismate synthaseChorismate mutasePrephenate aminotransferase and arogenate dehydrataseGenes involved in plant phenolic secondary metabolismsConclusionAcknowledgmentsReferences
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